Food Flavour Technology

Food Flavour Technology

ANDREW J. TAYLOR, Editor

Blackwell

Food Flavour Technology

Sheffield Food Technology Series Editors: B.A. Law and A.J. Taylor A series which presents the current state of the art of chosen sectors of the food and beverage industry. Written at professional and reference level, it is directed at food scientists and technologists, ingredients suppliers, packaging technologists, quality assurance personnel, analytical chemists and microbiologists. Each volume in the series provides an accessible source of information on the science and technology of a particular area. Titles in the series: Chemistry and Technology of Soft Drinks and Fruit Juices Edited by P.R. Ashurst Natural Toxicants in Food Edited by D.H. Watson Technology of Bottled Water Edited by D.A.G. Senior and P.R. Ashurst Environmental Contaminants in Food Edited by C.F. Moffat and K.J. Whittle Handbook of Beverage Packaging Edited by G.A. Giles Technology of Cheesemaking Edited by B.A. Law Mechanisation and Automation in Dairy Technology Edited by A.Y. Tamime and B.A. Law Enzymes in Food Technology Edited by R.J. Whitehurst and B.A. Law Food Flavour Technology Edited by A.J. Taylor

Food Flavour Technology Edited by ANDREW J. TAYLOR Professor of Flavour Technology Division of Food Sciences University of Nottingham, UK

Sheffield A c a d e m i c Press

CRC Press

First published 2002 Copyright © 2002 Sheffield Academic Press Published by Sheffield Academic Press Ltd Mansion House, 19 Kingfield Road Sheffield SI 1 9AS. UK ISBN 1-84127-224-8 Published in the U.S.A. and Canada (only) by CRC Press LLC 2000 Corporate Blvd., N.W. Boca Raton, FL 33431, U.S.A. Orders from the U.S.A. and Canada (only) to CRC Press LLC U.S.A. and Canada only: ISBN 0-8493-9783-9 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 or otherwise, without the prior permission of the copyright owner. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. Printed on acid-free paper in Great Britain by Antony Rowe Ltd. Chippenham, Wiltshire British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library

Library of Congress Cataloging-in-Publication Data: A catalog record for this book is available from the Library of Congress

Preface At this moment, food flavour is of great interest because consumers are demanding better-tasting foods, made from natural ingredients and at a reasonable price. This has not always been the case. Early in the history of the human race, flavour perception is believed to have protected us from foods that might have harmed us. Babies still retain the ability to discriminate sweet foods from bitter and demonstrate their disgust clearly when fed bitter foods. We learn flavours as we grow, leading to personal and cultural differences and prejudices. The desire for flavour has always been strong and the many adventurers who set out from Europe to obtain precious herbs and spices from remote corners of the world are testament to that need. Whether the flavours were used for their intrinsic flavour properties or to mask the strong, rancid notes associated with poor quality food preservation is not so clear! Even today, the failure of many new food products is attributed to poor quality flavour characteristics. A classic example is the desire to produce foods that better fit our nutritional requirements—such as low fat, low sugar, low salt and high fibre foods. Changing the composition has an immediate and profound effect on flavour and texture and, although some foods have been successfully modified (e.g. low fat spreads), many others still suffer from poor flavour quality. The challenge for the food and flavour industry, therefore, is to formulate flavours that will not only satisfy our diverse sensory expectations but also meet with our expectations on safety and origin. Flavour creation has always been an art but there is an increasing scientific and technological basis behind the various parts of the process. The aim of this book is to present the scientific and technical principles that underpin the industry and that are used increasingly by industrialists and academics working with flavours. Since flavour creation is the key, the book starts with a chapter describing the process of flavour creation. It is a personal and rare account of flavour creation, which links together science and art. It leads naturally on to the other chapters, which are grouped to cover the sources of flavour (chapters 2-4) , the delivery of flavour (chapters 5-7) and the analysis and regulation of flavour (chapters 8-10). Authors were asked to include the underpinning scientific background to their chapters. Each has taken a personal view and the chapters demonstrate the different possible approaches. I am indebted to the chapter authors for providing such excellent material and to the publishers for their help and encouragement. My job has been to arrange the material into a structured account and to add explanatory notes

vi

PREFACE

here and there. As ever, praise should be directed to the authors; any remaining mistakes must be the editor's responsibility! Andrew Taylor

Contributors Dr Daniel Benczedi

Firmenich SA, Corporate R&D, Rue de la Bergere 7, PO Box 148, CH-1217 Meyrin 2, Geneva, Switzerland

Professor Ralf G. Berger

Zentrum Angewandte Chemie, Institut fur Lebenschemie, Universitat Hannover, Wunstorfer StraBe 14, D-30453 Hannover, Germany

Dr Peter S. J. Cheetham

ZYLEPSIS Ltd., 6 Highpoint, Henwood Business Estate, Ashford, Kent TN24 SDH

Mr Jack Knights

Consultant, Wykes Lodge South, Whites Lane, New Duston, Northampton NN5 6UW

Dr Ulrich Krings

Zentrum Angewandte Chemie, Institut fur Lebenschemie, Universitat Hannover, Wunstorfer StraBe 14, D-30453 Hannover, Germany

Dr Josef Kerler

Quest International, Huizerstraatweg 28, 1411 GP Naarden, The Netherlands

Dr Rob Linforth

Samworth Flavour Laboratory, Division of Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics. LEI2 3RD, UK

Professor Ann C. Noble

Department of Viticulture and Enology, University of California, Davis, CA 95616, USA

Professor Gary Reineccius Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108 US A Dr Jacques P. Roozen

Laboratory of Food Chemistry, Department of Agrotechnology and Food Science, Wageningen University and Research Center, P.O. Box 8129, 6700 EV Wageningen, the Netherlands

Vlll

CONTRIBUTORS

Dr Saskia M. van Ruth

Nutritional Sciences, Department of Food Science, Food Technology and Nutrition, University College Cork, Western Road, Cork, Ireland

Dr Chris Winkel

Quest International, Huizerstraatweg 28, 1411 GP Naarden, The Netherlands

Mr John Wright

International Flavours and Fragrances, 706 Princeton Kingston Road, Princeton, NJ 08540. USA

Dr Holger Zorn

Zentrum Angewandte Chemie, Institut fur Lebenschemie, Universitat Hannover, Wunstorfer StraBe 14, D-30453 Hannover, Germany

Contents

1

Creating and formulating JOHN WRIGHT 1.1 1.2 1.3

1.4

1.5

1.6 1.7 1.8 1.9

1.10

Introduction 1.1.1 A little history Interpreting analyses Flavour characteristics 1.3.1 Primary characters Secondary characteristics Taste effects Complexity Flavour balance Unfinished work Applications 1.4.1 Ingredient factors 1.4.2 Processing factors 1.4.3 Storage factors 1.4.4 Consumption factors Flavour Forms 1.5.1 Water-soluble liquid 1.5.2 Clear water-soluble liquid 1.5.3 Oil-soluble liquid 1.5.4 Emulsion-based 1.5.5 Dispersed 1.5.6 Spray-dried Production issues Regulatory affairs A typical Commercial considerations 1.9.1 International tastes 1.9.2 Abstract 1.9.3 Matching 1.9.4 Customers Summary

flavours

flavours flavours flavours flavours flavours flavours

flavour

flavours

1 1 1 2 3 3 5 6 7 7 8 9 9 11 11 12 12 12 14 14 15 15 15 17 18 18 21 21 23 24 25 26

The basic chemistry and process conditions underpinning reaction flavour production JOSEF KERLER and CHRIS WINKEL

27

2.1 Introduction 2.2 General aspects of the Maillard reaction 2.2.1 Intermediates as flavour precursors 2.2.2 Carbohydrate fragmentation

27 27 28 33

CONTENTS 2.2.3 Strecker degradation 2.3 Important aroma compounds derived from Maillard reaction in food and process flavours 2.3.1 Character impact compounds of thermally treated foods 2.3.2 Character impact compounds of process flavours 2.4 Preparation of process flavours 2.4.1 General aspects 2.4.2 Factors influencing flavour formation 2.4.3 Savoury process flavours 2.4.4 Sweet process flavours 2.5 Outlook References

35 37 37 41 44 44 45 48 51 51 52

Biotechnological flavour generation RALF G. BERGER, ULRICH KRINGS and HOLGER ZORN

60

3.1 3.2 3.3 3.4

60 60 61 62 62 66 67 69 72 73 74 75 76 77 77 78 79 80 82 82 83 84 87 88 89 91 91

Introduction Natural flavours: market situation and driving forces Advantages of biocatalysis Microorganisms 3.4.1 Biotransformation and bioconversion of monoterpenes 3.4.2 Bioconversion of C13-norisoprenoids and sesquiterpenes 3.4.3 Generation of oxygen heterocycles 3.4.4 Generation of vanillin, benzaldehyde and benzoic compounds 3.4.5 Generation of miscellaneous compounds 3.5 Enzyme Technology 3.5.1 Liberation of volatiles from bound precursors 3.5.2 Biotransformations 3.5.3 Kinetic resolution of racemates 3.6 Plant catalysts 3.6.1 Plant cell, tissue and organ cultures 3.6.2 Callus and suspension cultures 3.6.3 Organ cultures 3.6.4 Plant cell biotransformations 3.7 Flavours through genetic engineering 3.7.1 Genetically modified microorganisms 3.7.2 Isolated enzymes from genetically modified microorganisms 3.7.3 Plant rDNA techniques 3.8 Advances in bioprocessing 3.8.1 Process developments in microbial and enzyme systems 3.8.2 Process developments of plant catalysts 3.9 Conclusion References

Plant-derived natural sources of PETER S. J. CHEETHAM

flavours

4.1 Introduction 4.2 Properties of flavour molecules 4.2.1 Flavour perception 4.2.2 Differences in sensory character and intensity between isomers

105 105 105 106 113

CONTENTS

5

6

XI

4.2.3 Extraction of flavours from plant material 4.2.4 Commercial, economic and safety aspects 4.3 Dairy flavours 4.3.1 Background 4.3.2 Cream and butter 4.3.3 Cheese 4.4 Fermented products 4.4.1 Hydrolysed vegetable proteins 4.4.2 Chocolate 4.4.3 Tea 4.4.4 Coffee 4.4.5 Beer 4.4.6 Wine 4.5 Cereal products 4.6 Vegetable sources of flavour 4.6.1 Vanilla 4.6.2 Spice flavours 4.6.3 Garlic, onion and related flavours 4.6.4 Brassica flavours, including mustard and horseradish 4.6.5 'Fresh/green/grassy' 4.7 Fruit 4.7.1 Apples 4.7.2 Pears 4.7.3 Grapefruit 4.7.4 Blackcurrant 4.7.5 Raspberry 4.7.6 Strawberry 4.7.7 Apricot and peach 4.7.8 Citrus and citrus processing 4.8 Other flavour characteristics 4.9 Conclusion References

114 119 120 120 121 122 125 125 126 128 129 130 133 134 135 135 136 138 140 140 142 142 143 144 144 145 145 146 146 149 150 150

Flavour encapsulation using polymer-based delivery systems DANIEL BENCZEDI

153

5.1 Introduction 5.2 Compatibility and cohesion 5.3 Sorption and swelling 5.4 Diffusion and release 5.5 Summary References

153 154 157 160 163 164

Delivery of flavours from food matrices

167

SASKIA M. VAN RUTH and JACQUES P. ROOZEN 6.1 Introduction 6.2 Flavour properties 6.3 Thermodynamic aspects of flavour delivery 6.3.1 Definition of gas/product partition coefficients and activity coefficients 6.3.2 Types of binding

167 168 168 168 170

Xli

CONTENTS 6.3.3 Lipid-flavour interactions 6.3.4 Carbohydrate-flavour interactions 6.3.5 Protein-flavour interactions 6.4 Kinetic aspects of flavour delivery 6.4.1 Principles of interfacial mass transfer 6.4.2 Liquid food products 6.4.3 Semi-solid food products 6.4.4 Solid food products 6.5 Conclusions References

7

8

171 172 174 175 175 178 179 179 181 181

Modelling flavour release ROB LINFORTH

185

7.1 Introduction 7.2 Equilibrium partition models 7.2.1 The air-water partition coefficient 7.2.2 Estimation of Kaw using QSPR 7.2.3 Effect of lipids on volatile partitioning 7.2.4 QSPR estimation of the air-emulsion partition coefficient 7.3 Dynamic systems 7.3.1 Modelling flavour release from a retronasal aroma simulator 7.3.2 Nonequilibrium partition modelling of volatile loss from matrices 7.3.3 Modelling the gas phase dilution of equilibrium headspace 7.3.4 Modelling the gas phase dilution of equilibrium headspace above emulsions 7.3.5 Modelling the rate of volatile equilibration in the headspace above emulsions 7.4 In vivo consumption 7.4.1 Emulsion dilution during consumption 7.4.2 Effect of gas flow on volatile equilibration above emulsions 7.4.3 Modelling volatile transfer through the upper airway 7.4.4 Nonequilibrium partition model for in vivo release 7.4.5 Modelling flavour release using time intensity data 7.4.6 QSPR of in vivo volatile release from gels 7.5 Conclusion References

185 186 186 187 189 190 192 193 194 196 198 199 200 201 201 202 203 204 205 207 208

Instrumental methods of analysis GARY REINECCIUS 8.1 Analytical challenges 8.2 Aroma isolation 8.2.1 Aroma isolation methods based on volatility 8.2.2 Aroma isolation methods using solvent extraction 8.2.3 Solid-phase microextraction 8.2.4 General considerations in preparing aroma isolates 8.2.5 Aroma isolation summary 8.3 Selection of aroma isolation method 8.3.1 'Complete'aroma profile 8.3.2 Key components contributing to sensory properties 8.3.3 Off-notes in a food product 8.3.4 Monitoring aroma changes in foods

210 210 12 13 20 22 24 25 26 26 27 27 28

CONTENTS 8.3.5 Using aroma profiles to predict sensory response 8.3.6 Summary comments on isolation methods 8.4 Aroma isolate fractionation prior to analysis 8.4.1 Fractionation of concentrates prior to analysis 8.5 Flavour analysis by gas chromatography 8.5.1 High-resolution gas chromatography 8.5.2 Gas chromatography-olfactometry 8.5.3 Specific gas chromatographic detectors 8.6 Flavour analysis by high-pressure liquid chromatography 8.7 Identification of volatile flavours 8.7.1 Gas chromatography 8.7.2 Infrared spectroscopy 8.7.3 Mass spectrometry 8.8 Electronic 'noses' 8.9 Summary References

Sensory methods of flavour analysis ANN C. NOBLE 9.1 Introduction 9.2 Types of test 9.2.1 Discrimination tests 9.2.2 Intensity rating tests 9.2.3 Time-intensity 9.2.4 Descriptive analysis 9.2.5 Consumer tests 9.2.6 Quality control tests 9.3 Sensory testing administration 9.3.1 Facilities 9.3.2 Test administration 9.3.3 Experimental design 9.4 Selection and training of judges 9.4.1 Human subject consent forms and regulations 9.4.2 Judge selection 9.4.3 Training 9.4.4 Reference standards 9.4.5 Evaluating judge performance 9.5 Statistical analysis of data 9.5.1 Difference tests 9.5.2 Analysis of variance 9.5.3 Power 9.5.4 Principal component analysis: an exploratory tool 9.6 Relating sensory and instrumental flavour data 9.7 Summary References

Xlll 229 229 229 230 234 234 235 239 240 240 241 242 243 246 247 247

252 252 252 252 254 255 258 259 260 261 261 262 262 264 264 265 265 265 266 266 266 266 267 268 269 271 27 i

xiv

CONTENTS

10 Flavour legislation JACK KNIGHTS 10.1 Introduction 10.2 Methods of legislation 10.3 Legislation in the United States 10.4 International situation: JECFA 10.5 Council of Europe 10.6 European Community 10.7 The future References

Index

276 276 276 277 279 280 283 293 293

297

1

Creating and formulating flavours John Wright

1.1

Introduction

There are many different approaches to flavour creation and no one approach has a monopoly on the truth. Any successful technique must simply recognise the fundamental structures of flavours and then proceed logically to the goal. Some flavourists rely totally on blotters (strips of filter paper that are used to assess the odour of a mixture by sniffing). Some never touch them and make everything up to taste. Some flavourists throw most of the ingredients in at the start and some prefer to build up the composition step by step. What all successful flavourists have in common is the ability to imagine the interactions between a very complex blend of raw materials and to use intuition and creative originality to fashion a work of art. Many successful flavourists trained as scientists, but scientific method alone, without the spark of creativity, would mean that a single flavour would be a lifetime's work. 1.1.1 A little history The flavour industry originated in the latter half of the nineteenth century with essential oil distillation and botanical extraction as the main sources of raw materials, often with a strong link to the pharmaceutical industry. Simple chemicals were available by the turn of the century and during the first half of the twentieth century the fledgling flavour industry was increasingly driven by chemical research. For the flavourist of those times (who was often a pharmacist or chemist turned flavourist) the task of making flavours was purely creative. Very little was known of nature, other than the major components of essential oils and a very limited number of chemicals that had been isolated from food and successfully identified. Most new chemicals that were synthesised had no possible value in flavours. The few that proved useful became the starting point for the synthesis of every possible related compound. Thus the available raw materials were concentrated in a few obvious areas. Flavours created in this era were often not very close to the character of the real food, but some of them displayed real creativity and became accepted standards in their own right. The advent of gas chromatography and mass spectroscopy marked a real turning point for the industry. For the first time it was possible to see, in some detail, the chemicals used by nature to flavour food. The advance was.

2

FOOD FLAVOUR TECHNOLOGY

understandably, treated with some caution. What had been a purely creative and artistic profession could possibly be reduced to analytical routine. The early analyses quickly dispelled all concerns. On reconstitution it was never possible to recognise anything more than a passing resemblance to the original target. Relieved flavourists quickly settled back to the old routine, but the more astute among them recognised a few diamonds in the mud. Among the first useful results from the new analytical techniques were pyrazines and unsaturated aliphatic alcohols. Chemicals such as trimethylpyrazine gave a true-to-nature roasted note to nut and chocolate flavours and were a quantum leap ahead of the old phenolic compounds. c/s-3-Hexenol gave an authentic green note to a multitude of fruit flavours, which had previously had to depend on methylheptine carbonate to achieve a modicum of freshness (tinged with melons and violets). Many of the failings of the early analytical techniques have now been overcome. Analyses are still not easy to interpret and different techniques can give very contradictory results, but they should form the starting point.

1.2 Interpreting analyses For virtually all flavours the nucleus is nature. We may or may not aim to reproduce nature accurately, but fully understanding nature is essential even for a caricature. Analysis is therefore the first step. Usually several different types of analyses will be available (see Chapter 8 for details of the different flavour analyses available). Headspace analyses emphasise the more volatile components and are relatively true to the character of the food being analysed. The quantification of headspace analyses can usually be improved by applying vapour pressure correction factors. Extract analyses are less accurate and contain more artefacts. They are often representative of a rather cooked character, but they do emphasise the less volatile components. Specialised analyses are often carried out to investigate the high-boiling components and also the sulfur and nitrogen compounds. The flavour of food will often vary depending on the variety and the growing or cooking conditions and many analyses will quantify these differences. In consequence, the flavourist will often first have to correlate a wealth of information about the target food. The correlated list can be daunting, often running into many hundreds of different chemicals. The quantification used by the flavourist should be derived from the best of the headspace results, corrected for vapour pressure, with extract results pressed into service for the less volatile chemicals. An impossibly complex problem on the face of it! The 'trick' of being a successful flavourist hinges on the ability to imagine the smell of complex mixtures, but a mixture of several hundred ingredients is far too complex to imagine. The first priority is to simplify the problem.

CREATING AND FORMULATING FLAVOURS

3

Simplification can be carried out in three stages. The first stage is relatively easy. Many of the chemicals that have been found will be present well below their threshold levels and it might seem safe to ignore them all. Some caution is needed because synergistic and additive effects are common. The best approach at this stage is to build in a comfortable margin of error and retain any questionable chemicals. The second stage of simplification is to eliminate those chemicals that are likely to be artefacts. Artefacts can be present in the original food, produced during the separation process prior to analysis or produced during the actual analysis. Again, in cases of doubt, retain rather than discard. The final stage of simplification is to reject those notes in the target food that are genuinely present but are not desirable. Examples would be the trace by-products of fermentation and enzymatic browning in fresh fruit. Even the simplified analysis will usually be of daunting complexity. At this stage it is beneficial to try to reconstitute the analysis by mixing the flavour components in the proportions identified by the various analyses and then smelling and/or tasting the mixture. The result is certain to be disappointing, but it will serve to clarify the key aroma characteristics of the target food and it should also be reanalysed to improve the quantification of the original analysis. Reanalysis will involve trying to recreate, as closely as possible, the conditions of the original analysis. It may highlight the odd errors of identification, but it will invariably give a much-improved quantitative base to start from. Reconstitution at this stage may now give a recognisable product, but not one that anybody would be remotely happy to buy. It is time to abandon the strictly scientific approach and move on to the more abstract creative approach. 1.3 Flavour characteristics Smelling and tasting the target food will give the flavourist a good idea of which aroma characteristics are important. Reconstituting the analysis will clarify this assessment even further and may well add a few unexpected notes. The aroma characteristics can be divided into two broad categories, primary and secondary characters. 1.3.1 Primary characters Primary characters are essential to the recognition of the target food. They constitute the basic skeleton of the flavour. Good examples are 'violet' (a-ionone) in raspberries and 'clove' (eugenol) in bananas. It is impossible to create a realistic flavour without some contribution from these notes. Secondary characters are not essential for recognition but contribute an optional descriptive characteristic. Good examples are 'leaf green' (cis-3hexenol) in strawberries and 'dried' (2-methylbutyric acid) in apricots. In both

4

FOOD FLAVOUR TECHNOLOGY

cases it is perfectly possible to make good, authentic flavours without these notes. Their effect is simply to vary the type of flavour to green strawberries and dried apricots respectively. Strictly speaking, the primary characteristics can also be regarded, in some circumstances, as having secondary characteristics as well. A raspberry flavour with unnaturally emphasised a-ionone will smell distinctly violet. This is not a problem because the object of this exercise is, once again, simplification. It allows the flavourist to balance the primary characteristics in isolation and leave the secondary characteristics for later. Flavours vary greatly in the complexity of their primary recognition characteristics. The simplest example, at first sight, is probably vanilla. On its own, the chemical vanillin smells recognisably of vanilla. For many vanilla flavours in common use worldwide, this is all the primary character needed. Where consumers are accustomed to a more complex flavour, such as the character of real vanilla beans, vanillin alone will not suffice to build a recognisable skeleton. Strawberry is a more complex flavour and a more complex mixture of notes is required to achieve a recognisable flavour. In this example 'peach' (y-decalactone), 'fruity' (ethyl butyrate), 'guava' (methyl cinnamate) and 'candy' (Furaneol), blended in the correct proportions, would be the primary characters for the strawberry flavour skeleton. Some of the primary characteristics will be simple and will be represented by just one chemical in the analysis. Others may be more complex and may be represented by several chemicals. An example is the 'peach' note in fruit flavours. Major contributors to this note in many fruit products are y-decalactone and y-dodecalactone. Both chemicals have a similar 'peach' odour, but the taste characteristics intensify (and the odour strength decreases) with increasing molecular weight. When several chemicals contribute, the balance between the different components may need to be adjusted from that indicated by the analysis. Fortunately, that task can often be deferred until the basic skeleton of the flavour has been devised. The flavourist is now ready to begin the real creative work. The objective is to achieve the best possible combination of what is now a reasonably limited number of chemicals to obtain a recognisable flavour skeleton. The analysis can be taken as a starting point, but it is no more than that. Even if the analysis is entirely quantitatively accurate, which is unlikely, it is still probably a long way from the optimum blend. It represents, at best, a specific example of the target food rather than one with every characteristic optimised—something that never quite occurs in nature! Ultimately, individual notes should be emphasised or reduced to make the flavour more attractive than the specific example of nature that has been analysed. It is possible, at this point, to try to take a relatively scientific approach and blend the two most important components first. The next step would be to

CREATING AND FORMULATING FLAVOURS

5

determine the best level for the third component, and so on. The problem with this approach is that the presence of the third component alters the ideal balance between the first two components. The scientific approach rapidly becomes unimaginably complex and impossibly time consuming. The best approach is to plunge in, taking the analysis as a starting point, and experiment with blends to understand the role of each of the primary characteristics. Speed is normally vital for commercial reasons, but it is also vital if the flavourist is to remain fresh and able to smell accurately. For that reason it is best just to use blotters at this stage and to experiment with large rather than cautious changes. If an addition is overdone it can be blended back very quickly. If it is underdone, it is a slow process to carry on adding small quantities and there is a very real risk that the nose will fatigue to the chemical being added. 1.3.2 Secondary characteristics Once the basic skeleton has been built, the flavourist has to concentrate on the more complex secondary characteristics. These can generally be worked on in groups. Green notes, for example, usually contain several sub-categories and many different chemicals. Our strawberry flavour would almost certainly contain the common 'leaf green' character m-3-hexenol, but it could also contain lesser quantities of 'fruity green' (cw-3-hexenyl acetate), 'apple green' (trans-2hexenal), 'melon green' (melonal), 'unripe green' (hexanal) and 'tropical green' (cw-3-hexenyl butyrate). Once again the empirical approach is used to optimise this blend. Working through all the secondary characteristics will probably take some time. It is still best to use a blotter at this stage and to experiment with large rather than small changes. By now the first stirrings of pride should be evident. It is time to taste the flavour. Tasting solutions should be simple and appropriate. If, for example, the target is a fruit and contains sugar and acid, then the taster should contain sugar and acid for the flavour to be appreciated accurately. Forget the end application at this stage. Two problems are apparent. The first, and most obvious, is that the balance between the components will seem a little different in aqueous solution from the way it appeared on the blotter. This is something flavourists learn to allow for when using blotters and is usually only a problem for trainees. Blotters offer three great advantages to the flavourist. They allow a very quick evaluation of each flavour. They also allow the simple comparison of many variants. Blotters uniquely offer a panorama of different aspects of your flavour as they air off and the more volatile components evaporate. This is a big advantage because it allows you to smell 'through' the flavour as it evaporates. The odour approximates that experienced in a simple taster for a relatively short time, usually about five to ten minutes after dipping.

6

FOOD FLAVOUR TECHNOLOGY

The second problem is that some of the real taste (as opposed to odour) characteristics may be partially or even totally missing. For some flavours, such as roast beef, the taste element is obviously vital. Even when it is not so obviously important, for example in bananas, it is still surprisingly vital to the realism of the flavour. Correcting the taste imbalance is the next step in the flavour creation process. 1.3.3 Taste effects Taste effects are normally confined to individual flavouring ingredients that are highly water-soluble or have a high molecular weight. Research on taste has lagged far behind that on odour, so natural extracts are still widely used to confer subtle taste effects. Maltol is a good example of a water-soluble taste effect ingredient. Maltol has a pleasant candyfloss odour, and a lingering sweet aftertaste, and is claimed to have flavour-enhancing properties. It forms an important part of the aroma of a number of flavours, but the use of maltol as a taste ingredient dwarfs its use as an odour ingredient. Ethylmaltol is stronger than maltol, has similar taste and odour characters, but is not found in nature. Furaneol is even stronger than ethylmaltol and is found widely in nature. The only drawback to the use of Furaneol is the fact that it can be easily oxidised. Vanillin is another watersoluble ingredient frequently used for its sweet taste effect and vanilla odour. Vanillin is widely found in nature and can be integrated into many flavour types. The taste effect of high molecular weight ingredients can be illustrated by the lactones in dairy flavours. The two most important lactones in all dairy flavours are 8-decalactone and S-dodecalactone. S-Decalactone provides an excellent creamy odour in dairy flavours. 8-Dodecalactone has a similar odour but has only about 10% of the odour strength of 8-decalactone. The two ingredients have similar costs and, if odour were the only consideration, it would not make any sense to use 8-dodecalactone. The higher molecular weight of S-dodecalactone gives it a noticeable creamy, oily taste. If cost were no object, the best combined taste and odour results would be achieved by a mixture of ten parts of S-dodecalactone and one part of 8-decalactone. Many high-boiling, nature-identical chemicals have been little used in flavours because of the historical emphasis on odour rather than taste. They can often play a very useful role in enhancing taste characteristics even though they have little or no effect on the odour of the flavour. A wide range of natural botanical extracts have useful taste characteristics. Kola nut extract has a good astringent character, ginger extract has a hot character, Saint John's bread extract has an attractive fruity sweetness and gentian extract has a lingering bitterness. All of these extracts also possess noticeable odours and care must be taken to blend in their odour when they are added to a flavour for their taste effect.

CREATING AND FORMULATING FLAVOURS

1.3.4

7

Complexity

Flavour formulations vary radically in complexity. The simplest flavour can be based on just one component. Many flavours, just like nature, contain hundreds of ingredients. Which is best? Very simple flavours have been popular since the earliest days of the flavour industry. Vanillin, isoamyl acetate and benzaldehyde have been the most popular single-component examples. Very simple flavours may represent an attractive caricature but they never taste like a real food. At the other extreme, very complex flavours often lack impact and can taste flat and characterless. Complex flavours can be deliberate (the result of slavishly following every detail of an analysis) or accidental (the result of lazy blending of flavours and intermediates). If a natural character is desired, then the optimum level of complexity is often the minimum number of components required to prevent the taster from perceiving the individual characters. This level of complexity can vary from perhaps as few as 15 components in simple fruit flavours to up to 100 in the most complex flavour of cooked food. There are, however, some important exceptions to this general rule. The key problem with complex flavours is the fact that a mixture of two chemicals usually smells weaker than the sum of its parts. The perceived intensity of flavour chemicals has a logarithmic rather than a linear relationship to concentration. At low concentrations, near the threshold, the logarithmic relationship does not hold because the chemical is not perceived at all until it reaches the threshold level. At high concentrations the relationship also does not hold because the nose fatigues to the stimulus. The lower extremes of the concentration scale explain synergistic effects, which otherwise appear to contradict the general rule that a mixture smells weaker than the sum of its parts. Traces of components that, tasted individually, would be well below their threshold level can thus have significant positive effects in mixtures. At the other extreme, it is unwise to use so much of any single ingredient that the taster will quickly become fatigued. A mixture of two or more chemicals with complementary odours can often give better results. 1.3.5 Flavour balance Evaluating the flavour in tasters may also involve quite a number of modifications to improve the overall balance of the flavour. Once you have something you are basically happy with, it is a good idea to try out variations of concentration of the flavour in the taster. This is a little known, but extremely critical, way of evaluating a new flavour. Most flavours in nature are not particularly sensitive to changes in concentration. If you add twice as many apricots to a yoghurt, apart from the added acidity and sweetness, the yoghurt just tastes twice as strongly of apricots.

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The flavour does not become unbalanced. Most flavours created by humans do not fare nearly so well. It is possible to draw an analogy with jigsaw puzzles. An ingredient that does not have a counterpart in nature, in the flavour being created, can be seen as a large misshapen piece in the jigsaw puzzle. Not only is that specific piece out of place, but it also forces many of the other components out of balance. It may be possible, with enough effort, to get this flavour to taste right in a specific application and at a specific dose rate. The flaws will immediately become obvious if the application or the dose rate is varied because the apparent strengths of the different components will not change in unison. A prime example of an 'alien' unbalancing ingredient is ethyl methyl phenyl glycidate (strawberry aldehyde). This chemical is seductively attractive to flavourists because it smells more like strawberries than any other ingredient they have. It is very hard to turn your back on something that seems likely to give your flavour such a great start! It is not found anywhere in nature and it is certainly not found in strawberries. As we saw earlier, the natural character recognition skeleton of strawberry is a combination of 'peach', 'fruit', 'guava' and 'candy' primary characteristics. Ethyl methyl phenyl glycidate has a very complex odour, with a little of each of these notes. 'Peach' and 'guava' dominate and the chemical also has a strong 'jammy' character. It follows that if ethyl methyl phenyl glycidate is used in a strawberry flavour it is impossible to build up the rest of the character recognition skeleton in the correct balance and it is also impossible to avoid some degree of 'jammy' character. This phenomenon is a powerful argument for using only those ingredients that are found in nature in the target flavour. This is undoubtedly the ideal, but, as long as the odour character of a potential raw material is close to that of a naturally occurring ingredient, it is often possible to use it effectively. This sort of substitution would be very desirable if the naturally occurring ingredient were prohibitively expensive, impossible to make, or very unstable. Tasting the flavour at double the optimum dose rate will make unbalanced components horribly obvious. Once those problems are corrected, the flavour should, at last, be something that is ready to show to other people. As with everything else involved in flavour creation, opinions vary radically about when and how to solicit opinions from other flavourists, nonflavourists and sensory panels. One thing is certain—I do not know of a single instance of a really successful flavourist who works in complete isolation. 1.3.6

Unfinished work

An old saying cautions that you should 'never show fools or children unfinished work'. Like many old sayings, it has an uncomfortable kernel of truth. It certainly highlights a real dilemma for the aspiring flavourist! Successful flavourists must be able to memorise and recognise a formidable range of raw materials. They must also have the ability to imagine the effect of

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complex mixtures and the creative spark to use these talents to make original flavours. A further essential requirement for this formidable being is an abundant helping of self-confidence. By self-confidence I certainly do not mean arrogance. Input from others is vital and it should never be treated with contempt. Selfconfidence is essential to keep the flavourist sane in the face of well-meaning, but often contradictory, suggestions and criticism. Help for a trainee during the early stages of the creation of a new flavour is really the preserve of a mentor who is deeply involved in the project. It may not be obvious to other flavourists in which direction the trainee is trying to go, and their advice in the early stages of a project is likely to be wildly contradictory. Advice once the flavour has taken shape can be sought from a wide variety of sources. Other flavourists can be very helpful in a number of ways. They can give quick, and often accurate, assessments based solely on blotters. They will often have original ideas of raw materials to try out. Some will work and some will not, but the extra source of ideas is invaluable. Other flavourists can often pick out mistakes that the originator has missed or, more frequently, has become too saturated with the flavour to notice. It is important to recognise that the advising flavourist is often making an impromptu suggestion based on a quick evaluation. However good the flavourist, their suggestions are not necessarily gold dust. Sensory panels, especially expert panels, can be a valuable source of guidance on matches, hedonic ratings of new flavours and profiling. Preference mapping, linked to profiling, can provide real insight into the best way to optimise a flavour for a specific consumer group. Other, noncreative, staff can also be a useful source of criticism. It is often helpful to involve applications and sales staff. It is, after all, very difficult for sales staff to sell something that has not first been sold to them. Sensory panels and noncreative staff can rarely comment on blotters or simple tasters. The flavour must first be applied to a realistic end product. 1.4

Applications

All flavours are used in end products that impose some requirements on the finished flavour because of interactions with the finished food. These interactions can be broadly grouped into four categories—ingredients, processing, storage, and consumption. 1.4.1 Ingredient factors The most important factor is the fat content of the finished product. Flavour chemicals vary in polarity and consequently in fat solubility. Taste thresholds in fat are much higher than in water. The partition of different components in a flavour may vary, and this can alter the balance of the perceived aroma. It is

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often possible to adjust the formulation, but a better approach is to avoid drastic differences in the polarities of the flavour components. In all foods containing fat, added flavour will slowly partition between the fat and the aqueous phases on storage. This effect can be partly avoided by adding separate flavours to the fat and the aqueous phases, but this is a laborious approach and will rarely avoid subsequent partition effects. Care must be taken in application trials to store the finished food sufficiently long before tasting to allow the partition of the flavour to be substantially completed. The lipophilic gum base in chewing gum has an effect similar to that of fat, but the problems are aggravated because the flavour is gradually extracted by chewing. If there are differences in the polarities of the flavour components, the chewing gum will appear to taste mainly of the most polar components at the start of chewing. Eventually only the nonpolar chemicals will be extracted. A completely fat-soluble flavour may be necessary for some applications. At the other extreme, entirely water-soluble flavours are essential for clear soft drinks. In both cases it is difficult to produce a balanced profile within a restricted range of polarity. Natural extracts and oils often contain chemicals with widely differing polarities. They can be processed by distillation, solvent extraction and chromatography to reduce these differences. The most common example of this type of process is the deterpenation of lemon oil. Lemon oil contains about 90% of terpene hydrocarbons, which are nonpolar, low-boiling and susceptible to oxidation, and contribute little to the overall flavour character. The oil also contains about 6% of oxygenated chemicals, which are polar, relatively high-boiling and less susceptible to oxidation, and provide most of the flavour character. The level of lemon oil that would be required to impart an acceptable flavour level to lemonade would result in a level of terpene hydrocarbons in the drink well in excess of their limit of solubility. The oxygenated chemicals would be readily soluble at this level, so a clear drink could be obtained by removing the hydrocarbons from the oil. Chromatography and solvent extraction are obvious possibilities. Distillation also works because of the difference between the boiling points of the terpene hydrocarbons and most of the oxygenated chemicals. Some loss of the true lemon character is inevitable owing to processing and the small flavour contribution made by the hydrocarbons. This is more than justified by the gain in stability to oxidation. Solvent extraction generally gives better results than distillation because this method retains the most volatile aliphatic chemicals, which are responsible for the fresh, juicy character of many citrus oils, especially orange oil. Solvent extraction is discussed in more detail later in this chapter. Distillation, if it is used to produce a terpeneless oil, also removes the high-boiling antioxidants that are present in cold-pressed citrus oils. Major components of a flavour may themselves cause problems in a finished food. These problems are often changes in texture or in the stability of emulsions. The solvents are the most likely culprits, and in many instances a change of

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solvent will provide a cure. Where flavour dose rates are very high, particularly in chewing gum, individual flavour chemicals may also be responsible. When this happens the flavour can often be modified, but sometimes the only possible solution is to modify the formulation of the application. This may also be necessary if the flavour contains large quantities of a food additive, for example, an acid. Carbohydrates in a finished food may have a binding effect under certain conditions, but this is not a frequent problem. The most obvious example is the loss of flavour in bread on storage because of flavour binding by helical starch molecules. Flavour binding by proteins is a more serious problem. Most protein molecules are folded in such a way that the nonpolar amino acid side chains are on the inside and the polar groups are on the outside. Flavour chemicals can interact with the nonpolar interior regions of the protein and cause it to unfold. They can be bound into the protein by absorption at the protein surface or inclusion in the nonpolar interior. Protein flavour binding is most evident in processes involving heat and is most pronounced with carbonyl flavour chemicals. Chemical interactions and partitioning effects make it essential to wait at least 24 hours before tasting some applications. 1.4.2 Processing factors Minor effects from processing include those from filtration, aeration and freezing, but by far the most important factor is heat. This may cause chemical changes in the flavour, but the main problem is the loss of volatiles. This may have the effect of reducing the fresh top note of a flavour. If the key recognition chemicals have widely different boiling points, heat could render the flavour unrecognisable. The choice of solvent can reduce this problem. In some instances volatile chemicals may be replaced by higher-boiling analogues. It is usually possible to change the balance of the flavour to allow for differential losses, but this solution gives a flavour that is suitable for only a limited range of applications. In processes involving considerable heat, such as bakery and extrusion, the best solution is multiple encapsulation. In this process a spraydried flavour is coated with a high melting point fat. This process protects the flavour until the fat melts. 1.4.3 Storage factors Some wines and cheeses improve with age, but they are the exceptions rather than the rule. Flavour stability in an application should ideally at least match the shelf-life of the food itself. When a flavour is added to food, some chemical changes, such as the hydrolysis of acetals, occur quite quickly. There are frequently subtle differences in

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flavour character after only one day. In the longer term, oxidation is responsible for most of the changes in flavour during storage. When most flavour chemicals oxidise, the effect is simply perceived as loss of flavour because the flavour chemical has a much stronger odour than its oxidation products. When some incidental components, such as the hydrocarbons in a lemon flavour, oxidise, the effect is often perceived as an off-note. Substitution of flavour components and the use of antioxidants usually reduce the problem to manageable proportions. Migration of flavour chemicals into or through food packaging materials can sometimes occur. It may lead to a detectable loss of flavour or crosscontamination problems. A change in packaging material is the best cure, but, if this is not possible, it may be practical to reformulate the flavour without the problem chemicals. This may change the profile of the flavour. Tea bags present a very specific packaging problem because of the size of the holes in the tea bags. Liquid flavour can be spread directly onto tea leaves, but this leaves the flavour very prone to oxidation and evaporative losses. Spraydried flavours need to be agglomerated to prevent them from falling through the holes in the tea bags. Tea used in tea bags is generally of small particle size and care needs to be taken to ensure that the size of the agglomerated flavour particles matches that of the tea. 1.4.4 Consumption factors Many of the processing problems can resurface when the food is consumed. This is particularly common when a powder flavour is used in a dry convenience food. The final factor that may influence the formulation of the flavour is the temperature at which the finished food is consumed. At temperatures below room temperature, as in the case of ice cream, the intensity of the whole flavour is reduced. The intensity of the most volatile chemicals is reduced relatively more that the rest of the flavour, and they may need to be increased. Caution should be exercised because the food can warm up in the mouth. Foods that are consumed hot are more difficult to flavour. The high temperature increases the intensity of the flavour, particularly the more volatile chemicals. As the same time it may cause a relatively greater loss of the same components. 1.5

Flavour forms

Liquid and powder flavours can be split into a number of major types, each of which poses some specific problems for the flavourist. 7.5.7 Water-soluble liquid flavours These are by far the most common type of flavour. The flavour chemicals and natural components are dissolved in a simple solvent, most commonly propylene glycol, triacetin or ethanol, with the possible addition of water.

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13

If the flavour contains significant amounts of solids, such as vanillin or maltol, then the quantities added must remain well within their limit of solubility. Storage conditions can be much harsher in real life than in a laboratory and a large safety margin should be built in. The same consideration should be applied to the nonsolid components of the flavour, but problems are not as common in this area. Propylene glycol is usually the solvent of choice. It is stable, virtually characterless in use, and confers some stability in applications involving heat processes. The drawbacks of propylene glycol are that it is not a strong solvent, that it is not natural, that the levels of use are restricted in some countries, and that it forms acetals and ketals quite readily with carbonyl flavour chemicals. Acetal and ketal formation can be inhibited by the addition of water to the flavour, but this makes an already weak solvent even weaker. Acetals and ketals can be useful in some applications because they may protect the parent carbonyl from oxidation, but later break down in the presence of water. Some acetals and ketals are not soluble in propylene glycol, so an initially clear flavour may gradually form two layers. Ethanol is also widely used, especially where there are no duty handicaps (many countries impose high taxes on ethanol). It is relatively stable, has a mild but pleasant character in use, is readily available in a natural form, and can be diluted significantly with water. The drawbacks are relative instability in applications involving heat processes, religious restrictions in some countries, and the formation of acetals and ketals. The last factor is less important because ethanol is a strong solvent and can still remain effective if sufficient water has been added to inhibit acetal and ketal formation. Triacetin is not a solvent of choice for most applications. It is not very water soluble and, when it does dissolve, it decomposes to glycerol and acetic acid. Triacetin has a slight bitter taste and acetic acid has a noticeable 'vinegar' odour. Triacetin can be the solvent of choice when water solubility is not critical (as in many confectionery applications), when propylene glycol is restricted, when the components of the flavour will not dissolve readily in ethanol or propylene glycol and, most importantly, when propylene glycol has an undesirable effect on the texture of the finished food. Chewing gum is the most important example. Propylene glycol hardens chewing gum, but triacetin acts as a plasticiser. Water is not added to triacetinbased flavours because it would hydrolyse the triacetin. Other solvents may be useful in specific cases. Triethyl citrate is similar in many respects to triacetin. It is poorly water soluble, but is odourless and confers heat stability. The major difficulty with triethyl citrate is the bitter aftertaste, which severely restricts the level of use. Diacetin is also similar to triacetin but is generally less effective. Glycerol is a very weak solvent but can be used effectively in conjunction with ethanol in natural extracts to confer some heat stability. Lactic acid is not generally a very effective solvent but can be useful, in mixtures, for some problematic raw materials, especially

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maltol. Benzyl alcohol has a faint floral character and is a good solvent but is prone to oxidation to benzaldehyde. Benzyl benzoate is stable and relatively odourless. It can be used in solvent mixtures, especially for oil-soluble flavours, but has an unpleasant flavour at high levels. Many of these lesser solvents are not universally recognised as solvents. They may be permitted as flavouring ingredients, but care must be taken of the level of use. 7.5.2

Clear water-soluble liquid flavours

This category is very similar except for the requirement that the end product, usually a beverage, should be crystal clear. Most flavour raw materials are entirely water soluble at their normal level of use. The exceptions are limited to a few chemicals that need to be used at relatively high levels (usually esters), chemicals that can form insoluble polymers on storage and terpene hydrocarbons. Terpene hydrocarbons are found in many natural essential oils. They have limited use as flavouring ingredients (there are exceptions, such as myrcene). and they are prone to oxidation. The hydrocarbons can be removed from essential oils by distillation, chromatography or solvent extraction. The most effective method is solvent extraction (often called 'washing') because it causes least change in the character of the original oil. The most effective solvent is a mixture of ethanol and water, but propylene glycol can also be used. The extraction is carried out by dissolving the oil (for example, orange oil) in ethanol, adding water to throw out the hydrocarbons (commonly called 'terpenes'), chilling the mixture and allowing it to stand for 2 days. The 'terpenes' float to the top of the mixture, which can then be drawn off and filtered. A little extra alcohol is added as the final stage to prevent the flavour becoming cloudy if it is stored in the cold. The process can be speeded up by the use of a coalescer, a metal mesh that coalesces the oil droplets. Propylene glycol 'washings' are difficult to make because of the viscosity of the solvent and the small amount of water that can be added. The use of a coalescer is virtually essential to make propylene glycol based 'washings'. A surprising, but effective, alternative way to produce clear beverages is through the use of low payload, small particle size emulsions. The flavour must contain only very limited amounts of terpene hydrocarbons for the process to work. This method is widely used for cola flavours. 1.5.3 Oil-soluble liquid flavours Oil-soluble flavours are needed where the end product is an oil or a fat. They are also used where the end product cannot tolerate water. Both ethanol and propylene glycol contain small amounts of water, so these solvents cannot be used in water-sensitive products such as chocolate. Natural or synthetic

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(medium-chain triglyceride) vegetable oils can be used as solvents. The problems are susceptibility to oxidation (for the natural oils) and poor solvent power. Many of the chemicals that are important for taste effects are highly polar and poorly soluble in oils. Some of the minor solvents discussed earlier, such as benzyl benzoate and triethyl citrate, can be particularly effective in oil-soluble flavours. They all have some drawbacks and may be more effective when used as mixtures. If this does not work, one possible solution is to dispense with traditional solvents altogether and use the major components of the flavour to dissolve the solids. This is not always possible without adding excess quantities of weak-tasting esters and, when it is possible, the resulting flavour may be highly concentrated and very difficult to use accurately in an industrial environment. Essential oils can be effective 'solvents' for some oil-based flavours. This is especially true of citrus flavours. The natural oil gives a realistic, complex background and added flavour ingredients give powerful specific character. 1.5.4 Emulsion-based flavours Emulsions, based for example on orange oil, are often used to give cloud to a beverage, but they can also be a cheap and effective way of delivering a flavour where cloud is not an issue. The water-soluble components, such as vanillin, can be dissolved in the gum solution (typically gum arabic or modified starch are used as emulsifiers) and the remaining components can be mixed together to form an oil phase, which is then emulsified. Problems include the separation of the oil phase and the microbiological stability of the emulsion. Ideally the oil phase should constitute around 5% and certainly not more than 10% of the emulsion. 7.5.5 Dispersed flavours Dispersions are a similar, cheap and cheerful, way of delivering flavours in powder form. If all the ingredients are solids, they may be mixed together and diluted with a carrier such as lactose. If some of the ingredients are liquids, they are mixed together and spread on the carrier before the solids are mixed in. This method works if all the ingredients are relatively high-boiling and not susceptible to oxidation. Even so, it produces flavours with a short shelf-life. 1.5.6 Spray-dried flavours Spray drying is the method of choice for powder flavours (see Chapter 5 for the mechanisms of flavour encapsulation). The flavour is typically emulsified in an aqueous gum solution, and then dried by spraying into a hot chamber.

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This method is preferable to dry mixing because the resulting flavour is stronger and much more stable to evaporation and oxidation. Spray drying works so well because the sprayed droplets form a semipermeable shell very quickly, long before most of the water has evaporated. The semipermeable shell allows water to pass, continuing the drying process, but is relatively impermeable to most flavour components. This is true of even the smallest flavour molecules, such as ethyl acetate. Only a small proportion of the ethyl acetate added to a flavour survives spray drying, but, without the effect of the semipermeable shell, logic would dictate that the loss on drying would be virtually 100%. Flavourists are not expected to be experts in the area of spray drying and the many variants of this technique, but they should know enough to get the best out of the process. The first issue is the way the flavour is added to the emulsion. The criteria are much the same as those for liquid emulsions, except that there is no need for the emulsion to be stable in the long term. The ingredients of the flavour should be split into water-soluble and oil-soluble keys. The watersoluble components should be dissolved in the gum solution. The oil-soluble components should be emulsified in the resulting mixture. This emulsion does not need to be stable for longer than it takes to dry the batch, but it should be emulsified to a reasonably small and uniform particle size. Poor emulsification will result in more surface oil, flavour loss and susceptibility to oxidation. Some solvents should not be used in the formulation of the keys. Ethanol will increase the flavour loss, propylene glycol (in more than trace amounts) will make the powder flavour hygroscopic. Triacetin works well in most instances. The maximum loading of the oil phase is around 30% of the dry weight, but drying losses increase steeply after 20%, as does the amount of surface oil. Twenty per cent is a good maximum to aim for. The second issue is the composition of the gum solution. Gum acacia is the most widely used material, although some modified starches can give equally good results. Gum acacia varies widely in quality and care should be taken to buy 100% pure gum from a reputable source. Cheap gum that has been cut back with filler is always poor value. The flavourist should be free to control the proportion of pure gum used. It is a waste of money to use 100% gum acacia as the carrier. In spray-dried flavours, 30% is the absolute maximum quantity of gum needed for even the most challenging applications. In many cases as little as 10% is all that is needed to form the semipermeable film during drying. The filler, usually maltodextrin, is important because a high dextrose equivalence is needed to make the shell of the spray-dried particle less permeable to oxygen. One unintentional advantage of using reduced levels of gum acacia is that the viscosity of the emulsion is lower. This allows the solid content of the emulsion to be increased, while still keeping to a viscosity level that can be handled readily. Higher solids mean more throughput and less energy costs.

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The third issue is the processing conditions. They should ideally be set for each flavour. The emulsion should not be warm because this will damage the flavour. The inlet temperature must be adjusted so that particles hitting the sides of the drier do not stick. In general the best results are obtained with the highest inlet temperatures and the highest throughput. The outlet temperature should be reduced as much as possible, but not so much that the spray-dried particles contain significant moisture when they leave the drier.

1.6 Production issues One of the most difficult challenges facing flavour companies is the link (or lack of it) between the creative flavourists and the production staff. It is possible to sidestep the issue by introducing a complete department to sort out problems, but it is obviously much better not to have them in the first place. The first issue is the total number of raw materials available for use. A sensible number can be reached by adding to the number of GRAS (Generally Recognised as Safe) raw materials (around 3000) the number of sensible variations of natural products (around 500) and the legal variations (natural, kosher, etc.) (around 1000). A sensible maximum is 5000. Not many companies can boast such a small list, but the cost and service problems associated with large raw material lists are formidable. The total number of flavour formulations is also often quoted by production as a problem area, but it is only a problem if the operations function is so inefficient that it is necessary to keep stock of finished flavours. Very few flavours are now sold off-the-shelf and the tailor-made flavour is becoming the standard. It makes much more sense to concentrate on controlling the number of raw materials. The second real issue is the number of raw materials in any individual formulation. It is simply not possible to justify more than 100 ingredients in a flavour. In many cases the best effect is obtained by around 30 ingredients. The cost of compounding and the service problems associated with very complex flavours are both serious issues. Accurate compounding in a production environment is very different from the situation in a laboratory. The use of solutions should be tailored to production needs and a single key may be helpful in some instances to separate out all the very low-volume items. Outside this restricted context, the use of keys and the blending of flavours in general are real headaches for production. They are also, frankly, indicative of lazy work on the part of the flavourist concerned. The correct compounding order may be obvious to the flavourist, but it must be specified in a formulation to assist production. Other important notes are the need to filter (which can often be avoided by better selection of raw materials or more careful formulation) and full details of any processes. The originating

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flavourist should always be involved in the quality control testing of the first batch in case there are problems scaling up the flavour. 1.7 Regulatory affairs Flavourists should receive extensive training on regulatory issues, not simply the widely varied global flavour regulations but also the implications for finished foods and labelling (see Chapter 10 for further information on the safety and legislation of flavours). With the current time scales for projects it is not practical to expect that a final regulatory check should be anything more than a safety net. A generally conservative approach should be taken and, wherever possible GRAS ingredients should be used. The regulations in Europe and the US are increasingly well harmonised, so this restriction is usually practical. For any country, the IOFI (International Organisation of the Flavour Industry) guidelines represent the minimum standard, irrespective of the lack of local regulations. Natural certification of raw materials should not be accepted without critical evaluation. Natural standards vary by country and common sense should be applied. 1.8 A typical flavour Raspberry flavour is a good learning tool. It is relatively simple, but not so simple that it does not contain a multitude of useful lessons. To illustrate the process of flavour creation we will work on an imaginary, but typical, customer project. The task at hand is to create a nature-identical flavour, with a profile, that the customer has described as true to nature, fresh and red. The end use is hard candy. Let us imagine that the flavourist has two analyses to work from. One derived from the analysis of an extract from the fruit and the other derived from the analysis of the headspace over the fruit. In these two, hypothetical analyses, 362 chemicals have been identified, 271 in the extract analysis and 203 in the headspace analysis. In both cases the quantification is expressed as a percentage of the total volatiles. The headspace analysis is also quantified with an added vapour pressure correction. Table 1.1 gives the quantification of those chemicals from the analyses that we will consider using to create a simple flavour. The first step is to decide which chemicals in either analysis represent primary characteristics. In the case of raspberries the violet note is clearly essential. The analyses contain both a-ionone and pMonone. a-Ionone has a clean 'violet' note and p-ionone has a 'violet' note in addition to a strong 'cedar' note. To keep things simple there is an obvious temptation to ignore the complications of ji-ionone and work with a-ionone alone. This simplification might work, but it is probably unwise. The 'cedar' note of (3-ionone generates a 'pippy' or 'seedy'

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Table 1.1 Flavour components identified in analyses of raspberry flavour Cast (in order of appearance)

Extract (%)

Headspace (%)

VP Adjusted (%)

a-Ionone fi-Ionone 4-Hydroxyphenylbutan-2-one Damascenone Dimethyl sulfide Acetylmethyl carbinol Ethyl acetate r/.v-3-Hexenol
4.00 1.80 0.50 0.05 0.02 0.50 5.00 8.00 0.02 0.60

0.70 0.50

8.000 9.500

_

-

0.02 1.50 0.20 9.80 0.60 0.04

0.150 0.001 0.002 0.040 0.030 0.010

_

-

effect in raspberry flavours. This note is hardly a primary characteristic, but it is normally attractive. If |3-ionone is ignored at this stage, then a later correction to add a 'seedy' note will necessitate a rebalancing of the 'violet' character. The next step is to establish an estimate of the correct concentration of the 70/30 mixture (these proportions are derived from the extract analysis) of a-ionone and p-ionone using a simple taster. A good starting level would be 0.25 ppm. The most common dilution of flavours in beverages is 0.05% rtd (ready to drink). This is equivalent to 0.035% a-ionone and 0.015% of (3-ionone in the flavour. The other primary characteristic is not quite so easy to identify. The flavour of a-ionone alone is simply 'floral, violet'. The missing character should confer a specifically 'berry' note. The only feasible candidate in the analyses is 4-hydroxyphenylbutan-2-one. This chemical has a distinct 'berry' aroma, even a specific hint of raspberries. Again trial and error can be used to establish a good balance between these two chemicals. A good starting level would be around 2% in the flavour, but later in the process this will prove to be too high (once other ingredients have been added) and the final level is 1%. At this stage we already have a recognisable raspberry skeleton. We can move on to the optional, secondary components. The customer wants a true-to-nature character, but also describes the target as 'red' and 'fresh'. Neither of these descriptors is very specific, so the flavourist has to try to guess the customer's wishes. This dilemma is very common and illustrates the need to work with customers to establish specific descriptors. 'Red' can reasonably be taken to mean red raspberries rather than black (so no musk character), ripe rather than unripe (so ripe, 'fruit' notes and restricted 'green' notes). 'Fresh' can be taken to mean an absence of 'jammy' or 'cooked' notes. It might also indicate high 'green' notes, which certainly confer freshness. A more moderate level of 'green' notes is probably a good idea because 'red' was also specified. High levels of 'green' notes, especially 'raw, green' notes give an unripe effect.

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FOOD FLAVOUR TECHNOLOGY

A good choice for the red 'fruit' note is the 'damson' character of damascenone. This chemical is found widely in nature and is, justifiably, a favourite with flavourists. The 'damson' character of damascenone adds richness and a deep 'fruity' character to our fledgling raspberry flavour. Taking the extract analysis as a guide (damascenone is fairly high-boiling), the levels of the ionones used in the flavour indicate a level of 0.0004% of damascenone in the flavour. This seems very low indeed. To obtain the correct character we must increase the level in the flavour to 0.04%. Dimethyl sulfide is also an excellent ripe 'fruit" note in dilution, although at high levels it has a 'cabbage' character and can make the flavour seem cooked and 'jammy'. The addition of dimethyl sulfide also improves our flavour dramatically, but 0.01 % is the most we can add before the character becomes slightly 'jammy'. This is, however, far more than the level indicated by the vapour pressure-corrected headspace analysis. The 'buttery' note of acetyl methyl carbinol will also, surprisingly, add to the 'ripe', 'red' character. The concentration that would be required in the flavour, based on the amount found in the extract analysis, relative to ot-ionone, is around 0.005%. This level works well and provides the required note. The final 'fruity', 'red' note is ethyl acetate. The headspace analysis indicates a very low level, but the extract analysis (which would be expected to give a low result) indicates a level broadly similar to that of a-ionone. Increasing that level a little to 0.10% in the flavour gives an attractive result. The most obvious green note is c/5-3-hexenol, but this chemical has a 'leaf, green' character, similar to fresh cut grass. Like damascenone, c/.s-3-hexenol is found very widely in nature and is often the first choice when a 'fresh' character is desired. We could add a low level of c/s-3-hexenol, but we would run the risk of introducing an 'unripe' note. A much better choice for this flavour would be c/s-3-hexenyl acetate, which has a softer 'fruity, green' character and very little unripe note. The analyses would indicate a low level of c/s-3-hexenyl acetate, but a higher level is necessary because we are not adding any m-3-hexenol. In practice the ideal level is 0.02% in the flavour. This flavour will smell reasonable but taste very thin. Only two components of the flavour so far have a significant taste effect—damascenone and 4-hydroxyphenylbutan-2-one. The flavour has a degree of 'berry' depth of taste, but needs added 'sweet' character. The addition of 2% of maltol (not found in the analysis) will help to solve this problem, but is obviously not the ideal solution. Maltol has a 'candyfloss' aroma and imparts a lingering sweet aftertaste. It adds depth, but is too simple a character. One other addition that will help to add depth is 5-decalactone. This chemical has a 'creamy' character and trial and error establishes an ideal concentration in the flavour of 0.05%. This level is higher than that indicated by the extract analysis. The flavour we have developed thus far is much too simple and will taste like an obvious mixture of separate notes. The addition of a small amount of jasmine absolute (0.02%) will add complexity and traces of desirable 'berry'

CREATING AND FORMULATING FLAVOURS

Table 1.2 Comparison of raspberry flavour analysis (from table 1.1) with formulation of a raspberry flavour suitable for hard candies Cast (in order of appearance)

Extract %

VP Adjusted %

Flavour %

a-Ionone p-Ionone 4~Hydroxyphenylbutan-2-one Damascenone Dimethyl sulfide Acetyl methyl carbinol Ethyl acetate ra-3-Hexenol cf'.v-3-Hexenyl acetate Maltol S-Decalactone Jasmine absolute

4.00 1.80 0.50 0.05 0.02 0.50 5.00 8.00 0.02 0.60 -

8.000 9.500 0.150 0.001 0.002 0.040 0.030 0.010 _ -

0.035 0.015 1 .000 0.040 0.020 0.009 0.200 _ 0.030 2.000 0.050 0.020

(from benzyl acetate), 'lavender' (from linalool), 'animalic' (from indole) and 'jasmine' (from methyl jasmonate) notes. The final stage in the creation of our very simple raspberry flavour is to make allowances for the processes involved in making hard candy. The only factor is heat, so the most volatile components must be increased to allow for the losses in processing. Ethyl acetate should be increased to 0.20%, dimethyl sulfide to 0.02%, acetyl methyl carbinol to 0.009% and cw-3-hexenyl acetate to 0.03%. In real life the process of developing this flavour would be much more complicated, but this simple example serves to illustrate the principles involved. The composition of the final flavour is compared to the two analyses in Table 1.2. The analyses help, but they are a long way from the quantification of the finished flavour. This example illustrates the high level of creative input, even when analyses are taken as the starting point.

1.9

Commercial considerations

/. 9.1 International tastes We are all accustomed to rapidly increasing globalisation, and with it the assumption that one product can be sold in all markets. There are cases where, with sufficient advertising, this is manifestly true. In most cases, however, the assumption does not hold. Regional tastes for most of the key flavour types still override global stereotypes. The regional tastes are often derived from historical familiarity and may fade in time, but, for now, they are very important. The main regional preferences for the most important flavour categories are summarised below.

22

FOOD FLAVOUR TECHNOLOGY

Beef. Roast beef is the preferred profile in the UK. Grilled beef reigns supreme in the US and in much of Asia boiled beef is the main profile. Cheese. Cheddar is far and away the most important type of cheese in terms of flavour sales. Blue cheese and Parmesan are very small categories compared with Cheddar. Cheddar can be broken down into two main types by region: sharp and mild. Sharp Cheddar is best defined (ironically) by aged US or Canadian Cheddar cheese and represents the target profile in Europe. The taste preferences of US consumers are very different. In this region a mild, creamy, buttery character is preferred. Cherry. In Europe the hawthorn note of Morello Cherries is preferred, but in the US benzaldehyde is the prominent character. Chocolate. Milk chocolate predominates in most markets outside Europe and the milk component often has a cooked character. Some popular milk chocolates also have an added signature note such as cinnamon or almond. Dark chocolate is popular in Europe and can have pronounced burnt and bitter characteristics in this market. Lemon. In the UK especially, but also in much of Continental Europe, a high citral level is liked. The European taste also likes an exaggerated level of jasmine character. In the US, lemon is milder and more floral and in much of the rest of the world citral is the defining note, often accompanied by the waxy character of oxidised oil. Mango. As with most other tropical fruits the situation is the exact reverse of berry flavours. The genuine character, with its strong terpene, skin note is preferred in Asia and Latin America. In Europe and the US a pale imitation flavour is preferred, with much-reduced skin and sulfur notes and an emphasis on melon and peach. Milk. Fresh milk and dairy flavours are optimum in the US and Europe. In Asia a boiled, condensed milk note is preferred and in Latin America the even more caramelised 'dulche de leche' is ideal. Orange. In Europe there is the strange contradiction that the flavour of processed juice is liked, presumably for nostalgic reasons, together with the pungent, fresh note of acetaldehyde. An exaggerated hint of violet is also liked in many orange flavours for confectionery. Fresh juice character is popular in the US and, for the rest of the world, cold-pressed orange peel oil is the most popular character.

CREATING AND FORMULATING FLAVOURS

23

Raspberry. In the US a strong violet character is preferred, but in Europe this note is muted and balanced by fresh and green characters. In Asia real raspberries are a rarity and an old-fashioned candy character is preferred. Strawberry. At first sight strawberry would seem to be easily standardised. Not so. It is one of the most difficult flavours to fit into its many regional variations. In the US strawberry is generally sweet and slightly jammy. Green notes are not liked. In most of Europe the preferred character is fresh and distinctly green. Within Europe the French taste is for a pronounced jasmine note and the Spanish taste is for strawberry jam. In Asia the preferences are more abstract and oldfashioned because of unfamiliarity with the real fruit. Vanilla. Alcoholic genuine vanilla extract, with rum and fruity overtones, defines the US taste. In France the taste veers towards a creamy hawthorn note. In Germany a hint of balsam is appreciated and in much of northern Europe a simple vanillin taste is preferred. The UK preference is for a distinctly buttery note. 7.9.2 Abstract flavours Most flavours have an obvious natural target. The flavour may be realistic or have a degree of abstraction, especially for children's products. Many flavours, such as lemon-lime, are blends of recognisable natural targets. Very few flavours are basically abstract. The most important examples are cola and tutti-frutti. Cola flavours are all quite complex blends. The main characters are distilled lime oil, cassia oil, nutmeg oil and vanilla extract. In some instances the caramel colour also contributes a characteristic flavour. The flavour ages very quickly in the bottling syrup because of the high level of phosphoric acid. Matching cola flavours is especially difficult because of the noticeable change in flavour in the syrup and the need to age the syrup before evaluation. Tutti-frutti flavours, in contrast, are quite widely varied. The main characters are banana, orange, pineapple, vanilla and berries. They can be classified in two broad types—those based on banana and those based on berries. The banana family is usually built around isoamyl acetate and the berry family is usually built around a-ionone. There are other interesting, but less commercially important, abstract flavours. Root beer was originally based on sassafras oil, vanilla and methyl salicylate, plus a host of minor ingredients. Sassafras oil has not been used for many years (because it contains salfrole over which there are safety concerns) and the substitutes vary in effectiveness. Sarsaparilla and Dandelion-and-Burdock are similar products. Cream soda flavours were similarly modified in line with regulatory requirements and now consist of vanillin with lactone-based hay and

24

cream notes. Cachou flavours are intensely perfumed and are often based on combinations of violet, rose and musk. Another, accidental, category of abstract flavours is the group of the best of the flavours from the early years of the industry. They were often not exactly recognisable, but were triumphs of artistry over paucity of raw materials and became standards in their own right. 7.9.3

Matching

No project is less welcome to the typical flavourist than one that requires matching. The very idea of matching someone else's work is profoundly unattractive. Improving on someone else's work is quite another matter and represents a real challenge, but simple matching is boring. Behind the flavourists' manifest hostility is not just the simple lack of challenge and novelty; there is also the commercial fact that matching work represents by far the least rewarding use of creative time. Most matching projects are obtained by novice sales staff as a way of gaining entry to the account. Most successful matches are simply used to pressure the existing supplier to reduce prices. Very few product managers will risk changing the flavour of a successful consumer product to save a few cents, especially when it must involve expensive consumer trials. Some very successful flavour companies will generally not accept matching projects, and it is not evident that their customer standing has suffered as a result. Despite all the objections, there are occasionally good reasons to carry out matching work. The customer may have become genuinely hostile to their current supplier and wish to change at any price. The need to carry out matching may also derive from a reduction in the number of suppliers in a core supplier programme. The customer may wish to duplicate a competitor's existing consumer product, although this type of project is more often directed towards beating the existing product. Matching generally starts with an analysis. Normally it will be a direct analysis of the existing flavour, if the customer is serious. In some cases it will be a consumer product. Once the analysis has been completed it should be reconstituted and reanalysed (in consumer product if necessary) to pick up errors in identification and quantification. This corrected analysis represents the starting point for the flavourist. The target flavour will often contain natural extracts and essential oils, so the first job is to allocate all the components derived from natural sources correctly. This is usually a matter of experience and the analyst should also be able to provide some guidance. The trickiest part is usually trying to determine which processes (solvent extraction, concentration, etc.) have been applied to the natural raw materials. Trial and error, plus quite a few reanalyses, overcome this hurdle. The major chemical components should be identified and quantified from the analysis. It is often helpful to cam' out

CREATING AND FORMULATING FLAVOURS

23

liquid chromatography of the main chemicals to improve the accuracy of the quantification. One issue that is often forgotten is the need to get the solvents in liquid flavours identified and quantified correctly. Flavourists sometimes assume that they will not be important in the end product. That is not always true, but incorrect solvent balance always hinders rapid evaluation of matches on blotters. All that remains are the trace components. Unfortunately, this is often where most of the problems lie. Traces of sulfur chemicals, for example, can be very difficult to pick up on analysis, but can be a vital part of the flavour. Matches are often carried out under severe time pressure and it is easy to become stale and run out of ideas. Involving other flavourists is a must and can be especially helpful in generating ideas about the identity of missing trace components. It is also useful to have available a bank of analyses of flavours from the same competitor. In many companies the same ideas are trotted out with surprising regularity. Sensory panel work is essential to validate the accuracy of the final match. It may also help persuade the customer to accept the change. Caution should be exercised about blindly accepting routine panel results. If a very close match is required, an expert panel may be needed. 1.9.4 Customers A flavour is, sadly, nothing, if it is not sold. Part of the tremendous 'buzz' of being a successful flavourist is the feeling of having created something really good. The other part of the 'buzz' is seeing your product on the supermarket shelves, enjoyed by thousands, or perhaps millions, of people. The best flavourists do not divorce themselves from customer involvement and the art of selling. Most successful flavourists reach the inescapable conclusion that they are the best judge of the market and the best flavour for a specific project. This conclusion has some basis in fact, but the sad truth is that customer involvement is usually the key to success. Customers usually do know their own market best and should be encouraged to guide the creative process. One additional advantage of this approach is the fact that the customer buys into the process and regards the resultant flavour as 'theirs'. The only barrier to this approach is the communication problem. If the project has passed through intermediaries (sales, marketing, etc.) then communication is very difficult, however thoroughly the project information has been gathered. The only practical solution is direct contact between the flavourist and the applications specialists in the customer's laboratories. Descriptive terms need to be defined and understood and simple examples (such as ds-3-hexenol for 'leaf green') can help a great deal. Knowledge of the customer's applications processes is also needed and the involvement of an applications specialist from the flavour company is often

26

FOOD FLAVOUR TECHNOLOGY

vital. Problems are often caused by the interactions between the flavour and the application ingredients and processes. In many cases a small change to the customer's formulation or process can save the day. Sensory evaluation can play a big part, particularly in respect of flavour profiles preferred by a target market segment. This is usually a specialised area where the flavour company probably has more depth of knowledge than the customer. Most projects go through a number of iterations before they are concluded successfully and it is vital in this process to remember that 'the customer is always right'. 1.10

Summary

Flavour creation is still more of an art than a science. Science provides a vital understanding of nature and a broad palette of raw materials. Science may also provide insights into the preferences of a target group of consumers and some understanding of the mechanisms of taste and smell, but science cannot yet replace the intuitive, creative skills of a good flavourist.

The basic chemistry and process conditions underpinning reaction flavour production Josef Kerler and Chris Winkel

2.1

Introduction

Maillard reaction technology is used by the flavour industry for the production of process/reaction flavours. Process flavours are complex aroma building blocks that provide similar aroma and taste properties to those found in thermally treated foodstuffs such as meat, chocolate, coffee, caramel, popcorn and bread. The Maillard reaction between a reducing sugar and a food-grade nitrogen source is the principal underlying reaction, which is responsible for flavour and colour development. This review provides a summary of general aspects of the Maillard reaction in flavour formation, as well as of important aroma compounds of thermally treated foodstuffs and process flavours. In addition, the patent literature and other publications relating to reaction flavour production and their process conditions will be discussed. 2.2

General aspects of the Maillard reaction

The Maillard reaction is of great importance for flavour and colour formation of thermally treated foodstuffs. The thermal generation of aromas in foods, process flavours and model systems has accordingly been the subject of many symposia and reviews (e.g. Parliment et al., 1989, 1994; Weenen et al., 1997; Tressl and Rewicki, 1998; Reineccius, 1998). A first milestone in the history of Maillard chemistry was the publication of the well-known Hodge scheme (Hodge, 1953). Although Hodge's studies focused on Maillard browning only, this scheme provided a framework that also covered important reaction routes for the formation of aroma compounds. The work of Hodge triggered a large number of studies on the elucidation of important intermediates and pathways of the Maillard reaction (see good reviews by Ledl and Schleicher, 1990; Tressl and Rewicki, 1998). Isotopic labelling of sugars and/or amino acids in conjunction with GC-MS analysis (Tressl et al, 1993; Gi and Baltes, 1995; Keyhani and Yaylayan, 1996) as well as trapping of reactive intermediates (Nevidek et al., 1992; Hofmann, 1999) are key techniques for the improved understanding of Maillard reaction pathways. Improvements and refinements of the Hodge scheme were presented by Tressl et al. (1995). Their scheme provides an excellent overview of Maillard reaction

28

FOOD FLAVOUR TECHNOLOGY

pathways leading to the formation of volatile compounds. For the optimisation of reaction flavours, however, a strong emphasis on those routes that are involved in the generation of key aroma compounds is required. This can be achieved by first evaluating the character impact compounds of a process flavour or model system using a combination of sensorial and instrumental analysis (e.g. based on the odour activity value concept). The mechanistic studies can then be focused on the key substances only. The work of Hofmann (1995) is an excellent example of such an approach. Figure 2.1 gives an overview of the pathways that are involved in the formation of important aroma compounds during Maillard reaction. There are three main routes involved in flavour generation. All three routes start with imine formation between a reducing sugar and an amino acid. The Amadori (derived from aldoses) or Heyns (derived from ketoses) rearrangement products are important intermediates of the early phase of the Maillard reaction. Route A (see also Section 2.2.1) leads to the formation of 1- and 3-deoxyosones, which upon cyclisation, reduction, dehydration and/or reaction with hydrogen sulfide result in heterocyclic aroma compounds. Route B (see also Section 2.2.2) is characterised by fragmentation of the sugar chain through retro-aldolisation or a- or ^-cleavage. By aldol-condensation of two sugar fragments or a sugar fragment and an amino acid fragment, heterocyclic aroma compounds are generated upon cyclisation, dehydration and/or oxidation reactions. Alternatively, the fragments can react with hydrogen sulfide and form very potent alicyclic flavour substances. Route C (see also Section 2.3) involves the so-called Strecker degradation of amino acids, which is catalysed by dicarbonyl or hydroxycarbonyl compounds. The reaction is a 'decarboxylating transamination' and the resulting Strecker aldehydes are potent flavour compounds. Strecker aldehydes can also be formed directly from Amadori or Heyns rearrangement products. 2.2.1 Intermediates as flavour precursors Amadori rearrangement products (ARPs) and Heyns rearrangement products (HRPs) are relative stable intermediates and have been detected in various heat-processed foods (Eichner et al., 1994). Since ARPs and HRPs can easily be synthesised (Van den Ouweland and Peer, 1970; Yaylayan and Sporns. 1987), their potential as flavour precursors has been evaluated in several studies. Doornbos et al. (1981), for example, found that the ARP derived from rhamnose and proline is a useful precursor for the generation of the potent caramel-like odorant 4-hydroxy-2,5-dimethyl-3(2//)-furanone. ARPs and HRPs have also been reported to be good precursors for Strecker aldehydes and, in the absence of oxygen, also for 1- and 3-deoxyosones (Hofmann and Schieberle, 2000). At higher pH values, ARPs and HRPs easily undergo cleavage of the carbohydrate chain, yielding fission products such as 2,3-butanedione and pyruvaldehyde (Weenen and Apeldoorn, 1996).

AMINO ACID

SUGAR

Amadori/Heyns rearrangement compounds

Fragmentation products Retro-aldol or a(p)-cleavage

-am/no acid

B

H&NH acid fra< Condensation products

1-and 3-Deoxyosones HSS

H2S, NH3

AMINO ACID

reduction Cyclisation products

Cyclisation products Strecker

HSS reduction dehydration

V

V Flavour substances

Figure 2.1 Major pathways for the formation of flavour substances during Maillard reaction. A, B and C denote the three key pathways.

30

FOOD FLAVOUR TECHNOLOGY

When cysteine is heated with reducing sugars, thiazolidine carboxylic acids (TCAs) are formed instead of ARPs or HRPs (de Roos, 1992). TCAs are relatively stable in anionic form, which is probably the main reason for the inhibitory effect of cysteine in Maillard reactions, especially at higher pH. This can also explain the more efficient formation of important meat sulfur compounds at low pH (Hofmann and Schieberle, 1998). A research disclosure (Anonymous, 1979), however, describes the use of TCAs as precursors for meat flavours. TCAs of glyceraldehyde, fructose or xylose were reacted as such or in conjunction with organic acids (e.g. succinic acid, malic acid, citric acid) or fatty acids (e.g. oleic acid, linoleic acid). Using a pH of 6-7 and temperatures between 50°C and 100°C, the TCA of glyceraldehyde and cysteine was reported to result in a beef-like aroma, whereas TCAs of fructose or xylose and cysteine yielded 'meaty/savoury' flavours. Other important Maillard reaction intermediates are the deoxyosones. In general, 1-deoxyosones are more important flavour precursors than are 3-deoxyosones. Although 1-deoxyglucosone has been synthesised by Ishizu et al. (1967), 1-deoxyosones are too unstable to be used as precursors. 3-Deoxyosones, however, are more stable and are easily obtainable from compounds like difructoseglycine (Anet, 1960). The structure and reactivity of various 3-deoxyosones have been extensively studied by Weenen and Tjan (1992,1994) and Weenen et al. (1998). By using various ' H NMR and I3C NMR techniques, the authors showed that 3-deoxypentosone and 3-deoxyglucosone consist almost exclusively of monocyclic and bicyclic (hemi)acetal/(hemi)ketal structures. 3-Deoxypentosones and 3-deoxyhexosones are good precursors for furfural and (5-hydroxymethyl)furfural under acidic conditions. Under basic conditions, they undergo cleavage of the carbohydrate chain and can form pyrazines. Hofmann and Schieberle (2001) recently showed that acetylformoin. which is formed from 1-deoxyhexosone, is an effective precursor for 4-hydroxy-2,5dimethyl-3(2//)-furanone (Furaneol). The amounts of furaneol obtained from acetylformoin were significantly enhanced in the presence of reductones such as ascorbic acid or methylene reductinic acid as well as the Strecker active amino acid proline. The reaction between acetylformoin and proline also resulted in high amounts of the cracker-like odorant 6-acetyltetrahydropyridine. A number of articles and patents report the production of meat-like aromas by reacting 4-hydroxy-5-methy 1-3(2//)-furanone (nor-HDF) with hydrogen sulfide or cysteine. Van den Ouweland and Peer (1968) were first to file a patent on the use of this precursor system to prepare 3-mercaptomethylfurans, which exhibit meat-like character. Later, several studies identified sulfur-containing aroma compounds derived from the reaction of nor-HDF and hydrogen sulfide (e.g van den Ouweland and Peer, 1975; Whitfield and Mottram. 1999). Whitfield and Mottram (1999) showed that this precursor system is capable of producing compounds like 2-methyl-3-furanthiol and 2/3-mercapto-3/2-pentanone in

THE BASIC CHEMISTRY AND PROCESS

31

relatively high amounts. These thiols are also key aroma compounds of heated meat (Kerscher and Grosch, 1998). Shu and Ho (1989) and Zheng et al. (1997) studied the reaction of the methyl homologue 4-hydroxy-2,5-dimethyl-3(2//)-furanone (furaneol) with hydrogen sulfide or cysteine, which also gave rise to meat-like aromas. Among the identified sulfur-containing volatiles, however, the methyl homologue of 2-methyl-3-furanthiol, (2,5-dimethyl-3-furanthiol), was not found in the reaction mixtures. Unilever patented processes for the preparation of savoury flavours using the precursor systems and reaction conditions shown in Figure 2.2 (Turksma, 1993; Rosing and Turksma, 1997). When 2,5-dimethyl-2-(2-hydroxy-3-oxo2-butyl)-3(2//)-furanone (diacetyloligomer; R and R' = CH3; process A in Figure 2.2), which can be obtained by heating 2,3-butanedione under acidic conditions (Doornbos et al., 1991), is reacted with cysteine and hydrogen sulfide, high amounts of 2,5-dimethyl-3-furanthiol (DMFT) are generated (Turksma, 1993). For example, almost 40% yield of DMFT was obtained after 1 h at 120°C using a polar organic solvent, acidic conditions and superatmospheric pressure (100-2500 kPa). DMFT, which was found to have a 'meaty taste and roasted meat aroma' was also formed from 2,5-dimethyl-3(2//)-furanone. Using similar conditions as for the diacetyloligomer, Rosing and Turksma (1997) reacted 4-hydroxy-2,5-dimethyl-(2-hydroxy-3-oxo-2-butyl)-3(2//)-furanone (fupre 2; R ! -R 3 = CH3, R4 = acetyl; process B in Figure 2.2) with cysteine and hydrogen sulfide. This process resulted in a flavouring with sweet, onion-like, meaty aroma, odours attributed to high amounts of 2,5-dimethyl-4-mercapto3(2//)-furanone and 2,5-dimethyl-4-mercapto-3(2//)-thiophenone. However, these two sulfur compounds were not found to significantly contribute to the flavour of heated meat. Mottram et al. (1998) filed a patent on flavouring agents that serve as precursors for generating cooked (e.g. cooked meat) flavours in foodstuffs in situ. The authors claim a long list of precursor substances that are capable of developing flavour during microwave cooking or conventional oven cooking with reduced cooking times. This list comprises several sulfur compounds such as hydrogen/ammonium/sodium sulfides, cysteine, thiamin, onion and garlic as well as 'non-sulfur-containing postrearrangement Maillard products' such as furanones (e.g. 4-hydroxy-5-methyl-3(2//)-furanone, 4-hydroxy-2,5~ dimethyl-3(2//)-furanone, 2-methyl-4,5-dihydro-3(2//)-furanone), pyranones (e.g. maltol, 5-hydroxy-5,6-dihydromaltol), 3-deoxyglucosone, ketones (e.g. cyclotene) and aldehydes. The precursor mixtures were encapsulated or spray dried and applied to the foodstuffs through dusting or inclusion prior to the heat treatment. In two other studies, a similar precursor system consisting of unsaturated aldehydes and hydrogen sulfide resulted in aroma blocks with deep fried notes (van den Ouweland, 1989; Zhang and Ho, 1989).

Precursor

o

Main aroma compound 0,

Cysteine + H2S

.SH

(a) glycerol 90-120°C 1-3h 100- 2500 kPa

R,R = CH3 or H

HO.

Cysteine + H2S

HO

,SH

(b) R1

R' 1

""

R =CH 3 , C 2 H 5 orH

glycerol 90-120°C 1-3h 100-2500 kPa

R1

R2 = alky I with 1-4 C-atoms or H R3 = alkyl with 1-5 C-atoms R4 = organic radical with 1-6 C-atoms, hydrogen and 0-2 O-atoms Figure 2.2 Precursors, reaction conditions and main aroma compounds of two savoury process flavours (according toTurksma. I993 (a) and Rosing and Turksma, I997(h)).

33

THE BASIC CHEMISTRY AND PROCESS

2.2.2 Carbohydrate fragmentation Carbohydrate fragments have been found to originate from deoxyosones, Amadori or Heyns rearrangement products (ARPs or HRPs) as well as from the sugar directly (Ledl and Schleicher, 1990; Weenen, 1998) (see also Figure 2.1). The extent of the sugar cleavage reactions depends on the pH and on the solvent of the reaction medium, with fragmentation favoured at higher pH values (pH > 7) and in aqueous systems. Using isotopic labelling of the sugar molecule in conjunction with GC-MS analysis, Cs/C], €4/02 and Cs/Ca fission reactions were established (see review by Tressl et al., 1995). The proposed cleavage routes involve retro-aldolisation, vinylogous retro-aldolisation, a- and (3-dicarbonyl cleavage (reviewed by Tressl and Rewicki, 1999; Weenen, 1998). Retro-aldolisation is by far the most accepted fragmentation route. Carbohydrate cleavage products have been analysed by several authors (Nedvidek et al., 1992; Weenen and Apeldoorn, 1996; Hofmann, 1999). Since these intermediates are reactive dicarbonyl and hydroxycarbonyl compounds, trapping agents such as 1,2-diaminobenzene and ethoxamine hydrochloride were used to transform them into stable quinoxaline and O-ethyloxime derivatives respectively. Weenen and Apeldoorn (1996) studied the formation of glyoxal, methylglyoxal, 2,3-butanedione and 2,3-pentanedione in both Maillard and caramelisation reactions. The results are shown in Table 2.1. The study revealed that sugar fragmentation is highest in the presence of a Strecker inactive amine functionality (cyclohexyl amine), followed by a Strecker active

Table 2.1 Formation of a-dicarbonyl products (according to Weenen and Apeldoorn, 1996) a-Dicarbonyl products (ug) Amine

Carbohydrate

No No No No No Alanine Alanine Alanine Alanine Alanine Cyclohexylamine Cyclohexylamine Cyclohexylamine Cyclohexylamine Cyclohexylamine

Glucose Fructose Xylose 3-Deoxyglucosone Fru-ala ARPa Glucose Fructose Xylose 3-Deoxyglucosone Fru-ala ARP Glucose Fructose Xylose 3-Deoxyglucosone Fru-ala ARP

a

Glyoxal 26 28 62 23 103 58 45 27 16 81 618 691 591 317 509

Methyl glyoxal 11 15 17 57 101 43 28 81 56 67 865 1104 925 583 454

2,3-Butanedione _ _. _ 98 41 22 28 11 81 227 265 614 146 232

2,3-Pentanedione 18 38 25 42 21 22 39 89 101 25 39

/V-l-(deoxy-D-fructosyl)-L-alanine (Amadori rearrangement product of glucose and alanine).

34

FOOD FLAVOUR TECHNOLOGY

amino acid (alanine). Without amine (caramelisation reaction), the extent of fragmentation was even lower and no detectable amounts of 2,3-butanedione and 2,3-pentanedione were observed. The yields of the pentanedione were relatively high in alanine model systems, indicating that the Strecker aldehyde, acetaldehyde, is involved in its formation. In addition, the ARP of glucose and alanine was found to be an efficient a-dicarbonyl precursor, whereas the 3-deoxyglucosone/alanine reaction mixture yielded only low concentrations of fission products (see Table 2.1). The latter result is in good agreement with the finding that 3-deoxyglucosone is also a poor pyrazine precursor (Weenen and Tjan, 1994). 2,3-Butanedione (diacetyl) and 2,3-pentanedione are aroma-active carbohydrate cleavage products, contributing a sweet-caramel odour to coffee (Grosch, 2001), for example in the presence of hydrogen sulfide or cysteine, they are precursors of important sulfur aroma compounds such as 2-mercapto-3-butanone and 2/3-mercapto-3/2-pentanone (Hofmann, 1995). Yaylayan and Keyhani (1999) investigated the origin of these two dicarbonyl compounds in glucose/ alanine Maillard model systems. Using labelled glucose or alanine, the authors showed that 90% of the formed pentanedione requires the participation of the C2/C3 atoms of alanine, whereas diacetyl was derived from the sugar chain only. This result is supported by the finding of Hofmann (1995) that 2,3-pentanedione is formed by reacting acetaldehyde and hydroxy-2-propanone. In another study, Yaylayan and Keyhani (2000) also investigated the origin of the sugar fragmentation products glycolaldehyde, methylglyoxal, hydroxy-2propanone and 3-hydroxy-2-butanone. Hofmann (1999) studied the time course of the formation of carbohydrate degradation products in thermally treated solutions of either xylose or glucose with alanine. The author showed that during the first 10 minutes of the Maillard reaction, glyoxal is the most abundant fragmentation product from both xylose and glucose. Its formation can be explained by retro-aldol cleavage of 2-xylosulose or 2-glucosulose. After 20 minutes of the Maillard reaction, the main fission products were found to be methyl glyoxal and hydroxy-2propanone, with xylose yielding higher amounts than glucose. Hofmann and Schieberle (2001) developed an elegant method for assessing the contribution of carbohydrate fragments to the formation of aroma compounds. The authors used a Maillard model system containing 1:1 mixtures of unlabelled and 13C6-labelled glucose as well as unlabelled proline and then measured the labelling pattern ( 12 C6, 13 C3 and I3 C6) of the caramel-like odorant 4-hydroxy-2,5-dimethyl-3(2//)-furanone (furaneol) by GC-MS. The relative abundance of the mass of 13C3-labelled furaneol was directly correlated with the amount of the furanone that was formed from C^-sugar cleavage products. The authors showed, for example, that under dry heating conditions furaneol was generated entirely via the intact sugar skeleton, whereas in aqueous solution 63% of the furanone was derived from CVfragments.

THE BASIC CHEMISTRY AND PROCESS

35

2.2.3 Strecker degradation In the presence of a- or vinylogous dicarbonyl compounds, a-amino acids can undergo a 'decarboxylating transamination', which results in the formation of aldehydes with one carbon atom less than the amino acid (Strecker aldehydes) (Strecker, 1862; Schonberg and Moubacher, 1952). The Strecker degradation of amino acids is a key reaction in the generation of potent aroma compounds during Maillard-type processes (Ledl and Schleicher, 1992) (see also Figure 2.1). Certain amino acids (leucine, valine, methionine or phenylalanine) are known to produce Strecker aldehydes with significant odour strength such as 3- methylbutanal, methylpropanal, methional or phenylacetaldehyde. These aldehydes have been confirmed as key contributors to many thermally processed foods (Hofmann et al., 2000). Besides aldehyde formation, Strecker degradation also contributes to flavour formation during Maillard reaction by reducing dicarbonyls to hydroxycarbonyls (e.g. formation of 1,4-dideoxyosone from 1-deoxyosone) (Nedvidek etal., 1992) or by generating a-aminocarbonyl compounds, which are pyrazine precursors (Weenen and Tjan, 1994). Weenen and van der Ven (1999) studied the formation of phenylacetaldehyde in Maillard model systems, including reactions of phenylalanine with various sugars, a-dicarbonyl and hydroxycarbonyl compounds as well as Amadori rearrangement products (ARPs). The authors found that methyl glyoxal was the most efficient dicarbonyl compound for the formation of phenyl acetaldehyde, followed by 3-deoxyerythrosone, glyoxal, 3-deoxyxylosone and 3-deoxyglucosone. Hydroxycarbonyl compounds such as dihydroxyacetone and glyceraldehyde also yielded high amounts of the Strecker aldehyde. Sugars were less reactive, with the reactivity decreasing in the order erythrose, xylose, fructose and glucose. In addition, the authors showed that the Glu/Phe ARP is a superior phenylacetaldehyde precursor to the corresponding sugar/amino acid mixture. This finding was also confirmed by Hofmann and Schieberle (2000). In addition, Hofmann and Schieberle (2000) revealed that the yields of phenylacetaldehyde formed from Glu/Phe ARP were significantly increased in the presence of oxygen and copper(II) ions. Based on the observation that 1,2-hexodiulose is also generated in high amounts under these conditions, they proposed a mechanism for the formation of phenylacetaldehyde from Glu/Phe ARP (Figure 2.3). Hofmann et al. (2000) additionally found that in the reaction of phenylalanine and glucose, considerable amounts of phenylacetic acid is generated. While the formation of phenylacetaldehyde showed an optimum pH of 5 and was not influenced by oxygen, the acid was most abundant at pH 9 in the presence of oxygen and copper(II) ions. The authors proposed a mechanism for the generation of phenylacetic acid that involves a similar oxidation step to that shown in Figure 2.3. Cremer and Eichner (2000) studied the influence of the pH on the formation of 3-methylbutanal during Maillard reaction of glucose and leucine. They showed

O2,

H20

\ OH

\

H

Figure 2.3 Formation of phenylacetaklehyde via an oxidativc degradation of A^-( l-dcoxy-D-fructosyl)-l.-phenylalanine (Glu/Phe ARP) (according to Hofmann and Schieherle. 2(XX)).

THE BASIC CHEMISTRY AND PROCESS

37

that the formation rate of the aldehyde was higher at pH 7 than at pH 5 or 3. This was consistent with the high degradation rate of the Glu/Leu ARP at pH 7, suggesting that the ARP was a good precursor for 3-methylbutanal. However, Chan and Reineccius (1994) showed that the optimal reaction conditions of different Strecker aldehydes vary, owing to differences in stability of the aldehydes.

2.3

Important aroma compounds derived from Maillard reaction in food and process flavours

In the flavour industry, process flavours are often developed by empirical means, i.e. the reaction conditions are optimised using organoleptic evaluation. However, this approach should be accompanied by analytical evaluation of the products. Knowledge of the important aroma compounds derived from the Maillard reaction in food and process flavours is essential in order to focus investigations into reaction mechanisms and flavour optimisation on the key aroma compounds. It is recommended that the evaluation of character impact aroma compounds should be based on a combination of instrumental and sensorial analysis. A wellestablished approach involves the determination of the odour activity values (ratio of concentration and odour threshold values) of aroma compounds (Grosch, 1994). This requires quantitative analysis of a selected number of aroma compounds, the importance of which has been screened by GColfactometry (GC-O) techniques. Based on GC-O of stepwise decreasing concentrations of solvent extracts or headspace, suitable screening techniques such as aroma extract dilution analysis or headspace dilution analysis are available (Ullrich and Grosch, 1987; Guth and Grosch, 1993a,b). As these methods do not consider interactions of different aroma compounds, they should be combined with organoleptic evaluations of reconstituted model mixtures. Our selection of important Maillard-derived aroma compounds (see Sections 2.3.1 and 2.3.2) is primarily based on results of GC-olfactometry techniques or other sensory evaluations or on quantitative data. 2.3.1 Character impact compounds of thermally treated foods Character impact compounds of thermally treated foods that are formed during Maillard reaction are summarised in Table 2.2. Their identification in foodstuffs such as meat, bread, coffee, cocoa, chocolate, sesame, popcorn, French fries, tea, fish and yeast is covered, as well as odour description and odour thresholds of the aroma substances. In the following discussion of Table 2.2, emphasis will be given to important aroma compounds of meat, yeast, coffee and bread.

Table 2.2 Important aroma compounds derived from the Maillard reaction in various thermally treated foods'1 No.

Compound

A.

Compounds containing sulfur

1 2 3 4 5 6 7 8 9 10 II 12 13

Odour description

Odour threshold in water(ug/kg) h

2-Methyl-3-furanthiol 2-Furfurylthiol

Meaty, sweet, sulfury Roasty, sulfury

0.007 (1) ().() 1 ( 1 )

3-Mercapto-2-butanone 3-Mercapto-2-pentanone 2.5-Dimethyl-3-furanthiol 2-Methyl-3-(methylthio)furan 2-Methyl-3-(methyldithio)furan 2-Methyl-3-(methyltrithio)furan 2-Furfurylmethyl disulfide 2-Methyl-3-fury Ithioucetatc l-(2-Methyl-3-furylthio)-ethanethiol 4-Hydroxy-2.5-dimethyl-3(2//)-thiophenone Methional

Sulfury, catty Sulfury, catty Meaty, sweet, sulfury Meaty, thiamin-like Cooked meat-like Cooked meat-like Roasty, brothy Meaty, onion-like, coffee-like Meaty, roast beef Caramel-like, fruity Cooked potato-like

3.0(1) 0.7 (1) 0.018(1) 0.05(15) ().(X)4 (16)

Roasty. popcorn-like, burnt

1.0 (25)

0.05 (20) 0.2 (18)

Detected in h

Meat (2-6), yeast (20,40), coffee (7,8) Meat (3-6,9), yeast (40), coffee (7,8), sesame (10), popcorn (11) Beef (12) Beef (3,4,13), chicken (3,14), yeast (40) Chicken (4) Yeast (15) Cocoa (17). chocolate (17). meat (41) Beef(12) Beef(12) Yeast (20) Yeast (20) Yeast (20) Beef (3,9,19). chicken (4,5). pork (6), coffee (7). French fries (21), potato chip (18), fish (22,23). bread (24). yeast (40) Beef (3,9), chicken (4,5), sesame (10). fish (22)

14

2-Acetyl-2-thia/oline

B.

Compounds containing oxygen

15

4-Hydroxy-2.5-dimethyl-3(2//)-furanonc (HDF)

Caramel-like, strawberry-like

10 (26)

Id

3-Hydroxy-4.5-diniethyl-2(5//)-furaiione (sotolon) 2-Kthyl-4-hydroxy-5-methyl-3(2//)-furanonc (HHMF) 3-Hydroxy-4-methyl-5-ethyl-2(5/y Muranone (ahhexon) 2/3-Methylbutaiial

Seasoning-like

0.3 (5)

Caramel-like, sweet

1.15(26)

Beef (3,9,19), chicken (6), coffee (7), beer (27), popcorn (27), bread (24). chocolate (17), sesame (28), French fries (21), tea (29), yeast (40) Beef (3,9,19), chicken (5). coffee (7), French fries (21). tea (29), chocolate (17), cocoa (17), yeast (40) Coffee (7)

Seasoning-like

7.5 (26)

Coffee (26). chocolate (17). cocoa (17)

Malty, cocoa-like

0.35 (5)

Meat (3,5). yeasi (40), chocolate (17). cocoa (17), coffee (7). French fries (21). bread (24). lea (29), beer (30)

17 18 19

20

Methylpropanal

Malty, fruity, pungent

21.

Phenylacetaldehyde

Honey-like, sweet,

22.

Acetaldehyde

Solvent-like

25 (9)

23.

2,3-Butanedione

Buttery

15 (32)

Buttery, green

30 (32)

0.7 (23) flowery

4(18)

24.

2,3-Pentanedione

C.

Compounds containing nitrogen

25.

2-Acetyl-l-pyrroline

Roasty, popcorn, bread-like

0.1 (33)

26. 27.

6-Acetyltetrahydropyridine 2-Ethyl-3,5-dimethylpyrazine

Roasty, cracker-like Earthy, roasty

1.6 (35) 0.16 (7)

28.

2-Ethyl-3,6-dimethylpyrazine

Earthy, roasty

0.4 (18)

29.

2,3-Diethyl-5-methylpyrazine

Earthy, roasty

0.09 (7)

30. 31. 32.

2-Ethenyl-3,5-dimethylpyrazine 2-Ethenyl-3-ethyl-5-methyl-pyrazine 2-Acetylpyrazine

Earthy, roasty Earthy, roasty Roasty, sweet, nutty

a

62 (38)

Beef (19), chicken (5), bread (24), chocolate(17) coffee (7), French fries (20), tea (29) Beef (2,19), cocoa (17). chocolate (17), bread (24), French fries (21), coffee (7), tea (29), yeast (40) Beef (9), chicken (5), coffee (8), French fries (21), fish (31) Beef (3,9), bread (24), coffee (7), fish (22,23). Chocolate (17), cocoa (17), yeast (40) Fish (22,23), coffee (7), bread (24)

Bread (24), rice (34), popcorn (11), sesame (10), beef (23), French fries (21), yeast (40) Bread (24), popcorn (11) Beef (19,25), chicken (14), coffee (7), sesame (10), bread (24), French fries (21), cocoa (17), chocolate (17), popcorn (36) Bread (24), French fries (21), cocoa (17), chocolate (17), popcorn (36) Beef (19,25), chicken (14), coffee (7), French fries (21). cocoa (17), chocolate (17), sesame (10), popcorn (36), bread (37), yeast (40) Coffee (7) Coffee (7), French fries (21) Sesame (28), popcorn (39), bread (36)

The sensory significance of the aroma compounds was assessed by quantitative data or by GC-O techniques. 'References: 1 Hofmann (1995); 2 Gasser and Grosch (1988); 3 Kerscher and Grosch (1997): 4 Gasser and Grosch (1990); 5 Kerler and Grosch (1997); 6 Gasser and Grosch (1991); 7 Grosch (2001): 8 Semmelroch and Grosch (1995); 9 Guth and Grosch (1994); 10 Schieberle (1993a); 11 Schieberle (1991a); 12 Mottram and Madruga (1994); 13 Guth and Grosch (1993a); 14 Kerler (1996); 15 MacLeod and Ames (1986); 16 Schieberle et al. (2000); 17 Schnermann and Schieberle (1997); 18 Guadagni et al. (1972); 19 Kerler and Grosch (1996); 20 Werkhoff et al. (1991); 21 Wagner and Grosch (1997); 22 Milo and Grosch (1993); 23 Milo and Grosch (1996): 24 Rychlik and Grosch (1996); 25 Cerny and Grosch (1993); 26 Semmelroch et al. (1995); 27 Schieberle (1993b); 28 Schieberle (1996); 29 Guth and Grosch (1993b); 30 Schieberle (1991b); 31 Milo and Grosch (1995); 32 Blank el al. (1992); 33 Buttery et al. (1983); 34 Buttery et al. (1982); 35 Buttery and Ling (1995); 36 Schieberle and Grosch (1987): 37 Schieberle and Grosch (1994); 38 Teranishi et al. (1975); 39 Schieberle (1995): 40 Munch and Schieberle (1998): 41 Madruga and Mottram (1995). fc

40

FOOD FLAVOUR TECHNOLOGY

The meaty character of boiled beef, pork or chicken is mainly due to sulfur compounds such as 2-methyl-3-furanthiol, 2-furfurylthiol, 3-mercapto2-butanone, 2/3-mercapto-3/2-pentanone, 2,5-dimethyl-3-furanthiol, methanethiol, hydrogen sulfide and methional (Gasser and Grosch, 1988,1990; Mottram and Madruga, 1994; Kerscher, 2000). The precursors of these aroma substances in meat are known to be free or bound C5-sugars such as ribose, ribose phosphate and inosine monophosphate as well as sulfur-containing compounds such as thiamin, cysteine, glutathione and methionine. After the publication of the patent of Morton et al. (1960), in which meat aroma formation established from the reaction of cysteine and ribose was described, a great number of patents and publication on meat-like process flavours followed (see Section 2.4.3). It is well known that species-specific differences in the aroma of cooked meats like beef and chicken are mainly due to concentration and composition differences in lipid-derived flavour substances. Kerscher and Grosch (1998) and Kerscher (2000) confirmed these findings and additionally showed that significant differences also exist for Maillard-derived aroma compounds. Cooked beef, for example, was found to contain higher amounts of the sulfur compounds 2-methyl-3-furanthiol and 2-furfurylthiol as well as the caramel-like 4-hydroxy2,5-dimethyl-3(2//)-furanone, whereas 3-mercapto-2-pentanone and methional were more important in cooked chicken. The character impact compounds of yeast extracts were found to be very similar to those of cooked meat, which is due to similar pools of Maillard precursors. Munch and Schieberle (1998) reported high odour activity values for the sulfur compounds 2-methyl-3-furanthiol, 2-furfurylthiol, 3-mercapto-2-pentanone and methional, the Strecker aldehydes 3-methylbutanal and phenylacetaldehyde, as well as the furanones 4-hydroxy-2,5-dimethyl3(2//)-furanone (furaneol) and 3-hydroxy-4,5-dimethy 1-2(5//)-furanone (sotolon). Werkhoff et al. (1991) additionally identified 2-methyl-3-(methylthio) furan, 2-methyl-3-furylthioacetate, l-(2-methyl-3-furylthio)ethanethiol and 4-hydroxy-2,5-dimethyl-3(2f/)-thiophenone (thiofuraneol) in yeast. The authors claimed that these sulfur compounds contribute significantly to the meaty character of yeast. The Maillard reaction is also a key reaction in flavour formation during roasting of coffee. The precursor pool in green coffee comprises a complex mixture of various soluble sugars such as glucose, fructose, galactose and sucrose. In addition, the amount of polymeric arabinose and rhamnose was found to decrease during roasting, which indicates that these sugars are also involved in caramelisation and Maillard processes (Tressl, 1989). The total amino acid content drops by about 30% during roasting. Especially the amino acids lysine, serine, threonine, arginine, histidine, methionine and cystine are degraded to a high extent during the roasting process (Belitz and Grosch. 1999). Semmelroch and Grosch (1995) reported the simulation of the aroma of arabica and robusta coffee brews using reconstituted mixtures of 23 aroma compounds. The authors showed that the Maillard products 2-furfurylthiol.

THE BASIC CHEMISTRY AND PROCESS

41

furaneol, sotolon, methanethiol, 2,3-butanedione, 2,3-pentanedione, 2-ethyl3,5-dimethylpyrazine (EDMP), 2,3-diethyl-5-methylpyrazine (DEMP), methylpropanal and 3-methylbutanal contribute to the flavour of coffee brews. They also investigated the aroma differences between arabica and robusta coffee. The more earthy/roasty and less caramel character of robusta was found to be due to higher concentrations of the pyrazines EDPM and DEMP as well as to lower amounts of furaneol and sotolon respectively. The flavour of cereal products, especially of bread, has been studied extensively, and the results have been reviewed (Grosch and Schieberle, 1997). 2-Acetyl-1 -pyrroline (ACP) and 6-acetyltetrahydropyridine (ACTP) are responsible for the pleasant roasty character of wheat bread crust and popcorn. However, these compounds are not important in wheat bread crumb and rye bread. Both ACP and ACTP are generated by a reaction of proline with reducing sugars or sugar breakdown products (Schieberle, 1990). When ornithine instead of proline was reacted, only ACP was formed. The fact that yeast contains relatively high amounts of oraithine explains why ACP concentrations in bread are strongly dependent on the amount of yeast used in the baking process (Grosch and Schieberle, 1997). Other important Maillard-derived aroma compounds of wheat and rye bread are furaneol and the Strecker aldehydes methional, 3-methylbutanal and methylpropanal. They contribute to the caramel-like and malty aroma of bread. 2.5.2 Character impact compounds of process flavours Meat-like process flavours are often prepared by reacting cysteine and/or thiamin with sugars, although pentoses such as xylose and ribose are preferably used. The products serve as building blocks in the creation of meat flavours (see also Section 2.4.3). The important sulfur-containing compounds derived from either cysteine/ribose or thiamin reaction systems are quite similar (Table 2.3). The aroma of both cysteine and thiamin based process flavours is determined by 2-methyl-3-furanthiol, 3-mercapto-2-butanone, 2/3-mercapto-3/2-pentanone, 2~methyl-3-thiophenethiol and 2-thenylthiol (Hofmann and Schieberle, 1995, 1997; Gtintert et al., 1992, 1996). The same compounds are also responsible for the meaty character of process flavours that are based on S'-inosine monophosphate (5'-IMP) and cysteine (Zhang and Ho, 1991; Madruga and Mottram, 1998). Although 5'-IMP is more abundant in raw meat than is ribose, it is a much poorer precursor for these sulfur compounds than ribose, when reacted with cysteine (Mottram and Nobrega, 1998). Other character impact compounds that are formed primarily in thiamin or thiamin/cysteine reaction systems are 2-methyl-4,5-dihydro-3-furanthiol, 1 -(methylthio)ethanethiol, mercaptoacetaldehyde and 2-methyl-l,3-dithiolane (Giintert et al., 1996). Many of the thiols mentioned above are also important aroma substances in glucose/cysteine or rhamnose/cysteine process flavourings (Hofmann and Schieberle, 1997). However, both reaction systems contain other characteristic

Table 2.3 Character impact compounds of process flavourings11 Odour description

Odour threshold |ug/kg] in water h

No.

Compound

A.

Compounds containing sulfur

1

2-Methyl-3-furanthiol

Meaty, sulfury, sweet

0.007(1)

2

2-Furfurylthiol

Sulfury, roasty, coffee-like

0.01 (1)

3 4 5

Mercaptoacetaldehyde Mercapto-2-propanone 3-Mercapto-2-butanone

Cabbage-like Sulfury, putrid Sulfury, catty

3.0(1)

6

3/2-Mercapto-2/3-pentanone

Sulfury, catty

0.7(1)

7 8

Methional 2-Methyl-3-thiophenethiol

Cooked potato-like Meaty, sulfury

0.2 (8) 0.02(1)

9 10

2-Methyl-4.5-dihydro-3-t'uranthiol bis(2-Methyl-3-furyl) disultide

Meaty, sulfury Meaty, sulfury

0.00002(10)

11

2-Thenylthiol

Sulfury. roasty

0.042 (1)

12 13 14 15 I (•>

5-Methyl-2-furfurylthiol 5-Methyl-2-thcnylthiol 2-( l-Mercaptoethyl)furanc 2-( l-Mercaptoethyl)thiophene 2-Methyltetrahydrothiophen-3-onc

Sulfury, roasty Sulfury. roasty Sulfury, burnt Sulfury. burnt Sulfury. burnt

0.048(1) 0.049(1) 0.022(1) 0.038(1)

17

I -(Methvlthiolethanethiol

Thiamin-likc. meaty

Detected in h Cysteine/ribose (2,3,6), cysteine/glucose (4), cysteine/rhamnose (4), glutathione/ribose (3), thiamin (3,5), thiamin/cysteine (5), cysteine/IMP (6,7), cysteine/ribose-5-P (6) Cysteine/ribose (2,3,6), cysteine/glucose (4), cysteine/rhamnose (4), glutathione/ribose (3), thiamin (3), cysteine/IMP (6,7), cysteine/ribose-5-P (6) Thiamin/cysteine (5) Cysteine/IMP (7) Cysteine/ribose (2,6), cysteine/glucose (4), cysteine/rhamnose (4), thiamin/cysteine (5), cysteine/IMP (6,7), cysteine/ribose-5-P (6) Cysteine/ribose (2,3,6), cysteine/glucose (4), cysteine/rhamnose (4). glutathione/ribose (3), thiamin (3,5). thiamin/cysteine (5), cysteine/IMP (6), cysteine/ribose-5-P (6) Methionine/ribose (9). thiamin/methionine (5) Cysteine/ribose (2,6). cysteine/IMP (6). cysteine/ribose-5-P (6). thiamin/methionine (5) Thiamin (3,5), thiamin/cysteine (5) Cysteine/ribose (2,3,6), glutathione/ribose (3), thiamin (5), cysteine/IMP (6). cysteine/ribose-5-P (6) Cysteine/ribose (2,6). cysteine/glucose (4), thiamin/cysteine (5), cysteine/IMP (6,7). cysteine/ribose-5-P (6) Cysteine/rhamnose (4) Cysteine/rhamnose (4) Cysteine/glucose (4) Cysteine/glucose (4) Cysteine/ribose (23), thiamin (5), thiamin/cysteine (5), cysteine/ IMP (6,7). cysteine/ribose-5-P (6) Thiamin/cysteine (5), thiamin/methionine (5)

18 19 20 21 22 23 24

2-Methyl-1,3-dithiolane Hydrogen sulfide Methanethiol Ethanethiol 2-Acetyl-2-thiazoline 5-Acetyl-2,3-dihydro-1,4-thiazine 4-Hydroxy-2,5-methyl-3(2//)thiophenone

B.

Compounds containing oxygen

25

4-Hydroxy-2,5-dimethyl-3(2//)furanone (furaneol) 4-Hydroxy-5-methyl-3(2#)furanone (nor-furaneol) 2-Ethyl-4-hydroxy-5-methyl-3(2//)furanone (homo-Furaneol) 3-Hydroxy-2-methyl-4(4//)pyranone (maltol) 3-Hydroxy-4,5-dimethyl-2(5//)furanone (sotolon) 3-Hydroxy-6-methyl-2(2//)pyranone

26 27 28 29 30 C. 31 32 33 34

Compounds containing nitrogen 2,3-Diethyl-5-methylpyrazine 2-Acetyl-l-pyrroline 6-Acetyltetrahydropyridine 2-Acetylpyridine

Sulfury Sulfury, egg-like Sulfury, putrid Sulfury, putrid Roasty, popcorn-like Roasty, popcorn-like Caramel-like, sweet, meaty

10(11) 0.2 (12) 1.0 (13) 1.25 (1) 24.0 (1)

Thiamin/cysteine (5) Cysteine/ribose (2), cysteine/glucose (4), cysteine/rhamnose Cysteine/ribose (2), cysteine/glucose (4), cysteine/rhamnose Cysteine/ribose (2), cysteine/glucose (4), cysteine/rhamnose Cysteine/ribose (2), cysteine/glucose (4), cysteine/rhamnose Cysteine/ribose (2), cysteine/glucose (4). cysteine/rhamnose Cysteine/glucose (4)

Caramel, strawberry-like

10(14)

Caramel-like, burnt chicory Caramel-like, sweet

8500 (1)

Cysteine/ribose (2), cysteine/glucose (4), cysteine/rhamnose (4), proline/glucose (15), proline/rhamnose (16). lysine/rhamnose (16) Cysteine/ribose (2), proline/xylose (16)

1.15(14)

Cysteine/rhamnose (4)

Caramel-like

35000 (17)

Serine/maltose (18), proline/lactose (18)

Seasoning-like

0.3 (19)

Cysteine/ribose (2). cysteine/glucose (4). cysteine/rhamnose (4)

Seasoning-like

15.0(1)

Cysteine/rhamnose (4)

Earthy, roasty Roasty, popcorn-like Roasty. burnt, caramel-like Roasty, caramel-like

0.09 (20) 0.1(21) 1.6(22) 19(23)

Cysteine/glucose (4), cysteine/rhamnose (4) Proline/glucose (15) Proline/glucose (15) Proline/glucose (15)

"The sensory significance of the aroma compounds was assessed by quantitative data or by GC-O techniques. •"References: 1 Hofmann (1995); 2 Hofmann and Schieberle (1995); 3 Gasser (1990); 4 Hofmann and Schieberle (1997); 5 Giintert et al. (1996); 6 Mottram and Nobrega (1998); 7 Zhang and Ho (1991); 8 Guadagni et al. (1972); 9 Meynier and Mottram (1995); 10 Buttery et at, (1984); 11 Pippen and Mecchi (1969); 12 Guth and Grosch (1994); 13 Cerny and Grosch (1993); 14 Semmelroch et al. (1995); 15 Roberts and Acree (1994); 16 Decnop et al. (1990); 17 Pittet etal. (1970); 18 Fickert (1999); 19 Kerler and Grosch (1997); 20 Grosch (2001); 21 Buttery et al. (I983); 22 Buttery and Ling (1995): 23 Teranishi et al. (1975).

(4) (4) (4) (4) (4)

44

FOOD FLAVOUR TECHNOLOGY

aroma compounds. For example, 2-(l-mercaptoethyl)furan and its thiophene derivative are only formed from glucose and cysteine, whereas rhamnose/ cysteine generates very high amounts of 4-hydroxy-2,5-dimethy 1-3(2//)furanone (furaneol) and 3-hydroxy~6-methyl-2(2//)-pyranone. The latter two substances are responsible for the strong caramel and seasoning-like character of rhamnose/cysteine process blocks, which render them very suitable for application in beef flavours. Another compound having seasoning-like character is 3-hydroxy-4,5-dimethyl-2(5f/)-furanone (sotolon). Sotolon was found to contribute to the aroma of various cysteine derived reaction flavours (Hofmann and Schieberle, 1995, 1997). In contrast to the other compounds mentioned above, the formation of sotolon is less influenced by the type of sugar. Furaneol, which is also a key ingredient of caramel-like process flavours can be efficiently prepared by reacting rhamnose or other 6- deoxyhexoses with lysine, proline or hydroxyproline (Decnop et a!., 1990). Another important caramel-like aroma compound, 3- hydroxy-2-methyl-4(4//)-pyranone (maltol), is a key substance of process flavours that are prepared from disaccharides (maltose or lactose) and proline or serine (Fickert, 1999). In thermally treated solutions of proline and glucose or fructose, 2-acetyl1-pyrroline (ACP) and 6-acetyltetrahydropyridine (ACTP) have been evaluated as important aroma compounds, contributing a roasty, popcorn-like odour to these process flavours (Roberts and Acree, 1994; Schieberle, 1995). ACP and ACTP have also been reported to be character impact compounds of various thermally treated cereal products (see also Section 2.3.1). In addition, Roberts and Acree (1994) showed that furaneol and 2-acetylpyridine additionally contribute to the flavour of the proline/glucose model system.

2.4

Preparation of process flavours

2.4.1 General aspects In Europe, flavourings that are obtained by thermal treatment of a reducing sugar and a food-grade nitrogen source such as amino acids, peptides, food proteins, hydrolysed vegetable proteins (HVPs) and yeasts are referred to as process flavours. These products have been designated as a separate class of flavours and are identified as complex mixtures that have been converted to flavours by heat processing. In the US, the term 'process flavours' does not exist in regulatory terms. Maillard reaction flavours are considered natural or artificial flavours, depending on whether the starting materials and process are considered natural or not. The International Organisation of the Flavour Industry (IOFI) has established a guideline for manufacturers of process flavours. This guideline is part of the lOFI's Code of Practice and defines the types of raw materials and general reaction conditions (for instance, a maximum temperature/time treatment

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of 180°C/15min, pH<8) (reviewed by Manley, 1995). The US Department of Agriculture (USDA), however, did not set a guideline for manufacture but established labelling criteria for materials used to produce a process flavour (Lin, 1995). Although processed flavours that are prepared according to the IOFI guidelines were considered GRAS (Generally Recognised as Safe) in the US in 1995, the regulatory status of Maillard reaction flavours still lacks clarity with respect to the GRAS specification. This is due to lack of information on whether processed flavours contain heterocyclic amines in amounts sufficient to affect their safe use in foods. Maillard reaction technology is commonly used by the flavour industry to produce complex building blocks that provide similar aroma and taste properties to thermally treated foodstuffs such as meat, chocolate, coffee, caramel, popcorn or bread. Although flavour formation during the Maillard reaction is quantitatively a minor pathway, process flavours are very important to the flavour industry. This is because these complex blocks exhibit unique flavour qualities, are difficult to copy and are relatively cheap, being based on low production costs and high flavour potency of the aroma compounds formed. 2.4.2 Factors influencing flavour formation The factors that influence flavour formation and, thus, the sensory properties of process flavours are the type of sugar and amino acid, pH, solvent, water activity as well as temperature and time (see reviews by Reineccius, 1990; Shibamoto, 1983). In general, the sensory quality of a process flavour is less influenced by the type of sugar than by the amino acid. Several authors (Herz and Schallenberger, 1960; Lane and Nursten, 1983; Yaylayan et al., 1994) studied the variety of odours produced in Maillard model systems comprising two or more components in reaction systems containing various sugars (or ascorbic acid) with each of the protein-derived amino acids. The flavour characters of some of these processed sugar-amino acid model mixtures are summarised in Table 2.4. Cysteine is the favoured amino acid to produce meat-like flavours, both upon heating with reducing sugars or alone (Lane and Nursten, 1983). These authors also obtained chicken and beef aromas by reacting respectively, cysteine and threonine with ascorbic acid. Chocolate flavours can be prepared by heating glucose with amino acids such as serine, glutamine, tyrosine, leucine, threonine or phenylalanine (Herz and Schallenberger, 1960; Lane and Nursten, 1983). Phenylalanine gives also rise to a floral aroma (in reaction with glucose or alone), whereas threonine yields nutty aromas when reacted with ribose or xylose. Proline is the favoured amino acid for the production of bread-like and baked flavours (Lane and Nursten, 1983; Yaylayan et al., 1994). However, Schieberle (1992) showed that the yeast-derived amino acids ornithine and citrulline are even more effective precursors for 2-acetyl-l-pyrroline, which is a

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Table 2.4 Flavour types of processed sugar-ammo acid model mixtures Sugar

Amino acid

Glucose Ribose Ascorbic acid Ascorbic acid Glucose

Cysteine Cysteine Threonine Cysteine Serine or glutamine or tyrosine Leucine Threonine Phenylalanine Threonine Proline Proline or hydroxyproline Alanine Lysine Lysine Lysine Valine Arginine Methionine Isoleucine Glutamine or asparagine

Glucose Glucose Glucose Ribose or xylose Glucose Glucose Glucose Glucose Xylose Ribose Glucose Glucose Glucose Glucose Glucose

Flavour description

References3

100-140 100 140 140 100-220

Meaty, beefy Meaty, roast beef Beef extract, meaty Chicken Chocolate

,6 ,2,6

100 100 100-140 140 100-140 180

Chocolate Chocolate Floral, chocolate Almond, marzipan Nutty Bread, baked

3 3 1 1 1 3,6

100-220 110-120 100 140 100 100 100-140 100

Caramel Caramel Caramel, butter.' Toast Rye bread Popcorn Cooked potatos Celery Nutty

1.6 4 5 1 3 3 1,3 1 6

Temperature ( C)

_h

a

l Lane and Nursten (1983): 2 Morton et al. (1960): 3 Herz and Schallenberger (I960): 4 McKenna (1988): 5 Apriyantono and Ames (1990): 6 Yaylayan et al. (1994). b Microwave heating (640 W for 2-4 minutes).

key aroma compound of bread crust. Herz and Schallenberger (1960) reported the generation of rye bread and popcorn aromas by heating valine or arginine with glucose. In addition, alanine and lysine were found to give caramel, and glutamine and arginine nutty flavours. Besides the type of sugar and amino acid, pH is another important factor determining aroma of process flavours. It is well known to the flavour industry that meat flavours are preferably prepared at low pH (4-5.5). whereas roast and caramel flavours are obtained under neutral or slightly basic conditions. Madruga and Mottram (1995) as well as Hofmann and Schieberle (1998) showed that important sulfur-containing compounds in meat such as 2-methyl3-furanthiol, 2-furfurylthiol and 2-methyl-3-(methyldithio)furan are preferably formed at a pH of 3-4. Sensorial evaluations of thermally treated model mixtures of ribose or 5'-inosine monophosphate (5'-IMP) and cysteine revealed that the highest scores for boiled meat character were obtained when the reactions were carried out at pH 4.5 (ribose) and 3 (5'-IMP) (Madruga and Mottram, 1998). Solvents and water activity of the Maillard reaction systems are additional factors that influence aroma generation. Besides buffered aqueous solutions.

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solvents such as propylene glycol, glycerol, triacetin or fats and oils as well as their emulsions or mixtures with water are used. Vauthey et al. (1998), for example, filed a patent on the generation of roast-chicken aroma using a cubic phase system. This system was prepared by introducing a melted monoglyceride (saturated in Ci6 and Qg) into an aqueous phosphate buffer solution. Compared to the same reaction in phosphate buffer, the flavour formed in the cubic system was more intense, corresponding to higher amounts of sulfur compounds like 2-methyl-3-furanthiol. Shu and Ho (1989) investigated the reaction of cysteine and 4-hydroxy-2,5-dimethyl-3(2//)-furanone in varying proportions of water and glycerol. They found that a superior roasted/meaty character was obtained in the aqueous system. The influence of the water activity on pyrazine formation during Maillard reaction was studied by Leathy and Reineccius (1989a). The authors observed that pyrazine formation was optimal at an Aw of about 0.75. Schieberle and Hofmann (1998) compared the character impact compounds of cysteine-based process flavours formed in aqueous solution and under dry heating conditions. In a cysteine/ribose model system, dry heating yielded higher amounts of key odorants with roasty notes such as 2-furfurylthiol, 2-acetyl-2-thiazoline and 2-propionyl-2-thiazoline as well as 2-ethyl- and 2emenyl-3,5-dimemylpyazine, whereas the meat-like sulfur compounds 2-methyl-3-furanthiol and 3-mercapto-2-pentanone were found in comparable or lower concentrations respectively. Their study also revealed that the amounts of the 3-deoxyosone derived compounds 2-furfural and 5-methylfuran2-aldehyde were significantly higher in the dry-heated model systems, whereas the formation of the 1-deoxyosone-derived 4-hydroxy-2,5-dimemyl-3(2//)furanone was enhanced in aqueous solution. An explanation for this finding could be that, under dry heating conditions, caramelisation processes are favoured relative to Maillard reaction. In caramelisation processes, 1,2enolisation of the sugar molecule is preferred over 2,3-enolisation, leading to the formation of high amounts of 3-deoxyosones (Kroh, 1994). Temperature and time have a major effect on the sensory properties of process flavours. Knowledge of the reaction kinetics of aroma compounds helps to explain the influence of temperature and time on their formation (Reineccius, 1990). Although flavour formation is a multistep reaction sequence, Arrhenius kinetics have been found to describe flavour formation well in model and real food systems. Stahl and Parliment (1994) used an ingenious device to obtain clear time/temperature conditions for model systems and determined the activation energies of flavour compounds. Leathy and Reineccius (1989b) showed that the sensory quality of a product is less influenced by temperature/time in a model system that is designed to result in similar key aroma compounds (e.g. pyrazines). This can be explained by the fact that these compounds have similar activation energies. However, their study focused on dialkylpyrazines and did not consider the more potent trialkylpyrazines, which were suggested

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to have different formation pathways (Amrani-Hemaimi, 1995; Schieberle and Hofmann, 1998). As a result, flavour generation during Maillard reaction is in most cases strongly influenced by temperature and time, also because Maillard reaction flavours are complex mixtures of different classes of key aroma compounds. Lee (1995) proposed a different approach, based on differential equations, to describe Maillard kinetics. The model produced was able to simulate the Maillard reaction not just as a function of time and temperature but also as a function of reactant concentrations and pH. From this simulation, the relative amounts of reactants could be plotted against time. Examples given include the concentrations of aldose and Amadori compounds over a 10-hour reaction and the time profiles of enolisation compounds from both the 1,2 and 2,3 pathways under defined pH conditions. The following sections give a survey of the patent literature in the area of savoury and sweet process flavours. Emphasis will be given to meat-like process flavours. 2.4.3 Savoury process flavours The great majority of patents based on Maillard reaction technology have been directed to the production of meat-like process flavours. Most of these reaction flavours indicate cysteine and thiamin as the essential sulfur-containing precursor compounds. In 1960, the basic concept of Maillard flavour technology was beginning to emerge with the Unilever patent of Morton et al. (1960). The authors disclosed Maillard processes for the production of cooked beef and pork flavours by reacting ribose or mixtures of ribose and glucose with cysteine and either additional amino acids or deflavoured protein hydrolysates from cod fish flesh, casein, groundnut or soya. The group of additional amino acids consisted of (3-alanine, glutamic acid, glycine, a-alanine, threonine, histidine, lysine. leucine, serine and valine. All flavours were prepared in water at a pH between 3 and 6 and a temperature around 130CC. Using similar reaction conditions. May and Morton (1960) and May (1961) also prepared meat flavours through reacting cysteine with glyceraldehyde or furfural in combination with deflavoured cod fish hydrolysate or amino acid mixtures. Jaeggi (1973) patented processes for boiled and roasted beef flavours, which were also based on the reaction of ribose and cysteine. However, the inventor used methionine and proline as additional amino acids and carried out the reaction in glycerol or groundnut oil instead of water. Methionine as the sole sulfur source was also reported to result in beef flavourings when reacted with xylose and cysteine-free hydrolysed plant protein (van Pottelsberghe de la Potterie, 1973). Tandy (1985) prepared process flavourings with white chicken meat character using leucine and cysteine in combination with the reducing sugars arabinose and glucose. In addition, he found that the use of rhamnose

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instead of glucose (as well as the addition of serine) provided a more aromatic, characteristic white meat chicken flavour. International Flavors & Fragrances (IFF) filed a number of patents on the preparation of chicken, beef and pork flavours using cysteine in combination with thiamin, often in carbohydrate-free systems. This demonstrates that thiamin is capable of replacing carbohydrates by providing similar intermediates and aroma compounds as found in Maillard systems. Intense beef flavours, for example, were obtained by refluxing cysteine, thiamin and carbohydrate-free vegetable protein hydrolysate (HVP) in water or water-ethanol mixtures (IFF, 1965). The addition of beef tallow was found to result in a beef flavour with a 'pan-dripping' character. In addition, chicken flavourings were prepared by heating cysteine, thiamin and HVP in combination with other ingredients such as p-alanine, glycine and ascorbic acid, whereas pork flavours required the addition of methionine and lard. Similar processes for chicken, beef and pork flavours were disclosed in other IFF patents (IFF, 1967; Giacino, 1968a, 1969; Katz and Evers, 1973). Chicken aroma was found to be improved, for example, by adding diacetyl and hexanal (Giacino, 1968a) or mercaptoalkanones (Katz and Evers, 1973) to the processed flavours. Dihydroxyacetone, pyruvic acid or pyruvic aldehyde in combination with thiamin and HVPs were claimed to result in beef flavourings with improved cooked note (Giacino, 1968b). Kerscher (2000) investigated the analytical assessment of the species-specific character of beef, chicken and pork. His results (see Section 2.3.1) cannot explain the choice of the ingredients for the preparation of chicken, beef and pork flavours that are disclosed in the IFF patents mentioned above, but are in agreement with the findings of Chen and Tandy (1988). The authors developed species-specific beef and chicken flavourings through oxidation of oleic and linoleic acid respectively. Their processes involved heat treatment of oleic or linoleic acid in the presence of air at high temperatures of about 300°C as well as trapping of the resulting aroma fraction in cold traps. The authors also claimed blocks that resembled roast, grilled, bloody and braised beef in different fractions of the distillate of oxidised oleic acid. In terms of alternative sulfur sources to cysteine and thiamin, Giacino (1970) found that process flavours with similar characters were obtained when cysteine was replaced by taurine. Patents of the Corn Products Company also describe the production of Maillard reaction flavours using taurine in combination with HVPs and xylose (Corn Products Company, 1969; Hack and Konigsdorf, 1969). From a scientific point of view, however, the finding that cysteine can be replaced by taurine has to be questioned, because there are no studies that report the generation of important meat aroma compounds from taurine. In addition, Tai and Ho (1997) could detect only trace amounts of volatile sulfur compounds in a Maillard model system containing cysteinesulfinic acid and glucose.

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Broderick and Linteris (1960) used derivatives of mercaptoacetaldehyde such as 2,5-dihydroxy-1,4-dithiane, diethyl- or dithio-acetals and hemi-mercaptals as precursors to impart meat-like flavour to canned simulated meat and vegetable products upon sterilisation. The patent of Heyland (1977) involved the use of hydrolysed onion, garlic and cabbage in combination with HVP, ribose and beef fat for the preparation of beef flavours. Other flavour companies developed meat flavourings with sulfur sources such as hydrogen, sodium or ammonium sulfides (Gunther, 1972; Godman and Osborne, 1972), methionine (van Pottelsberghe de la Potterie, 1972) or egg white (Theron et al., 1975). Yeast extracts or yeast hydrolysates have traditionally been used either as precursors for the thermal generation of meat flavourings or as taste-enhancing ingredients, in blends with process flavours. The advantage of yeast extracts is that they are a relatively cheap, natural source of amino acids and thiamin. In addition, their high content of glutamate and 5'-ribonucleotides, particularly inosine 5'-monophosphate and guanosine 5'-monophosphate, provides complexity, body and flavour enhancement. Nestle, for example, filed several patents in which the preparation of beef and chicken process flavours using yeast extracts is disclosed (Nestle, 1966; Rolli et a/., 1988; Cerny, 1995). Cerny (1995) also developed bouillon flavours using yeast cream, which is enriched in hydrogen sulfide. Such a yeast cream was obtained by incubating baker's yeast with elemental sulfur. In order to obtain complete meat flavouring products, process flavours are blended with several other ingredients, which provide aroma, taste, taste enhancement, mouth feel and body. Besides topnote flavours, yeast extracts, hydrolysed vegetable proteins and the monosodium salts of glutamate, inosinate and guanylate, ingredients such as onion, garlic, celery and/or caramel powder, animal or vegetable fats, gelatin and spices are often used in meat flavour compositions. The use of some yeast extracts, however, is limited owing to their undesirable 'yeasty' character. Therefore, research groups have developed processes for manufacturing yeast hydrolysates with improved meat-like taste, in which the yeasty notes are absent. De Rooij and Hakkaart (1992). for example, improved the meaty character of yeast hydrolysates prepared from several yeast species by combining the enzymatic degradation of yeast cells with an additional fermentation step, which was carried out using lactic acidproducing microorganisms or additional yeasts. Hyoky et a!. (1996) developed a method for the production of yeast extracts in which undesirable bitter and yeasty flavour notes were removed. The authors evaluated several non-ionic and slightly basic macroporous polymeric adsorbents as well as activated carbon for their ability to bind bitter and other undesirable flavouring substances of yeast hydrolysates, without binding yeast peptides, amino acids or nucleotides. The best results were obtained with Amberlite XAD-16 and Amberlite XAD-765. which are a non-ionic styrene/divinylbenzene copolymer and a weakly basic phenolformaldehyde polymer respectively.

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5i

2,4.4 Sweet process flavours A few patents and articles on the generation of chocolate and caramel flavours will be discussed here. Rusoff (1958) prepared artificial chocolate flavours by heating partially hydrolysed proteins with sugars. The Maillard reactions were performed between protein hydrolysates derived from casein, soy, wheat gluten or gelatin and mixtures of pentoses and hexoses. The reaction medium contained 30% water and the reaction temperature ranged between 130 and 150°C. These 'base chocolate flavours' were rounded off by adding ingredients such as caffeine, theobromine and tannins before or after the heating process. The need for hydrolysed proteins to generate chocolate flavours through Maillard reaction was stressed by Rodel et al. (1988). The authors based their investigations on precursor studies of Mohr et al. (1971, 1976), who found that only mixtures of peptide and free amino acid fractions isolated from fermented raw cocoa beans develop cocoa aroma upon thermal treatment. The study of Rodel et al. (1988) covered a complete range of parameters such as source of protein, rate of hydrolysis, source of enzyme, amount of sugar, water content as well as temperature and time. The Maillard reaction flavours obtained were evaluated organoleptically and analytically. The authors revealed that gelatin that is enzymatically hydrolysed by more than 20% is an appropriate protein source for producing cocoa flavours through Maillard reaction. The quality of the cocoa aroma was further affected by water and sugar contents, whereas the source of enzyme had no influence. A water content of at least 5% and a sugar content of 20 g per lOOg hydrolysed protein gave a positive effect on flavour. In addition, temperature and time, which were the most sensitive parameters, were optimised at 144°C and 21 minutes. Pittet and Seitz (1974) disclosed processes for the preparation of various flavours resembling chocolate, sweetcorn, popcorn, bread, cracker and carameltoffee. The flavours were prepared by heating cyclic ketones such as furaneol, maltol or cyclotene with amino acids in propylene glycol or glycerol. The temperatures ranged between 120 and 205°C. Chocolate flavours, for example, were obtained when valine or leucine was reacted with maltol or furaneol, whereas proline (in combination with furaneol or maltol) yielded cracker-like, popcorn, sweetcorn and bread aromas. In addition, caramel-toffee and burnt sugar flavours were established from proline and cyclotene or ethyl cyclotene. Gilmore (1988) also developed a caramel butterscotch flavour by heating a mixture of sugar syrup and butter in the presence of ammonia at a pH of 7 and a temperature of about 100°C. 2.5

Outlook

Flavour formation is a minor but important pathway within the complex cascade of chemical reactions occurring during Maillard processes. A strong focus on

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the key flavour compounds, their precursors and reaction routes is required for the optimisation of process flavours. The better understanding of the influence of various process parameters on the formation of both the key aroma compounds and their precursors is still a challenge for future research. Experimental design and kinetic parameter estimations are good tools for limiting the amount of experiment required. In addition, the evaluation of important taste compounds derived from the Maillard reaction, the elucidation of their formation pathways as well as the understanding of how the interaction of aroma and taste compounds affects the sensorial quality of the flavours are certainly research areas that are worth investigating.

References Amrani-Hemaimi, M., Cerny, C. and Fay. L.B. (1995) Mechanisms of formation of alkylpyrazines in the Maillard reaction. / Agric. Food Chem., 43 2818-2822. Anet, E.F.L.J. (1960) 3-Deoxyhexosones. J. Am. Chem. Soc., 82 1502-1504. Anonymous (1979) Foodstuff with flavor precursor. Research Disclosure. 177-188 17918. Apriyantono. A. and Ames, J.M. (1990) Volatile compounds produced on heating lysine with xylose, in Flavour Science and Technology (eds Y. Bessiere and A.F. Thomas). Wiley. Chichester. pp 117-120. Belitz, H.-D. and Grosch. W. (1999) Food Chemistry, 2nd edn. Springer. Berlin. Blank, I.. Sen. A. and Grosch, W. (1992) Aroma impact compounds of Arabica and Robusta coffee. Colloq. Sci. Int. Cafe, 14 117-129. Broderick, J.J. and Linteris, L.L. (1960) Flavoring agents and process for imparting a meat-like flavor to an edible composition. US Patent 2 955041. Buttery, R.G., Haddon. W.F., Seifert, R.M. and Turnbaugh. J.G. (1984) Thiamin odor and bis( 2-methyl3-furyl) disulfide. J. Agric. Food Chem., 32 674-676. Buttery, R.G. and Ling, L.C. (1995) Volatile flavor components of corn tortillas and related products. J. Agric. Food Chem., 43 1878-1882. Buttery, R.G., Ling. L.C. and Juliano, B.O. (1982) 2-Acetyl-l-pyrroline: an important aroma compound of cooked rice. Chem. Ind. (London), 23 958-959. Buttery. R.G.. Ling, L.C., Juliano, B.O. and Turnbaugh, J.G. (1983) Cooked rice aroma and 2-acetyl1-pyrroline. J. Agric. Food Chem., 31 823-826. Cerny, C. (1995) Precede de transformation du soufre elementaire en sulfure. European Patent 778 350. Cerny, C. and Grosch.W. (1993) Quantification of character-impact odor compounds of roasted beef. Z Lebensm.-Unter. Forsch.. 1% 417-122. Chan, F. and Reineccius. G.A. (1994) The reaction kinetics for the formation of isovaleraldehyde. 2-acetyl-l-pyrroline. di(H)di(OH)-6-methylpyranone, phenylacet-aldehyde. 5-methyl-2-phenyl2-hexenal, and 2-acetylfuran in model systems, in Maillard Reactions in Chemistry, Food and Health (eds T.P. Labuza, G.A. Reineccius. V. Monnier. J. O'Brien and J. Baynes). The Royal Society of Chemistry, Cambridge, pp 131-139. Chen, T.-K. and Tandy. J.S. (1988) Production of flavour. European Patent 295509. Corn Products Company (1969) Process for the production of seasonings with a flavor similar to meat extract. GB Patent 1148449. Cremer. D.R. and Eichner, K. (2000) The influence of the pH value on the formation of Strecker aldehydes in low moisture model systems and in plant powders. Eur. Food Res. Technol., 211 247-251. Decnop. C., van Dort. J.M. and de Hey. J.T. (1990) Hydroxyfuranone preparation. European Patent 398417.

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De Rooij, J.F.M, and Hakkaart, MJ.J. (1992) A process for the preparation of food flavours. European Patent 191513. de Roos, K.B. (1992) Meat flavor generation from cysteine and sugars, in Flavor Precursors: Thermal and Enzymatic Conversions (eds R. Teranishi, G.R. Takeoka and M. Giintert), American Chemical Society, Washington, DC, pp 203-216. Doornbos, T, Haring, P.G.M. and Van der Heijden, A. (1991) Use of diketone in food products. European Patent 293 957. Doornbos, T., van den Ouweland, G.A.M. and Tjan, S.B. (1981) Amadori compounds, derived from 6-deoxy sugars, as flavour precursor, in Progress in Food and Nutrition Science. Maillard Reactions in Food (ed. C. Eriksson), Pergamon Press, Oxford, pp 57-63. Eichner, K., Reutter, M. and Wittmann, R. (1994) Detection of Amadori compounds in heated foods, in Thermally Generated Flavors: Maillard, Microwave, and Extrusion Processes (eds T.H. Parliament, M.J. Morello and R.J. McGorrin), American Chemical Society, Washington, DC, pp 42-54. Fickert, B. (1999) Untersuchungen zur Bildung von Aromastoffen bei der Mlilzung von Getreide. PhD thesis, Technical University of Munich, Garching, Germany. Gasser, U. (1990) Fliichtige Verbindungen aus gekochtem Rind- und Huhnerfleisch, Fleischaromen and Fleischextrakt. PhD thesis, Technical University of Munich, Garching, Germany. Gasser, U. and Grosch, W. (1988) Identification of volatile flavor compounds with high aroma values from cooked beef. Z. Lebensm.-Unters. Forsch., 186 489-494. Gasser, U. and Grosch, W. (1990) Primary odorants of chicken broth. A comparative study with meat broths from ox and cow. Z. Lebensm.-Unters. Forsch., 190 3-8. Gasser, U. and Grosch, W. (1991) Aroma von gekochtem Schweinefleisch Lebensrnittelchemie, 45 15-16, Gi, U.-S. and Baltes, W. (1995) Model reactions on roast aroma formation, 15. Investigations on the formation of pyrido[3,4-d]imidazoles during the Maillard reaction. Journal of Agricultural and Food Chemistry, 43 2226-2230. Giacino, C. (1968a) Poultry flavor composition and process. US Patent 3 3940] 7. Giacino, C. (1968b) Product and process of reacting a proteinaceous substance with a sulfur-containing compound to provide a meat-like flavor. US Patent 3 394015. Giacino, C. (1969) Meat flavor compositions. GB Patent 1146337. Giacino, C. (1970) Meat flavor compositions. US Patent 3 519437. Gilmore, L.T. (1988) Process for preparing a caramel butterscotch flavor syrup. US Patent 4 753814. Godman, J.L. and Osborne, D.R.D. (1972) Edible products. GB Patent 1285568. Grosch, W. (1994) Determination of potent odourants in foods by aroma extract dilution analysis (AEDA) and calculation of odour activity values (OAVs). Flavour Fragrance J., 9 147-158. Grosch, W. (2001) Flavour of chemistry III: Coffee and volatile compounds, in Coffee, recent developments (eds O. Vitzthum and R.J. Clarke), Blackwell Science, Oxford, pp 68-89. Grosch, W. and Schieberle, P. (1997) Flavor of cereal products—a review. Cereal Chemistry, 74(2) 91-97. Guadagni, D.G., Buttery, R.G. and Turnbaugh, J.G. (1972) Odor thresholds and similarity ratings of some potato chip components. J. Sci. Food Agric., 23 1435-1444. Giintert, M., Bertram, H.-J., Hopp, R., Silberzahn, W, Sommer, H. and Werkhoff, P. (1996) Thermal generation of flavor compounds from thiamin and various amino acids, in Recent Developments in Flavor and Fragrance Chemistry (eds R. Hopp and K. Mori), VCH-Verlagsgesellschaft, Weinheim, pp 215-240. Guntert, M., Brtiing, J., Emberger, R., Hopp, R., Kopsel, M., Surburg, H. and Werkhoff, P. (1992) Thermally degraded thiamin: A potent source of interesting flavor compounds, in Flavor Precursors: Thermal and Enzymatic Conversions (eds R. Teranishi, G.R. Takeoka and M. Guntert), American Chemical Society, Washington, DC, pp 140-163. Gunther, R. (1972) Flavoring compositions produced by reacting hydrogen sulfide with a pentose. US Patent 3 642 497.

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Kerscher, R. (2000) Objektivierung tierartspezifischer Aromaunterschiede bei erhitztem Fleisch. PhD thesis, Technical University of Munich, Garching, Germany. Kerscher, R. and Grosch, W. (1997) Comparative evaluation of potent odorants of boiled beef by aroma extract dilution and concentration analysis. Z. Lebemrn.-Unters. Forsch., 204 3-6. Kerscher, R. and Grosch, W. (1998) Quantification of 2-methyl-3-furanthiol, 2-furfurylthiol, 3-mercapto2-pentanone, and 2-mercapto-3-pentanone in heated meat. /. Agric. Food Chem., 46 1954-1958. Keyhani, A. and Yaylayan, V.A. (1996) Elucidation of the mechanism of pyrazidone formation in glycine model systems using labeled sugars and amino acids../. Agric. Food Chem., 44 2511-2516. Kroh, L.W. (1994) Caramelisation in food and beverages. Food Chem., 51 373-379. Lane, MJ. and Nursten, H.E. (1983) The variety of odors produced in Maillard model systems and how they are influenced by reaction conditions, in Maillard Reaction in Food and Nutrition (eds G.R. Waller and M.S. Feather), American Chemical Society, Washington, DC, pp 141-158. Leathy, M.M. and Reineccius, G.A. (1989a) Kinetics on the formation of alkylpyrazines: effect of pH and water activity, in Thermal Generation of Aromas (eds T.H. Parliament, R.J. McGorrin and C.-T. Ho), American Chemical Society, Washington, DC, pp 196-208. Leathy, M.M. and Reineccius, G.A. (1989b) Kinetics on the formation of alkylpyrazines: effect of amino acid type and type of sugar, in Flavor Chemistry (eds R. Teranishi, R.G. Buttery and F. Shahidi), American Chemical Society, Washington, DC, pp 76-91. Ledl, F. and Schleicher, E. (1990) New aspects of the Maillard reaction in foods and in the human body. Angew. Chem., Int. Ed. Engl., 29 (6) 565-706. Ledl, F. and Schleicher, E. (1992) The Maillard reaction in food and human body—new results in chemistry, biochemistry and medicine. Angew. Chem., 102 597-626. Lee, P. S. (1995) Modeling the Maillard reaction—a computer-simulation and a discussion of its application to Maillard reaction analysis and design, in Flavor Technology: Physical Chemistry, Modification, and Process (eds. C.-T. Ho, C. T. Tan and C. H. Tong), American Chemical Society, Washington DC, pp 74-97. Lin, L.J. (1995) Regulatory status of Maillard reaction flavors, in Savory Flavors (ed. T.W. Nagodawithana), Esteekay Associates, Wisconsin, pp. 7-15. MacLeod, G. and Ames, J. (1986) 2-Methyl-3-(methylthio)furan: a meaty character impact compound from cooked beef. Chem. Ind., 3 175-177. Madruga, M.S. and Mottram, D.S. (1995) The effect of pH on the formation of Maillard-derived aroma volatiles using a cooked meat system. J. Sci. Food Agric., 68 305-310. Madruga, M.S. and Mottram, D.S. (1998) Sensory analysis of a model system using 5'-IMP and cysteine at different pH. Ciencia e Tecnologia de Alimentos, 18(4), 397-404. Manley, C.H. (1995) Process flavors and precursor systems, in Savory Flavors (ed. T.W. Nagodawithana). Esteekay Associates, Wisconsin, pp. 16-25. May, C.G. (1961) Flavouring substances and their preparation. GB Patent 858333. May, C.G. and Morton, I.D. (1960) Process for the preparation of a meat flavor. US Patent 2 934436. McKenna, A.B. (1988) Influence of heating on the formation of caramel flavour, brown colour and hydroxymethylfurfural in reconstituted concentrated skim milk. NZ J. Dairy Sci. TeclmoL. 23 363-372. Meynier, A. and Mottram, D.S. (1995) The effect of pH on the formation of volatile compounds in meat-related model systems. Food Chem., 52 361-366. Milo, C. and Grosch, W. (1993) Changes in the odorants of boiled trout (Salmofario) as affected by the storage of the raw material. J. Agric. Food Chem., 41 2076-2081. Milo, C. and Grosch, W. (1995) Detection of odor defects in boiled cod and trout by gas chromatographyolfactometry of headspace samples. /. Agric. Food Chem., 43 2076-2081. Milo, C. and Grosch, W. (1996) Changes in the odorants of boiled salmon and cod as affected by the storage of the raw material. J. Agric. Food Chem., 44 459-462. Mohr, W., Rohrle, M. and Severin, Th. (1971) Uber die Bildung des Kakaoaromas aus seinen Vorstufen. Fette, Seifen, Anstrichmittel, 73 (8) 515-521.

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Biotechnological flavour generation Ralf G. Berger, Ulrich Krings and Holger Zorn

3.1 Introduction Aromas and flavours possess antimicrobial, medicinal and signalling properties, as well as acting as food preservatives. They even preserved human corpses in ancient Egypt. Above all, it is their alluring sensory properties that promised financial profits high enough to prompt the Phoenicians, Arabs, and, later, the Portuguese, Dutch, Spanish and Venetians to discover and conquer new countries and continents. Today's captains are scientists, their ocean is the metabolic flow, their ships and weapons are gene shuttles and biochemical knowledge, and their targets are no longer leaves, fruits and seeds, but cells, enzymes and genes. This chapter will describe: •

• • • •

How microorganisms transform flavour precursors directly into flavour molecules (biotransformation) or produce flavours along multistep processes (bioconversion and de novo synthesis) How enzymes catalyse hydrolytic or other chemical reactions leading to flavours How single plant cells in sterile culture may replace field-grown flavour producers How the dissemination of methods of genetic engineering fertilises bioflavour research How laboratory developments have been successfully transferred into industrial applications.

No mention will be made of flavours derived from traditional and genetically engineered starter cultures, because informative reviews already exist—e.g. on fermented meat flavours (Hammes and Hertel, 1998), on dairy flavours (Berger. 1995a; Henriksen et al., 1999; Mollet, 1999), on bread flavours (Randez-Gil et al., 1999) and on wine flavours (Pretorius, 2000). 3.2

Natural flavours: market situation and driving forces

Beverages are the largest market segment for added flavours, accounting for about one-third of the worldwide flavour sales. Around 90% of the flavours added to beverages in the EC are natural, and about 80% in the US; savoury products contain about 80% naturals in both the EC and the US. while the

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percentages for flavoured dairy products are 50% in the EC and 75% in the US (the remaining proportions are nature-identical and artificial flavours). These numbers clearly reflect a strong preference of the average food consumer for naturalness. (This scientifically unfounded, vague chemophobia has been extended also to furniture, clothing and cosmetics (Cheetham, 1999), as if the Garden of Eden still existed). As a result, the attribute 'natural' is an excellent marketing point, and the use of 'natural' flavour sources is the last bastion for food manufacturers to suggest the naturalness of their product explicitly. According to the definitions of the Code of Federal Regulations in the US (CFR, 1993) and to the mandatory guidelines of the Council of the European Communities (88/388/EWG of 22 June 1988; 91/71/EWG and 91/72/EWG of 16 January 1991) aromas generated by biotechnology are classified as natural if the starting materials used were 'natural' (Grundschober, 1999). The flavour industry, accounting for some legal uncertainties, has adopted a product-oriented point of view based on the GRAS (Generally Recognized as Safe) procedure and has set its own standards. The guidelines of the International Organisation of the Flavour Industry (IOFI) demand that the bioprocessing of flavours is in accordance with Good Manufacturing Practice and with the general principles of hygiene of the Codex Alimentarius. Bioflavours shall comply with national legislation, and safety must be 'adequately established'. Doubts about contamination with microbial toxins or the like have been resolved as the volatile nature of aroma compounds facilitates efficient downstream processing and exclusion of recombinant nucleic acids or other undesired nonvolatiles by distillation or lipophilic extraction. This is thought to provide sufficient protection of the consumer's health. The 'all-natural' mania of the consumer and the favourable legal situation are, however, only two of the forces driving the development of bioflavours. Cheetham (1997), in an impressively comprehensive approach, has collated a bundle of driving forces and has subdivided them into two sectors, 'business pull' and 'technical push'. Among the business factors listed are the decline of availability of some traditional raw materials, an increasing market size, modern processes and ingredient formulations, and the growing importance of functional, ethnic and exotic products (Bauer, 2000). Technical factors mentioned include improved analytical methods (Steinhart et al, 2000), improved bioprocessing techniques, genetic engineering, and an improving understanding of structure-activity relationships of flavours and fragrances (for example, 'olfactophore models', Kraft et al., 2000). 3.3 Advantages of biocatalysis If a chemosynthetic route results in a mixture of products or isomers, a subsequent separation may be more expensive than a comparable but more selective

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bioprocess. Trace impurities of a chemosynthetic compound may adulterate the sensory character of a product, and sensory activity is often dependent on an exact stereochemical structure, as the sense of smell is chiral. Biocatalysts not only provide high regiospecificity and stereospecificity but show high reaction velocity even at low molar fraction. Although biocatalysts are not free from inherent drawbacks, such as operational instability (isolated enzymes), processing cost and sometimes occurrence of side reactions, the advantages of biocatalysts, such as ecological compatibility or multistep synthesis, cannot be matched by a chiral chemical catalyst. While the closing of mass cycles and sustainable production have gained high priority in the chemical industries, nature itself sets the example. Biocatalysis will not provide immediate solutions for all fine chemicals, but highly prized flavours and fragrances appear to present a particularly suitable playground for biotechnologists. Many authors have discussed one class or various groups of aroma compounds under quite different aspects. For an introduction to the literature until 1997 and a description of some processes already operating, the reader is referred to Berger (1995b), Etievant and Schreier (1995) and Berger (1997). More recent biotechnological advances, which will broaden and facilitate the use of biocatalysts for industrial production of volatile chemicals, are described in this chapter. 3.4

Microorganisms

Intact microbial cells possess active transport systems, enzyme arrangements optimised by evolution, and they regenerate their biocatalytic molecules together with the co-factors required; costs arise for the equipment needed to maintain a suitable biochemical environment, but costs for isolation and stabilisation of the biocatalyst are inapplicable. Bacterial pathways to volatile flavours are often based on strong hydrolytic properties. Incomplete substrate oxidation and Strecker degradation of amino acids yield a limited spectrum of products (Montel et al., 1998). Yeasts, as unicellular nonfilamentous fungi, are more complex eukaryotic cells that show a much more diverse biochemistry including a broad range of volatile metabolites, such as esters, lactones, aldehydes and phenolics (Labuda et al., 1997). The most developed fungal species, the class of Basidiomycetes, show a complicated sexual cycle, pseudo-tissue formation, and the distinct ability to degrade native cellulose or lignin aerobically. Volatile flavours from all chemical classes were found in basidiomycete fruit bodies and cell cultures, with a particular emphasis on volatile phenylpropanoic and phenolic compounds (Krings and Berger, 1998; Lomascolo et al.. 1999a). 3.4.1 Biotransformation and byconversion of monoterpenes Many essential oils are dominated by monoterpene hydrocarbons. Because of their low sensory activity, low water solubility and tendency to autoxidise and

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polymerise, they are usually rectified from the oil and regarded as processing waste. These properties (and their role as physiological precursors of highvalued oxyfunctionalised terpenoids) turn terpene hydrocarbons, such as limonenes, pinenes and terpinenes, into ideal starting materials for microbial transformations. The amount of /?-(+)-lirnonene separated from cold-pressed citrus peel oil was estimated at 36000 tonnes per year, and the steam distillation of pine oils delivered 160000 tonnes of ot-pinene and 26000 tons of (3-pinene (Nonino, 1997; Ohloff, 1994). The first systematic studies on terpene hydrocarbon transformation date back to the early 1960s (Bhattacharyya et al, 1960; Prema and Bhattacharyya, 1962), and since that time a vast number of publications on the subject have appeared. 3.4,1.1 Ally lie hydroxylation From a biotechnological point of view the allylic hydroxylation is one of the most important transformation reactions leading to compounds with direct or indirect economic importance (carveol, borneol, verbenol, nootkatol). Concurrent rearrangements of double bonds are explained either by radical transition states, as occur at the end of the cytochrome P450 monooxygenase cycle, or by radical-cationic intermediates, as are typical of peroxidase-mediated reactions. Transformation organisms range from bacteria, such as Pseudomonas through deuteromycetes, such as Penicillium or Aspergillus to higher fungi, such as the basidiomycetes. A number of frequently occurring transformation products of limonene and a-pinene are compiled in Figure 3.1. High regioselectivities have been observed. While some strains preferentially attacked ring carbons, other strains hydroxylated exocyclic allylic positions. Because of its abundance, limonene has been the most popular transformation substrate in recent years. Its regiospecific hydroxylation by a strain of Pseudomonas putida yielded up to 3g of perillic acid per litre within 5 days (Speelmans et al., 1998). The success of this transformation was based on the selection of a limonene-tolerant microorganism, biphasic operation, and careful optimisation of reaction conditions including the co-substrate glycerol. The same strategy of pre-screening microbial strains, using substrate-enriched nutrient media, succeeded in the case of Pseudomonas alcaligenes, which transformed (+)-limonene into the hydration product ot-terpineol (Teunissen and De Bont, 1995). However, when 120 Gram-positive bacterial strains were selected by their growth on carvone as the sole source of carbon, none of them transformed limonene efficiently to carvone (Van der Werf and De Bont, 1998). Other regiospecific transformations of (+)-lirnonene were observed for the basidiomycete Pleurotus sapidus, which produced mainly carveols and carvone (Onken and Berger, 1999a), and for a nonconventional Hormonema black yeast to yield fra/w-isopiperitenol (Van Rensburg et al., 1997). In the latter case, a varying morphological appearance was associated with unstable product yields. The enantiospecific transformation of racemic limonene to (+)-a-terpineol by Penicillium digitatum (Tan et al..

64

FOOD FLAVOUR TECHNOLOGY ,OH

IV

Figure 3.1 Products of allylic oxidations: (I) carveol; (II) perillyl alcohol: (III) p-mentha-1.8-diene-4ol; (IV) p-Mentha-2.8-diene-l-ol: (V) isopiperitenol; (VI) verbenol: (VII) myrtenol.

1998) encouraged these researchers to immobilise the cells in calcium alginate and to perform an airlift reactor study (Tan and Day, 1998). While good molar conversion was achieved, the absolute yields remained in the lower milligram per litre range. Starting with a report on the microbial hydroxylation of a-pinene (Bhattacharyya et al., 1960), the history of pinene transformation now spans more than four decades. Work on the transformation of the pinenes, as well as other bicyclic monoterpenes, such as cineoles, camphor and carene, was summarised by Trudgill (1994). Recently, classical UV mutagenesis preceded the selection of strains ofAspergillus, which converted a-pinene to either verbenol (Agrawal et al., 1999) or to verbenone (Agrawal and Joseph. 2000). As long as the public concern about genetic engineering applications in the food sector persists, this random approach will continue to claim scientific interest. The transformation of monoterpene monoalcohols, such as citronellol, geraniol and nerol, using Botrytis cinerea followed the same general routes (Bock et al., 1988). Cystoderma carcharias, a basidiomycete, transformed citronellol to 3,7-dimethyl-l,6,7-octanetriol and several minor products, among them the flavour impact compounds E/Z-rose oxide (Onken and Berger. 1999b). Nerol or citral were transformed by isolates of Penicillium andAspergillus to a number of other monoterpenes and to the fruity smelling C2-shortened 6-methyl-5-hepten2-one (Demyttenaere and De Footer, 1998; Demyttenaere and De Kimpe. 2000).

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A microcultivation method in conjunction with solid-phase microextraction was used to speed up the process monitoring on a laboratory scale. Another recent study confirmed the minor role of yeasts in the formation of terpenes of wine and beer flavour (King and Dickinson, 2000): Neither Saccharomyces cerevisiae nor Kluyveromyces lactis or Torulospora delbrueckii accumulated significant amounts of the transformation products, linalool, a-terpineol and terpin hydrate. Low yields and the lack of stereoselectivity for linalool and a-terpineol suggest that chemical transformation overlapped with the biocatalysed routes. 3.4.1.2 Oxidation of nonactivated carbons This reaction is the Holy Grail of many biotechnologists. However, there will be a low chance of incidence with oligoisoprenoid compounds, as most of them carry carbon double bonds, where attack will be favoured. However, aliphatic bicyclic compounds, such as fenchol, borneol and 1,8-cineole were shown by the former group of Kieslich to be selectively converted to mono- and diols by a Zygomycete, Diplodia bisporm and by Bacillus cereus (Abraham et al., 1988). A chemically related reaction is the terminal hydroxylation of saturated fatty acids catalysed by Torulopsis yeasts, a reaction permitting access to macrocyclic musks (Williams, 1999). 3.4.1.3 Epoxidation Epoxidations are typical cytochrome P450-mediated reactions. The reaction products were either isolated as stable products or considered to be intermediates, based on the vicinal diols identified. Obviously the pH of the incubation medium governs the extent of hydrolysis. Asymmetric dihydroxylation of alkene moieties was obtained when strains of Pseudomonas putida were exposed to isoprene or to related dienes (Boyd et al., 2000). Further diol oxidation was successfully controlled by addition of propylene glycol as an inhibitor. Substrates with high ring tension, such as a-pinene epoxide, were often transformed by fungal strains to a number of products, among them campholenic aldehyde, frarcs-sobrerol, and £7Z-carveols. In such cases, bioatalysis appears at first glance not to have any advantage over a chemical transformation. The same epoxide substrate, however, was efficiently degraded by Pseudomonas fluorescens and Nocardia strains to a small set of products belonging to one specific pathway (Griffiths et al. 1987; Best et al, 1987; Figure 3.2). An unusual

COOH

COOH

Figure 3.2 Degradation pathway of a-pinene in Pseudomonas fluorescens.

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(4/?, 8/?)-limonene 8,9-epoxide was the only transformation product when a Xanthobacter isolate selected on cyclohexane as the sole carbon source was exposed to (4/?)-limonene (Van der Werf et al, 2000). Many other transformation pathways of limonene were described by the same authors in a compilation worth reading (Van der Werf et al., 1999). 3.4.1.4 Oxidation of alcohols Interest in the widespread oxidation of primary and secondary terpenols rests on the fact that many terpene aldehydes and ketones are more volatile and more powerful (in flavour terms) than the corresponding alcohols. Yeasts and some higher fungi may deliver the target compounds in yields close to 100%. Further oxidation to carboxylic acids is rarely observed (Figure 3.2 shows one of the few exceptions), because the free acyl moieties are quickly transformed to activated conjugates for further metabolism through ^-oxidation and related pathways. Another pathway along which terpene acids are formed is the Baeyer-Villiger oxidation: Pseudomonasputida strains were able to insert an oxygen atom next to a carbonyl function, and the subsequent ring opening yielded a degradable acid intermediate (Abraham et al., 1988). 3.4.1.5 Hydration Hydration of a double bond or of a ring bridge may proceed as an acid- or enzyme-catalysed reaction. A cell-free control experiment is indispensable to assess the extent of chemical side reactions. As a rule of thumb, troublesome chemical hydration occurs at ambient temperature only at a pH<4. Useful information on this aspect is sometimes hidden in reports on off-flavour formation in acidic food matrices, such as citrus juices (Haleva-Toleo et al., 1999). The required biocatalytic activity appears to be located mainly in bacteria, such as Escherichia coli, Barillas stearothermophilus and Pseudomonos gladioli, but also in Penicillium digitatum. Candida tropicalis transformed a-pinene to a-terpineol in a previously unmatched yield of 77% (Chatterjee et al., 1999a). 3.4.2 Byconversion ofCjj-norisoprenoids

and sesquiterpenes

The same fundamental transformation steps as for monoterpenes apply for larger oligoisoprenoids and related compounds. The increased structural complexity, however, allows more substructures of the molecules to fit into the active sites of enzymes, resulting in a mixture of products with a broad range of concentrations. Cn-Norisoprenoids of the ionone/damascone group have received particular attention, because they belong to the small group of highly potent character impact components with pleasant floral-fruity flavours. No cell-based concerted approach for the biogeneration of any of these compounds has become known, although, using a- or (3-ionone or damascene as substrates instead, regioselective insertion of oxygen was found with several fungal species. A

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broad screening of Streptomyces strains showed that some strains transformed a-ionone regioselectively to 3-hydroxyionone without many side products (Lutz-Wahl et al., 1998). Racemic a-ionone was selectively transformed to trans-(3R, 6R) and (35,65) alcohols. In contrast, there was limited transformation of p-ionone to the 4-hydroxy product with no formation of the 3-hydroxy product. Strains of Aspergillus niger metabolised (3-ionone much better and yielded almost 100% conversion to 3- and 4-hydroxy products in a batch-fed process (Larroche et al., 1995). An advanced mathematical model of the mass transfer rates in this two-phase system was presented recently (Grivel et al., 1999). Because of the observation that the growth inhibition of Botrytis cinerea by patchoulol faded with time, the metabolic fate of the fungicide was followed (Aleu et al., 1999). Numerous hydroxy compounds were found, indicating first steps of a detoxification pathway. As these hydroxy compounds are substrates for further degradation, only transient accumulation of volatile oxidation products can be expected. The group of Miyazawa has issued a series of papers looking, for example, at the conversion of substrates, such as (+)-cedrol, (+)-aromadendrene, (-)-a-bisabolol, (3-selinene, (—)-globulol, y-gurjunene, and farnesol isomers (Miyazawa et al., 1997, 1998; Nankai et al, 1998, and references therein). A plant pathogenic fungus, Glomerella cingulata, was the strain of choice, and the problem of low substrate solubility was partially circumvented by using monoalcohols instead of the hydrocarbon compounds. As a general outline, exomethylene carbons and isopropyl substituents were oxidised nonstereoselectively, while the oxidation at ring positions proceeded stereoselectively, indicating a more restricted conformational situation at the active site of the enzyme. Some evidence is accumulating that p-ionone and other terpenes (De-Oliveira et al., 1999) and sesquiterpenes (Sime, 2000) are quite efficient inhibitors of cytochrome enzymes. This would mean that not only low substrate solubility was responsible for the low product yields reported in the past, but also the inhibition of the first step of substrate detoxification, particularly if the substrate was administered to the cells all at once. 3.4.3 Generation of oxygen heterocycles A number of oxygen-containing heterocycles, such as the well-known flavour compounds maltol and Furaneol (and some alkanolides) have received much attention, because, besides possessing flavour themselves, they may also affect the smell, taste and umami impressions of other flavour compounds. Furaneol (2,5-dimethyl-4-hydroxy-2//-furan-3-one) was obtained by converting 6-deoxyhexoses along a Maillard-imitating soft chemistry route (Whitehead, 1998). This approach suffers, however, from the lack of economic sources of the precursor 6-deoxy sugars. Furaneol and the related 2- (or 5-) ethyl-5- (or 2-)

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methyl-4-hydroxy-2//-furan-3-one have been frequently found in oriental food fermented in the presence of certain yeasts (Hayashida et al, 1998). Concentrations of the two compounds (in the mg per litre range) accumulated after supplementing Zygosaccharomyces rouxii in simple fermentation media with an autoclaved mixture of a single amino acid and reducing sugars (Hayashida et al, 1999). Hexoses favoured the formation of Furaneol, while pentoses, such as ribose, stimulated formation of the ethyl-substituted furanone with a nonlinear correlation. Glutamate was the most efficient source of nitrogen. The authors, well aware of the competing chemical pathway of formation, described the formation of furanones in aging miso and simple fermentation media as a yeast-dependent reaction and suggest that 'many amino acids form compounds able to act as Furaneol precursors'. The same experiments did not result in the formation of significant amounts of furanones. when the heating of the precursor combination was replaced by a filter sterilisation (0.22 um) protocol. Even higher concentrations of 2- (or 5-) ethyl-5- (or 2-) methyl-4-hydroxy2//-furan-3-one (74.3 mg per litre in the presence of L-alanine) were reported subsequently (Sugawara and Sakurai, 1999). The occurrence of volatile lactones, such as 4,5-dimethyl-3-hydroxy-5//furan-2-one (sotolon) in higher fungi (Lizarraga-Guerra et al.. 1997) underscores the potential of fungal cells as catalysts in bioprocesses. The most common pathway is |3-oxidative degradation of hydroxy fatty acids followed by intramolecular esterification to the respective 4- and 5-alkanolides with fruity and fatty odour notes, but a number of different pathways have also been reviewed (Gatfield, 1997). 6-Pentyl-a-pyrone is a lactone with coconutlike flavour, and its formation by Trichoderma harzianum, Trichoderma viride and other fungi is well documented. Various surface and submerged fermentations (Kalyani et al, 1999) and solid-state fermentation on sugarcane bagasse (Sarhy-Bagnon et al, 2000) have yielded up to almost one gram of volatile product per litre. The lactone inhibits the growth of some phytopathogenic fungi and may be formed as a part of the chemical self-defence system of the Trichoderma fungi. T. harzianum was also able to convert castor oil into 4-decanolide (Serrano-Carreon et al, 1997). Using a 14-litre bioreactor. rheological studies on the shear-sensitive mycelial cells were performed. A lactone yield of 5.3g per kg of castor oil was calculated according to the principle of extractive fermentation. Indeed, the age of industrial bioflavours started with a patented process to convert ricinoleic acid (12-hydroxyoleic acid) to 4-decanolide using Candida yeast (Farbood and Willis, 1983). Initially, the market price was $20 000 per kg, which created a lot of enthusiasm among biotechnologists. Process development was facilitated by access to an inexpensive precursor substrate, by the widespread occurrence of the pathway in fungi, and by relatively simple bioprocessing conditions. An alternative access was opened by the transformation of 3-decen-4-olide to the reduced lactone using baker's yeast (Gatfield and Sommer. 1997). Among the more recently described

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producers were Sporidiobolus yeasts (Haffner and Tressl, 1998; Dufosse et al, 2000). Today 4-decanolide commands a market price 40-fold lower than initially. A survey of the pathways to 4-decanolide was presented by Krings and Berger (1998). Less work has been devoted to sulfur-containing heterocycles. A recent patent (Bel Rhlid et al., 1997a,b) claims a bioprocess based on baker's yeast that converted cysteamine or cysteine and hydroxy- or oxopropanoic acid (or derivatives) to a flavour mixture containing 2-methyl-3-furanthiol, mercaptopentanone, and 2-acetyl-2-thiazoline. The composition is used to intensify meat flavour of food and petfood. 3.4.4 Generation of vanillin, benzaldehyde and benzoic compounds Vanillin is the world's number one flavour chemical. Vested with a unique flavour characteristic, the compound also exhibits antioxidant, flavour-enhancing and bitterness-masking properties. The enormous difference of the market prices of chemosynthetic and Vanilla pod-derived vanillin has again triggered a lot of research. Vanilla planifolia is an orchid and fixes CO2 according to the rather uncommon CAM (crassulacean acid metabolism) pathway. Isotope analysis of vanillin will therefore be able to discriminate various 'natural' vanillins according to the origin of the vanillin precursor substrates (Hener et al., 1998). Among the vanillin precursors listed in recent reviews (Rabenhorst, 2000; Ramachandra Rao and Ravishankar, 2000) are ferulic acid, eugenol and isoeugenol, vanillylamine, methoxytyrosine, coniferyl aldehyde, coumaric acid and others. More than two dozen microbial strains, mainly soil bacteria and higher fungi, and at least two different enzymes, converted these substrates to vanillin with yields of up to several grams per litre. The process with the highest productivity (> 11 g per litre in 30 h) is based on an actinomycete of the Amycolatopsis family and a batch-fed dosage of ferulic acid (Rabenhorst, 2000). This retro-Claisenlike cleavage also resulted in metabolite overflow, subsequent secretion, and accumulation of gram amounts of vanillin by Streptomyces setonii (Muheim and Lerch, 1999). A Pseudomonasputida strain was cultivated by the same authors. Although ferulic acid catabolism was fast, vanillin did not accumulate, as it was oxidised faster than ferulic acid. Accumulation of vanillin also failed in growing cultures of a Nocardia strain (Li and Rosazza, 2000). A purified carboxylic acid reductase from the same strain, however, quantitatively reduced vanillic acid to vanillin. Isoeugenol from various essential oils is another inexpensive precursor substrate. Owing to its considerable cytotoxicity, the development of a high-yielding bioprocess towards vanillin was prevented for a long time. With the aid of enrichment techniques, isoeugenol tolerant strains have now been selected. Cell-free extracts of a Bacillus strain yielded 0.9 g vanillin per litre (Shimoni et al., 2000), and Rhodococcus rhodochrous tolerated 15 g of isoeugenol per litre, which was converted with a molar yield of about 60% to vanillin (Chatterjee et al, 1999b).

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The phenylpropanoid pool provides precursors for benzoic volatiles in flavour extracts from both cell cultures (Venkateshwarlu et al., 2000) and fruit bodies (Rosecke and Konig, 2000). Benzaldehyde, mandelates, phenylacetates and their derivatives occur regularly. Multiple pathways of the degradation of phenylpropanoid compounds appear to exist. Although less likely to occur than with terpenes, mere chemical conversion cannot be ruled out: a cell-free extract from Lactobacillus plantarum deaminated phenylalanine to phenylpyruvic acid, which was then chemically oxidised to benzaldehyde and other compounds at pH 8 if certain metal cations were present (Nierop Groot and De Bont, 1998). A Pseudomonasfluorescens strain, able to grow on ferulic acid as the sole carbon source, contained high levels of an inducible feruloyl-CoA ligase and a vanillin dehydrogenase (Narbad and Gasson, 1998). Acetyl-CoA was verified as a cleavage product by I3 C NMR, but the mechanism of the cleavage steps remained open. The mandelate pathway was demonstrated in the related Pseudomonas putida by feeding benzoylformate (Simmonds and Robinson, 1998), and also in the higher fungus Gloeophyllum odoratum by analysing volatile and nonvolatile (trimethylsilylated) constituents of a hot-water extract from the fruit bodies (Rosecke and Konig, 2000). Flavour chemists and natural products chemists often stick to their selective nonpolar and polar extraction solvents respectively, and are thus prone to overlook the respective nonsoluble intermediates of a given pathway, which might impede biogenetic understanding. Originating from the Strecker degradation of phenylalanine, the rose-like compound 2-phenylethanol is a common volatile in yeast fermentation flavours (Fabre etal, 1998). Metabolites of labelled L-phenylanaline in the higher fungus Bjerkandera adusta were the nonvolatile (£>cinnamic, phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids, and volatiles such as benzaldehyde and benzyl alcohol (Lapadatescu et al., 2000). The direct (3-oxidation of cinnamic acid to benzoic acid was concluded from the occurrence of acetophenone. the product of the spontaneous decarboxylation of the supposed intermediate ^-oxophenylpropanoic acid. Pycnoporus cinnabarinus. another white-rot fungus, was the subject of optimisation studies resulting in a high-density culture yielding >1.5g of vanillin per litre (Oddou et al., 1999: Stentelaire et al., 2000). Vanillin formation was favoured by reduced concentration of dissolved oxygen, high carbon dioxide, gentle agitation, low specific growth rate, and the application of a nonselective adsorbent. Partly on the basis of this well explored model, 5-2H-labelled ferulic acid was fed to the same fungus (Krings et al., 2001; Figure 3.3). The major labelled phenolic compounds identified were four lignans, the methyl esters of ferulic and vanillic acid. (£>coniferyl aldehyde and alcohol, vanillic acid, vanillin and vanillyl alcohol. Analogously to the study of Lapadatescu et al. (2000), labelled 4-hydroxy3-methoxyacetophenone occurred, suggesting the decarboxylation of free 4-hydroxy-3-methoxybenzoylacetic acid. Detailed mass spectrometric examinations revealed traces of 4-hydroxy-3-methoxybenzoylacetic acid methyl

71

BIOTECHNOLOGICAL FLAVOUR GENERATION Lignans R = H, CH3 CHO

COOR

CH2OH CH2OH

Polymers

OH

OH

Figure 3.3 p-Oxidation-like degradation of ferulic acid by Pycnoporus cirmabarinus.

ester and 3-hydroxy-(4-hydroxy-3-methoxyphenyl)propanoic acid methyl ester in the culture medium. Hence, the fungal degradation of the phenylpropenoic side chain, a principal key step of lignin decomposition, should proceed like the oxidation of fatty acids. Raspberry ketone (4-(4-hydroxyphenyl)butan-2-one) is one of the character impact components of raspberry flavour. If the compound were isolated from raspberry fruit, natural raspberry ketone would cost 6 million dollars per kg (cost of fruit only), compared to $8-10 per kg for the synthetic product. An enzymatic pathway was proposed involving the (3-glucosidase-catalysed hydrolysis of the naturally occurring betuloside from the European white birch (Betula alba) to release betuligenol, which is transformed by an Acetobacter alcohol dehydrogenase into the ketone. Plant cell culture or biomimetic acyl anion transfer reactions using aldolases were also suggested, but none of the known routes appears to have met industrial processing requirements (Whitehead, 1998).

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The basidiomycete Nidula niveo-tomentosa synthesised traces of the ketone and its corresponding alcohol de novo starting from simple nutrients, such as glucose and amino acids. A systematic attempt was made to improve the productivity of this fungus in submerged culture (Boker et al., 2001). Variation of the composition of the nutrient medium supported by a factorial experimental design yielded a 50-fold increase in metabolite concentrations. 3.4.5 Generation of miscellaneous compounds Short-chain methyl-branched fatty acids are not only important to cheese and other fermentation flavours, but are also added, for example, to strawberry flavours. Their generation from fusel oil constituents by strongly oxidising bacteria is feasible. Gluconobacter species preferentially produced (S)-2methylbutanoic acid from 2-methylbutanols of known enantiomeric composition (Schumacher et al, 1998). Mechanistic aspects were studied in detail (Gatfield et ai, 2000). Esterification of such fatty acids with ethanol using Geotrichum yeast removes the necessity to substitute inactive lipase during reverse-hydrolytic enzyme-based processes (Daigle et al., 1999). Work on the production of l-octen-3-ol, one of the mushroom flavour impact compounds, through the lipoxygenase/hydroperoxide lyase pathway is continuing (Assaf et al., 1997). Enantioselective reduction of 2-octanone by Lactobacillus fertnentum and others afforded (5)-2-octanol with high enantiomeric excess (Molinari etal, 1997). An extended screening of food-grade yeast species showed that some of them accumulated elevated concentrations of a-hydroxy ketones (acyloins). Zygosaccharomyces bisporus cells, for example, accepted a wide variety of aldehyde substrates and linked them to pyruvate to form a-hydroxy ketones (Neuser et al, 2000a). The enantiopurity of the products depended strongly on the substrate structure. Odour qualities and threshold values of 34 acyloins were evaluated using a GC-olfactometry dilution technique, and 23 of them possessed novel and pronounced flavour properties (Neuser et al., 2000b). This catalytic property is known to rely on a pyruvate decarboxylase, and the respective enzyme was isolated from Zygosaccharomyces bisporus and compared with crude preparations from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus. Conversion rates of >50% showed that the potential of this type of enzyme to catalyse the formation of aliphatic acyloins has been underestimated. A 1856 bp cDNA coding for the monomeric unit of the enzyme was amplified and sequenced (Neuser et al., 2000c). Aliphatics with sulfur functions often possess low odour detection threshold values. Of interest for cheese flavour, for example, is methanethiol. Species commonly present in ageing cheese, such as Lactococcus, Lactobacillus, and Brevibacterium degraded L-methionine through methionine aminotransferase or y-lyase activities to methanethiol (Dias and Weimer. 1998). The enzyme

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activities were quantitatively assessed, but no attempts at further purification were made. Particularly active formers of sulfur compounds are strains of Geotrichum candidum, which develop early in cheese ripening (Berger et al., 1999; Demarigny et al, 2000). Methanethiol, as well as di- and trisulfides, and several thio fatty acids with straight and branched chains were identified. From results obtained with L-[(S>rae?/zy/-2H]methionme, two different pathways for the generation of the sulfur compounds were suggested, but isolation and characterisation of the enzymes will have to follow. Whether an odorous molecule is perceived by humans as a positive or negative (off-flavour) sensation depends on the actual concentration, the composition of the matrix, and the presence of other complementing volatiles. Hence, offflavours attract the same industrial attention as pleasant volatile flavours. How the production of volatiles by yeast can be affected by bioprocess conditions has become evident during the development of continuous brewing processes (Van lersel et al., 2000). Immobilised yeast showed slower growth and decreased amino acid metabolism, and, thus, released increased concentrations of oxo acids into the fermentation medium. High levels of undesirable aldehydes may occur due to the activity of pyruvate decarboxylase, a problem present in the dealcoholisation of beer. As these oxo acids are the precursors of the abovementioned acyloins, this problem may be turned to advantage if the generation of acyloins is aimed at. Strains of Penicillium, Trichoderma, Aspergillus, Mucor, Monilia, and one of Streptomycetes were isolated from cork (Caldentey et al, 1998). Growth on cork resulted in the formation of off-flavours, but growth on malt extract medium did not. Many aliphatic alcohols and carbonyls, sesquiterpenes and some halogenated aromatics were identified, but no structural assignments of the off-notes were reported. The phenolic offflavour of fermented soy products is now clearly attributable to 4-ethyl- and 4-vinylguaiacol (Suezawa et al, 1998; Karmakar etal, 2000). Bacteria, mainly strains of Bacillus, Pseudomonas and Staphylococcus, that degrade phenylpropanoid precursors are responsible for this problem.

3.5 Enzyme technology If a single chemical step is to be biocatalysed, it appears to be a waste to maintain all of the thousands of concurrent metabolic steps of a living cell that are not wanted. A single enzyme, whether in free form or included in gel or microcapsules or bound onto solid supports, could perform the desired reaction just as well. Some problems associated with the use of enzymes may include location of the activity needed, isolation and purification, maintenance of activity during preparation and use, and, for kinases, methyl transferases and oxidoreductases, inevitable co-factor requirements. A review on enzymes in flavour biotechnology by Schreier (1997) shows how some of these disadvantages can

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be overcome. Most enzymes currently used in the food industry stem from only about 25 microorganisms. Many possible sources of enzymes, for example marine organisms (Chandrasekaran, 1997), have remained almost unexplored so far. With the rapid progress in basic enzymological and genetic knowledge even NAD+-dependent redox enzymes gain interest, particularly if a synthetic electron sink, such as dichlorophenol indophenol is accepted and high stereoselectivity is observed (Van der Werf et al, 1999). 3.5.1 Liberation of volatiles from bound precursors The application of glycosidases and, less frequently, of lyases, for the liberation of preformed flavours from nonvolatile precursors (Winterhalter and Skouroumounis, 1997) should not be confused with the maceration of recalcitrant plant materials, when enzymes serve merely as processing aids (for example, Sakho et al., 1998). The majority of reports deals with wine flavour (Table 3.1). p-D-Glucopyranosidase, a-L-arabinofuranosidase, (5-apiosidase, aL-rhamnosidase and a cysteine conjugate P-lyase were shown to enhance the level of volatiles in musts, wines and fruit juices. While strains of Aspergillm and yeasts are typical sources of these enzymes, the lyase was from Eubacterium limosum (Tominagaef a/., 1998). Flavour enhancement in common fruits and in vanilla bean (Pu etai, 1998) was often performed using fungal p-glucosidases. Tropical fruits and black tea leaves, however, also contain less abundant glycosides, such as primeverosides, vicianoides and rutinosides, which may not be sufficiently hydrolysed by side activities of the standard glycosidases. The long history of enzyme-modified cheese is discussed in reviews (Kilcawley et al,.

Table 3.1 Hydrolases of oenological relevance Flavour precursor

Enzyme

Reference

Glycoconjugated aroma compounds Mono- and diglycosides of terpenols (linalool, ct-terpineol. citronellol, nerol. geraniol) Monoglycosides of terpenols Apiofurananosylglucosides of geraniol and linalool a-L-Rhamnopyranoside Glycosides of nerol. geraniol. linalool. y-terpinene. 2-phenylethanol. benzyl alcohol S-Cysteine conjugates of volatile thiols (4-mercapto-4-methylpentane-2-one. 4-mercapto-4-methy lpentan-2-ol. 3-mercaptohexan-l-ol)

Endo-. exogenous glycosidases a-L-Arabinofuranosidase. p-D-glucopyranosidase

Winterhalter and Skouroumounis (1997) . (1998)

P-D-Glucosidase P-Apiosidase

Yanai and Sato (1999) Guoetal. (1999)

a-L-Rhamosidase p-Glucosidase

/. (1999) Gueguen et al. (1997)

Cysteine conjugate P-lyase

Tominaga et al. (1998)

BIOTECHNOLOGICAL FLAVOUR GENERATION

75

1998; Klein and Lortal, 1999). The proteolysis of minced fish tissue to produce a 'seafood flavour' (Imm and Lee, 1999) was likewise adopted from traditional food biotechnology. The reverse reaction may also be useful: lyophilised cells of Xanthomonas campestris and of Stenotrophomonas maltophilia synthesised L-menthyl a-D-glucopyranoside anomer-selectively from L-menthol (Nakagawa et al., 2000). An impressive molar yield of >99% in 48 hours was reported. The glucoside obtained is slowly hydrolysed in the oral cavity, thereby acting as a flavour depot. 5.5.2

Biotmnsformations

The interfacial enzymology of lipid hydrolases is useful in flavour generation, and there are, as with all carbonyl reactions, two options for shifting the reaction equilibrium: first, towards the liberation of odorous fatty acids from triacylglycerols and related esters, and, second, towards the synthesis of a variety of volatile esters from different acyl and alkyl precursor moieties in a microaqueous environment. A broad range of reaction conditions including organic media is tolerated and can be varied to achieve the desired selectivities (Saxena et al., 1999). A good example is the lipase from Candida antarctica, which is not only a stable, versatile enzyme with broad substrate specificity but has also been characterised by X-ray crystallography (Anderson et al., 1998). Some of the more than 170 lipases have been analysed down to the 1.5 A level of resolution; thus, the position of the helical lid that buries the active triad and conformational changes exerted on this by bipolar chemicals have been well established (http://www.rcsb.org/pdb/). The acetates of common acyclic monoterpenols are used as flavours and fragrances. These volatiles can be produced either by direct esterification or by transesterification using the Candida antarctica lipase SP435 in microaqueous n-hexane as reaction solvent (Claon and Akoh, 1994a,b). Whole cells of Hansenula saturnus or Pichia species in an interface reactor and n-decane (Oda et a/., 1995; Oda and Ohta, 1997) and dry mycelium of Rhizopus delemar in n-heptane (Molinari et al., 1998a,b) were successfully used for the same purpose. The reverse-hydrolytic process runs on an industrial scale and constitutes one of the fundamentals of modern flavour biotechnology. Lipases of Candida antarctica and ofRhizomucor miehei formed esters in high yields under solvolytic conditions and vacuum, where the substrate itself acted as a solvent (Chatterjee and Bhattacharyya, 1998a,b). The choice of the reaction solvent also appears to affect the degree of enantioselectivity. The synthesis (and hydrolysis) of (—)-citronellyl oleate with >98% of the ee form from racemic citronellol was achieved using a Candida cylindracea lipase in supercritical carbon dioxide, but only in a narrow window of pressure and temperature (Ikushima, 1996). Some contradictory results suggest, however, that a generalisation of these findings should be treated with caution (Michor et al, 1996).

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The synthesis of (3Z)-hexenyl butanoate in n-hexane or in solvent-free medium was likewise achieved using the Mucor or Candida Upases (Bourg-Garros et al., 1997; Kim-Jung-Bae et ai, 1998). Aliphatic alcohols from fusel oil, a side-product of the distillation of spirits, were reacted with dodecanoic acid (De Castro et al., 1999). Yields dropped with decreasing chain length of the aliphatic acyl moiety. Recent work also modified the structure of the alkyl moiety: Phenylethanol was transformed to its acetate, a koji style wine flavour impact, using supercritical carbon dioxide and lipase PS (Wen et al., 1999), and thioethyl, thiobutyl and thiohexyl propanoate, butanoate and valerate were produced using different immobilised lipases (Cavaille-Lefebre and Combes, 1997). One of the rare examples of a successful redox process was the oxidation of vanillylamine to vanillin by an amine oxidase from Aspergillus niger, or by a monoamine oxidase from E. coli (Yoshida et al., 1997). 3.5.3 Kinetic resolution ofracemates The preferred cleavage of one enantiomer of a racemic (usually ester) mixture is one possible route to enantiopure products, an approach patented for the generation of the bulk flavour chemical L-menthol in the early 1970s. Many subsequent papers and patents confirm the competitiveness and industrial usefulness of this mature bioprocess. This classical approach was followed to resolve (/?)- and (S)-karahanaenol produced from ring enlargement of limonene epoxide (Roy, 1999). The racemic monoterpenol acetate was resolved using a Pseudomonas cepacia lipase, and the 4/?-isomer alcohol was preferentially obtained through alcoholysis (Figure 3.4). Kinetic resolution in the course of the esterification of menthol was a novel aspect arising from work with lipase from Candida rugosa originally focusing on the formation of methyl esters as a flavour depot (Shimada et al., 1999). When a menthol racemate was esterified with oleate in an emulsion containing 30% water, 96% esterification and an enantiomeric excess of 88% of the L-enantiomer menthyl ester were found. The operational stability of some lipases is remarkable. In a study undertaken to separate racemates of ibuprofen and 1-phenylethanol, the commercial enzyme remained partly active even after 14 hours at 140=C and 15MPa (Overmeyer et al. 1999). These

Lipase

OAc

\

OH

\

OAc

Figure 3.4 Enantiospecific alcoholysis of karahanaenol acetate by Pseudomonas cepacia lipase: (I) terpinolene oxide; (II) (/?)-karahanaenol: (HI) (S)-karahanaenol acetate (after Roy. 1999).

BIOTECHNOLOGICAL FLAVOUR GENERATION

77

Table 3.2 Kinetic resolution of racemates Flavour compound

Enzyme

Reference

Lactones and epoxides 1 -Phenylethanol

Reductase Immobilized lipase (Mucor miehei) Lipase (Pseudomonas sp.) Lipases Commercial lipase (Novozym®) Lipase (Pseudomonas sp.) Lipase (Rhizomucor miehei)

Danchet et al. (1998) Fringsetal. (1999)

1 -Phenylethanol 2-Phenyl- L-propanol Ibuprofen, 1 -phenylethanol Y-. 8- Lactones 2-Methylbutanoic acid methyl ester

Ceynowa and Koter (1997) Goto et al, (2000) Overmeyer et al. (1999) Enzelberger et al. (1997) Kwonetal. (2000)

examples together with Table 3.2 show the broad range of substrates amenable to hydrolysis with various lipases. The theoretical modelling of the energy differences between the transition states of the diastereomeric enzyme-substrate complexes will aid in further refinement of kinetic resolution processes. There remains the drawback that the resolved substrates are usually derived from chemosynthesis; hence, the produced flavours are nonnaturals. 3.6

Plant catalysts

Most of the natural flavours currently processed by the flavour and fragrance industry are obtained by extraction or distillation of parts of field-grown plants. Their huge biosynthetic potential represents an attractive starting point for plant aroma biotechnology (Fu, 1999). At the same time, the high degree of structural and biochemical organisation of a plant cell impedes the biotechnological approach: specialised plant cells tend to dedifferentiate in vitro, they demand complex nutrient media and low-shear bioreactors, and usually do not accumulate and excrete elevated levels of volatile flavours. The enzymes of a phototrophic metabolism are not easily inducible by carbon substrates, which explains why plant cells in sterile culture are not as responsive to precursor substrates as are most microorganisms. Numerous attempts at the production of flavour and aroma compounds by plant cell, tissue and organ cultures have been described (Scragg, 1997; Berger, 1995b; Dorneburg and Knorr, 1996, Kumar et al., 1998). A collection of promising recent results obtained on the laboratory scale is presented in the following sections. 3.6.1 Plant cell, tissue and organ cultures Plant tissues, excised from differentiated and surface-sterilised materials and placed on phytoeffector-containing agar medium, start to divide again and

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form nondifferentiated cell clusters ('callus'). After transfer to liquid medium, a fine suspension of single cells and smaller cell aggregates develops. This propagation of plant cells is based entirely on mitotic events; the full genetic potential to form flavours is retained in each cultured cell. However, these cells dedifferentiate during subculturing and do not contain flavours, even if isolated from a flavour-bearing source tissue. The few observations of de novo flavour synthesis are exceptions to the rule. Low yields of volatile oil were reported from callus cultures of pot marjoram (Origanum vulgare L.) (Svoboda et al., 1995). Microdistillation of the callus gave only three major essential oil constituents. 3.6.2 Callus and suspension cultures Exceptionally high amounts of odorous mono- and sesquiterpenes (0.34% total oil content in cells plus medium) were recovered from callus cultures of the Brazilian snapdragon (Otacanthus coeruleus), an ornamental pot plant from east Brazil (Ronse et al., 1998). The amount of essential oil extracted from the nutrient media was higher than the intracellular amount. High sucrose treatments (>40g/l) increased the quantities of oil found in the medium due to the high osmotic stress. In cell cultures of two genotypes of rosemary, the concentrations of calcium ions, sucrose and plant growth regulators significantly affected the yields of camphene, 1,8-cineole, linalool, camphor, borneol and bornyl acetate (Tawfikera/., 1998). Photomixotrophic callus cells of grapefruit (Citrus paradisi Macf.), lemon (C. limon (L.) Burm.), and lime (C. aurantiifolia (Christm. et Panz.) Swingle) generated monoterpenes, although no cytodifferentiation was found by electron microscopy (Reil and Berger, 1996a). Chlorophyll content and the formation of oligoisoprenoid volatiles were positively correlated with high light intensities. An optimisation of the growth medium and the light regime resulted in the identification of more than 40 mono- and sesquiterpenes and aliphatic aldehydes in grapefruit callus. The maximum yield was 186 mg of volatiles per kg callus accumulated in four weeks, representing about 5% of the volatiles accumulated in flavedo tissue in about 13 months. A similar correlation existed for white diosma (Coleonema album Thunb.) photomixotrophic cell cultures (Reil and Berger, 1997). An extended photoperiod and certain concentrations of phytoeffectors were the conditions for the formation of volatiles including limonene and phellandrenes. The light conditions also affected the production of compounds by vanilla (Vanilla planifolia) cultures, particularly of 4-hydroxy3-methoxybenzyl alcohol (vanillyl alcohol) (Havkin-Frenkel et a/., 1996). Microbial infections, whether caused by an endogenous infection of the source tissue or by a secondary infection, present a serious experimental problem, because most microorganisms simply overgrow the slower plant cells. Otherwise, microbial infections are often accompanied by the formation of elicitors. components involved in plant chemical communication and defence.

79

BIOTECHNOLOGICAL FLAVOUR GENERATION

As volatile flavours may be part of a plant's response to amicrobial attack, flavour biogenesis can be elicited. A persistent contamination with Pseudomonas mallei was reported for cell cultures of rosemary (Rosmarinus officinalis L.) producing cineole and a-pinene (Shervington et al., 1997). An (unintended) elicitation may explain this observation. Strawberry tissue cultures (Fragaria x ananassa) synthesised 2,5-dimethyl-4-hydroxy-2//-furan-3-one (DMHF) and 2,5-dimethyl-4-methoxy-2//-furan-3-one (mesifuran) after they were treated with Methylobacterium extorquens. It was suggested that endogenous strawberry 1,2-propanediol was oxidised to 2-hydroxypropanal by the Methylobacterium; the lactaldehyde was then condensed with dihydroxyacetone phosphate by the strawberry cells to form the furanone progenitor 6-deoxy-D-fructose I-phosphate (Zabetakis, 1997). A combination of photomixotrophy and elicitation led to the excretion of volatiles by cell cultures of parsley (Petroselinum crispum (Mill.) Nym.). Treatment with autoclaved homogenate of the wooddestroying basidiomycetes Polyporus umbellatus or Tyromyces sambiiceus elicited a spicy odour, imparted by elemicin (5-allyl-l,2,3-trimethoxybenzene), 3-n-butylphthalide, (Z)- and (£>butylidenephthalide, sedanenolide and (Z)ligustilide (Reil and Berger, 1996b) (Figure 3.5). 3.6.3 Organ cultures There is abundant experimental evidence for the correlation of cytodifferentiation and the formation of secondary plant products. Visible differential gene activity and biochemical specialisation occur with morphological differentiation. Nonembryogenic cell lines derived from immature juice vesicles of sweet orange (Citrus sinensis (L.) Osbeck) failed to form the characteristic flavour constituents, but organised embryogenic cells emitted a fruity aroma and produced 420mg of volatiles per kg tissue (Niedz et al,, 1997). Some hairy root cultures, derived from the transformation of aseptic plantlets with Agrobacterium rhizogenes, were shown to be capable of forming volatile flavours. The essential oils of hairy root cultures of anise (Pimpinella anisum L.) differed significantly

3-n-Butylphthalide

(Z)- Butylidenephthalide

Sedanenotide

Figure 3.5 Flavours elicited in cell cultures of parsley.

(Z)-Ligustilide

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FOOD FLAVOUR TECHNOLOGY

from those of the fruits (Santos et al, 1998; Andarwulan and Shetty, 1999). While the major components of the essential oil from the hairy root cultures were the anethole precursor (£)-epoxypseudoisoeugenyl 2-methylbutanoate, zingiberene, p-bisabolene, geijerene and pregeijerene, the terpene spectrum of the fruits was dominated by (£)-anethole (Figure 3.6). The maintenance of morphological stability with no dedifferentiation or greening remains an experimental challenge (Santos et al, 1999). Matsuda et al. (2000) generated mutant hairy roots of musk melon (Cucumis melo L.) by means of t-DNA insertion. Out of more than 6500 clones (!) five fragrant hairy root clones were selected. The volatile compounds were identified as (Z)-3-hexenol. (£)2-hexenal, 1-nonanol and (Z)-6-nonenol, which also determine the flavour of the fruits. Aroma production was stable for more than three years. The yield of aroma compounds was about 6.5-fold higher than in ripe melon fruit. 3.6.4 Plant cell biotransformations B ^transformations of monoterpenoid alcohols, aldehydes, ketones and oxides by plant and microbial cell cultures have been reviewed by Shin (1995), and the conversion of monoterpenes, steroids and indole alkaloids using cell cultures was summarised by Hamada and Furuya (1999). Particular attention was dedicated to the regio- and stereospecifity of the reactions and to immobilisation techniques. Immobilised and free cells of yam (Dioscorea deltoidea) and of kangaroo apple (Solanum aviculare) oxidised (—)-limonene to (Z)- and (E)-carveol and to carvone. The preferred formation of either carvone or (Z)- and (E)-carveol

H3CO

(£>Epoxypseudoisoeugenyl 2-methylbutanoate

Zingiberene

/9-Bisabolene

Pregeijerene

Geijerene

Figure 3.6 Secondary metabolites produced by hairy root cultures of anise.

BIOTECHNOLOGICAL FLAVOUR GENERATION

81

depended on the immobilisation medium (Vanek et al., 1999). The same allylic oxidation of a terpene hydrocarbon was performed by grapefruit suspension cells (Reil and Berger, 1996a): exogenous valencene was converted to nootkatone via the 2-hydroxy derivative. Low, but stable concentrations were reached with stationary phase cells. The synthesis of menthol by peppermint (Mentha piperita L.) cell suspension cultures has been investigated thoroughly (Park et al., 1997). Distinct hydroxylation activity towards terpenes was found. Application of (—)-(4/?)- and (-f)-(45')-isopiperitones, for example, yielded the corresponding 7-hydroxyisopiperitones followed by conversion to the respective glucopyranosides. The common pattern of slow flavour synthesis was observed with various Allium tissue cultures (onion, garlic and chive) (Mellouki et al., 1996). Based on the good knowledge of the biosynthetic pathway in Allium species, the low yields were attributed to the low concentrations of the flavour precursors, the (+)-.£-alk(en)yl-L-cysteine sulfoxides rather than to a lack of C-S lyase activity (Prince et al., 1997). Addition of cysteine, glutathione or methionine increased the yields of methyl- and propenylcysteine sulfoxides. S-Ethyl-L-cysteine sulfoxide, a nonnaturally occurring compound, accumulated after application of ethanethiol to the root cultures, indicating the importance of cell differentiation and a possible use for the production of novel homologous metabolites. Chilli pepper (Capsicum frutescens) suspension cells accumulated vanillin, vanillic acid and ferulic acid after supplementation with isoeugenol (Ramachandra Rao and Ravishankar, 1999). Biotransformation rate was improved by the simultaneous addition of a-cyclodextrin and conversion substrate, by immobilising cells with sodium alginate, and by application of fungal elicitors. Attempts to further optimise the yields were reported recently (Ramachandra Rao and Ravishankar, 2000), but it will be difficult to compete with the very advanced microbial processes described above. Another transformation of commercial interest is the demethylation of methyl /V-methylanthranilate by peroxidases from various plant sources to produce the Concord grape and citrus flavour impact methyl anthranilate (Van Haandel etai, 2000). The enormous potential of unsaturated fatty acids as flavour precursors is well recognised (Gill and Valivety, 1997). So-called green-note compounds, such as (2£)-hexenal, were derived from a reaction catalysed by a highly active hydroperoxide lyase from mung bean (Phaseolus radiatus L.) (Rehbock and Berger, 1998; Rehbock etal, 1998). The comprehensive analysis of all volatile and nonvolatile oxylipins created by this reaction has contributed to the optimisation of yields. The initially formed fatty acid hydroperoxide was converted to the corresponding hydroxyacid by a batch-fed reaction with cysteine to yield almost enantiopure 13(,S)-hydroxy-(9Z, 1 l£)-octadecadienoic acid (Elshof et al., 1998). The resulting fatty acid derivatives may serve as precursors of certain lactones.

82

3.7

FOOD FLAVOUR TECHNOLOGY

Flavours through genetic engineering

Investigation of the pathways of flavour formation in bacteria, fungi and plants is a prerequisite for the re-routing of metabolites. Genetic engineering provides the tools to turn this biochemical knowledge into modified organisms with properties regarded (from an anthropocentric point of view) as improved. These tools comprise simple gene deletions or amplifications, DNA rearrangements in a species, trans-species gene transfer, and bioanalytical screening and monitoring techniques. The food industry of the new millennium is well aware of the tremendous prospects and is prepared to offer tailor-made products as soon as the public concern has been overcome (Pridmore etal., 2000). Another obstacle was presented in recent years by the uncertain legal situation. This has been removed in Europe by the guideline No. 50/2000 of the European Commission, which deals with the labelling of food and food additives containing ingredients and flavours from genetically modified organisms (GMOs). The guideline, in article 3, demands the labelling of GMO-derived flavours, if a measurable content of proteins or DNA is present as the result of genetic engineering. The International Organisation of the Flavour Industry set pragmatic standards some years ago to ensure the safety of the consumer. A breakthrough of novel and cheaper flavours is predicted, once the first successful products are available and accepted in the marketplace (Muheim et al., 1998). 3.7.1 Genetically modified microorganisms Most of the recent work on genetically modified microorganisms has focused on cinnamic acid metabolism. A strain of Pseudomonas fluorescens grew on ferulic acid as the sole carbon source and contained a gene coding for an enoylSCoA hydratase/lyase enzyme for the conversion of feruloyl-SCoA to vanillin (Gasson etal., 1998) (Figure 3.7). Heterologous expression of the gene in E. coli proved its function. The results suggest a degradation route different from the (3-oxidation-like chain shortening reaction mentioned above for Pycnoporus cinnabarinus. A transposon mutant of the same species grew on p-coumaric acid as the sole carbon source and was not only biochemically elucidated but used to produce extraordinary amounts of vanillin in the upper gram per litre range (Civolani et al., 2000). The group of Steinbiichel has put much effort into elaborating the same degradation process and confirmed the involvement of genes of a Pseudomonas sp. by gene disruption and characterisation of the resulting mutants (Overhage et al., 1999a). The inactivation of genes coding for vanillindegrading enzymes by insertion of omega elements was pursued to achieve accumulation of the intermediate vanillin (Overhage et al., 1999b). Based on the evaluation of genetic and enzymatic properties, a similar pathway was identified in the above-cited Amycolatopsis sp. (Achterholt etal., 2000). Patent protection for a series of enzymes and genes was acquired (Steinbiichel et al., 1997).

83

BIOTECHNOLOGICAL FLAVOUR GENERATION O SCoA

Feruloyl-SCoA

OCH,

H,O

Enoyl-CoA-hydratase/lyase

OH

O SCoA

4-Hydroxy-3-methoxyphenyl-phy d roxy propiony l-SCo A

0

SCoA

SCoA

CH3COSCoA Vanillin

Vanilloyl-SCoA

O

QCH (4-Hydroxy-3-methoxybenzoyl) acetyl-SCoA

Figure 3.7 Conversion of ferulic acid to vanillin; microorganisms vs vanilla (modified after Gasson et al., 1998; Achterholt et al, 2000).

Mutants of Yarrowia lipolytica were created by disrupting one or several acyl-CoA oxidase coding genes (Wache et al, 2000). The oxidase isoenzymes were believed to be involved in the biotransformation of methyl ricinoleate to 4-decanolide, as they catalyse the initial acyl-CoA dehydrogenation to (2£)-enoyl-CoA acids. A clear correlation of gene disruption and lactone formation was demonstrated. The same methodical approach was applied to a Saccharomyces cerevisiae strain to accumulate ethyl hexanoate in sake (Asano et al., 2000). A fatty acid activation gene, coding for a synthase acting on exogenous fatty acids, was disrupted. As a consequence of the de-repression of the endogenous pathway, an increased de novo production of fatty acids and of the desired ester compound was found. 3.7.2 Isolated enzymes from genetically modified microorganisms The well-understood enzymology of fermented food is a solid starting point for attempts to improve flavour generation. Smit et al. (2000) dealt with proteolysis and further degradation of amino acids in cheese, and Rijnen et al. (1999) characterised a lactococcal aminotransferase. A heterologous glutamate

84

FOOD FLAVOUR TECHNOLOGY

dehydrogenase was transferred into Lactococcus lactis to evaluate the impact of this enzyme in a cheese model using radiolabelled amino acids (Rijnen et al., 2000). Increased production of a-ketoglutarate and a general improvement of flavour development was found. A wine yeast was supplemented with an Aspergillus nidulans endoxylanase gene to increase fruity flavour notes (Ganga et al., 1999). In the reverse of this process, A. nidulans received an endoglucanase gene from Trichoderma longibrachiatum to release flavours from grape macerates (Villanueva et al, 2000). An endogenous S. cerevisiae alcohol acetyltransferase was overexpressed to increase acetate ester formation in wine and distilled beverages (Lilly et al., 2000). Constitutive expression at high levels was observed by Northern blot hybridisation in yeast transformants. While ethyl octanoate and decanoate levels remained unaffected, a profound increase of ethyl acetate, isopentyl acetate, and 2-phenylethyl acetate was reported. In some cases, substrate transport to the site of enzymatic conversion may be rate limiting in flavour formation. The citrate uptake of Lactococcus lactis, for example, determined the production of acetoin anddiacetyl (Drideretal., 1998). Analysis of the lactococcal transport system on the mRNA level has revealed a considerable degree of complexity. 3.7.3 Plant rDNA techniques New expectations for high-yielding cell cultures have been raised by the penetration of recombinant DNA (rDNA) techniques into the plant sciences (Van Berge, 1998) and by an ever-increasing knowledge of the biochemical backgrounds of flavour formation. The basic understanding of plant enzymes and regulatory mechanisms has benefited from stable-isotope precursor techniques and the concerted modification of enzymes (Mosandl et al., 2000). Leahy and Roderick (1999) and Takeoka (1999) have gathered a wealth of information on fruit and vegetable specific principles of flavour genesis from nonvolatile precursors. Genes coding for these enzymes are well accessible from cell cultures and can either be used to specifically modify food plants or be transferred into a suitable microbial expression system. Since the introduction of the FLAVRSAVR™ tomato in 1994, rDNA technology has developed rapidly, but most efforts were directed towards improved pest or pesticide resistance of field crops. Flavour quality was neglected, although sensory improvement was obviously a primrose path to gain better consumer acceptance of genetically modified food (Havkin-Frenkel et al, 1999). 3.7.3.1 Flavours from genetically engineered food plants ? In a single Agrobacterium-mediated transformation three genes from daffodil (Narcissus pseudonarcissus) and from Erwinia uredovora were introduced to the genome of rice to express the entire provitamin A pathway (Ye et al, 2000). The resulting 'golden rice' forms a,a-carotene from endogenous geranylgeranyl

BIOTECHNOLOGICAL FLAVOUR GENERATION

85

diphosphate. Intended to fight off vitamin A deficiency in Third World countries, the example shows what genetic engineering could do in the flavour area: carotenes are precursors not only of vitamins, but also of flavours, such as the Ci3-norisoprenoid ionones (Winterhalter and Skouroumounis, 1997; Winterhalter, 1996). Changes of the fatty acid profile were monitored after overexpressing a yeast A-9-desaturase gene in tomato fruit (Lycopersicon esculentum Mill.) (Wang et al., 1996). A concomitant increase of the concentrations of (Z)-S-hexenal and (Z)-3-hexen-l-ol, derived from linolenic acid peroxidation and degradation, was found in aroma extracts from the transgenic fruit. Speirs et al. (1998) transformed tomato using constructs containing a tomato alcohol dehydrogenase (ADH) cDNA in a sense orientation relative to the tomato polygalacturonase promoter to allow fruit-ripening specific expression of the cDNA. The transformed fruits displayed enhanced ADH activities which affected the redox balance between some of the aldehydes and the corresponding alcohols associated with tomato flavour. A maceration method simulating human consumption of tomato was used to measure the emanation of volatiles from the recombinant fruit by headspace analysis (Prestage et al., 1999). The levels of lipoxygenase (LOX) mRNA and of lipoxygenase activity of tomato were decreased by an antisense construct (Griffiths et al., 1999). This rDNA contained a fruit-specific promoter, a highly conserved 1.2 kb antisense fragment of the cDNA of tomato lipoxygenase a and no terminator. In contrast to expectations, the transgenic fruit did not show a significantly reduced formation of the lipoxygenase-derived volatile flavours. Another construct that contained a terminator sequence and a 400 bp antisense fragment was even less effective. The authors suggested either that the amount of LOX in the fruit tissue was not rate-limiting in flavour formation, or that some key isozymes of LOX remained unaffected by the antisense approach. The results show that, in this field of flavour research, the command of powerful molecular biological methods has advanced faster than our knowledge of the enzymatic details. The group of Croteau has studied enzymes of the plant terpene biosynthesis for many years. Recent papers report the isolation of oil gland cDNAs from spearmint and peppermint coding for regiospecific limonene hydroxylases (Lupien et al, 1999; Haudenschild et al., 2000). The cDNAs were overexpressed in E. coll and S. cerevisiae to further characterise the enzymes. The cytochrome P450 enzyme of spearmint introduced the hydroxy group in 6-position to yield (—)-trans-carveol, while the enzyme from peppermint led to the 3-allylic hydroxylation product, (—)-?ra/w-isopiperitenol. Both enzymes showed a high degree of sequence homology. The data constitute a valuable model for studying structure-activity relationships in cytochrome P450-catalysed reactions. (S)-linalool synthase from Clarkia brewed (Onagraceae), an annual plant native to California, was the subject of a patent (Pichersky, 1997). Expressing the gene coding for the synthase enhanced the

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accumulation of fragrant volatiles in appropriate host plants (Dudareva and Pichersky, 2000). The detailed paper gives a good account of the subcellular sites of formation of volatile monoterpenes, sesquiterpenes and phenylpropanoids. Ripening of climacteric fruits is controlled by ethene (ethylene) produced from methionine by the key enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (AGO). Cantaloupe charentais melon (Cucumis melo var. cantalupensis. Naud. cv Vedrandais) was transformed with an ACO antisense gene (Bauchot et al., 2000). The expected strong reduction of ethene synthesis led to delayed ripening and suppressed the biosynthesis of flavours. Application of exogenous ethylene restored an aroma profile similar to that of the control fruit (without antisense ACO). 3.7.3.2 Genetically engineered plant enzymes Enzymes involved in the biosynthesis of terpenoids in plants, such as acetoacetylCoA thiolase (EC 2.3.1.9), isopentenyl-diphosphate isomerase (EC 5.3.3.2). mevalonate kinase (EC 2.7.1.36) and 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) are well known (Van der Heijden et al., 1998; McCaskill and Croteau, 1997). Synthases catalysing the cyclisation of the intermediate linear isoprenoid diphosphates into mono- and polycyclic hydrocarbons are of particular interest, because these are in turn the metabolic starting points from which a large variety of oxyfunctionalised, commercial derivatives branch off. Monoterpene synthases, such as (—)-4-(S)-limonene synthase from spearmint, (-f)-bornyl diphosphate synthase from sage (Salvia officinalis L.), and ( —)pinene synthase from grand fir (Abies grandis) were cloned and expressed in E. coli to elucidate mechanistic details of the cyclisation reaction (Schwab et al., 2000). Plants possessing specific cyclase genes can thus be engineered by a back-transfer of these genes into a target plant host (Beale and Phillips, 1999). The formation of volatile aliphatic and aromatic esters in strawberry fruit (Fragaria sp.) depends on specific enzymes, such as aminotransferase, pyruvate decarboxylase, thiolase, alcohol dehydrogenase or acyl transferase (Aharoni et al., 2000a). DNA sequences that code for these activities were isolated from strawberry fruit, characterised and cloned. The comprehensive patent describes further acyltransferases and esterases isolated from apple (Mains domestica Borkh.), mango (Magnifera indica L.) and banana (Musa sp.). Microarray cDNA assays support rapid studies of the expression profile of large subsets of genes in given tissues (Aharoni et al., 2000b; Lemieux et al., 1998). This elegant tool was combined with appropriate statistical analyses to identify a novel strawberry acyltransferase. Another approach to isolate ripening-related genes utilised the differential screening of a high-quality cDNA library (Manning, 1998). Twentysix ripening-related cDNAs were identified from strawberry fruit. Cultured plant cells constitute a convenient, inexhaustible pool of homogeneous nucleic acids. A kinetin-treated cell suspension culture of vanilla (Vanilla planifolia Andr.) served as the mRNA source for constructing a cDNA library

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to isolate clones coding for 4-coumarate-CoA ligase (4CL) (EC 6.2.1.12) and caffeic acid 0-methyltransferase (EC 2.1.1.6), key enzymes of the phenylpropanoid metabolism (Xue and Brodelius, 1998; Brodelius and Xue, 1997). Downregulation of 4CL by an antisense technique was supposed to result in a redirection of phenylpropanoid precursors from lignin biosynthesis to vanillin and related compounds. Studies on tobacco (Nicotiana tabacum L.), alfalfa (Medicago saliva L.) and soybean (Glycine max (L.) Merr.) plants and cell cultures showed the potential of genetic engineering to alter the complex interrelations of phenylpropanoid biosynthesis (Dixon et al., 1999). The activity of hydroperoxide (HPO)-lyase is thought to be rate limiting for the degradation of polyunsaturated fatty acids along the lipoxygenase pathway. The gene coding for this enzyme was cloned from banana and heterologously expressed in yeast cells to generate a continuous source of active lyase (Muheim et al., 1997). Another plant HPO-lyase was purified 300-fold from tomatoes (Suurmeijer et al., 2000). The tomato enzyme cleaved only 13-hydroperoxides from linoleic acid and a-linolenic acid, whereas HPO-lyase from alfalfa (Medico sativa L.) also accepted 9-hydroperoxides as substrates to form the respective volatile Ccj-aldehydes (Noordermeer et al., 1999). The HPO-lyase gene from Arabidopsis was expressed after transfer to E. coli (Bate et al., 1998). Expression was confirmed by analysis of the volatile products. Molecular characterisation of the gene showed a wound-inducible expression in the plant. The short-term aim appears to be to express active lipoxygenase and HPO-lyase in a recombinant microorganism. Mint oil or other traditional plant sources of leaf alcohol and other C6 and C9 flavours would then be replaced by microbial sources. Physical characterisation of alliinase (E.G. 4.4.1.4), the key enzyme in onion flavour formation, and molecular analysis of the respective cDNA showed that two genes were expressed in onion bulb tissue. The sequences of these genes were highly homologous, but there were differences in the two protein subunits they coded for, probably due to a different pattern of glycosylation (Clark et al, 1998). Enzymatic release of flavour from nonvolatile glycosides, another form of flavour precursors, not only enhances the concentration of perceivable flavour in foods, but turns waste materials, such as peelings, skins and stems, into a renewable source of natural flavours. The practical application, however, is limited owing to low activity at neutral pH and strong inhibition by glucose. The construction of chimeric genes and their overexpression in different hosts is the subject of current research (Winterhalter and Skouroumounis, 1997; Winterhalter, 1996 and (references therein). 3.8 Advances in bioprocessing Many laboratory-scale bioprocesses for volatile flavours show productivities high enough for study of the conversion of a substrate and analysis of inducible

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enzymes and volatile products. To operate the same process successfully on an industrial scale requires an appreciable increase of productivity. Mass and energy transfer in the bioreactor need to be improved, inhibitory effects need to eliminated, and product recovery should be integrated. A sufficient level of oxygen, for example, may not be easily achieved by increasing the inlet pressure, because substrate and product may be prone to gas stripping. Like their metabolic products, most flavour precursors are medium polar to lipophilic. which entails problems of low substrate solubility and high volatility and cytotoxicity. Lipophilic substrates dissolve into the membranes of cells or interact with the active conformation of enzymes. Solvent-tolerant recombinant bacteria hold much promise for alleviating these persistent problems of bioprocessing of lipophilic chemicals (Isken and de Bont, 1998). 3.8.1 Process developments in microbial and enzyme systems It is always a good idea to start with a cheaply available precursor substrate. Apple pomace, soybean products, sugar beet pulp, cassava bagasse and all kinds of processing wastes of the food and agricultural industries may contain enough precursor substrate to be used as such (Christen et al., 1999; LesageMeessen et ai, 1999; Pandey et al., 2000a,b). The many different effects of immobilisation may lead to a net gain of productivity, as in the case of benzaldehyde synthesis by basidiomycetes (Lapadatescu et al., 1997). Organic/aqueous biphasic systems are a bioengineering response to the different solubilities and sensitivities of substrate, product and catalyst (De Carvalho et al., 2000). Microemulsions present an intriguing extension of this approach because of the much larger area of mass exchange (Orlich and Schomacker, 1999). Flavour development in blue cheese depends on the action of Penicillium roqueforti metabolism. Fatty acids liberated from triacyl glycerols can undergo decarboxylation as a result of an overflowing ^-oxidation. Decarboxylation of the intermediate free 2-oxoacids leads to methyl ketones. such as 2-heptanone. 2-nonanone and 2-undecanone. A combination of immobilisation and biphasic reaction supported the conversion of short-chain alkyl esters of hexanoic. octanoic and dodecanoic acid to 2-alkanones by spores of Penicillium roqueforti (Park et al., 2000). In the case of ethyl dodecanoate the substrate itself served as a second phase. Free octanoic acid was cytotoxic and therefore remained unchanged. The problem of a lacking metabolic trait can be solved by the consecutive action of two biocatalysts. A significant improvement of yields was reported for the combined action of a Hpase from Candida cylindracea and spores of Penicillium roqueforti in a simple phosphate buffer supplemented with copra oil (Chalier and Crouzet, 1998). A comprehensive patent describes (mainly prokaryotic) microorganisms for the degradation of the propenoic side chain of ferulic acid, and a second set of (mainly eukaryotic ) microorganisms for converting vanillic acid into vanillin (Cheetham etal, 1999). The same two-step

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process was successful using Aspergillus niger for the first step and Pycnoporus cinnabarinus for the second one (Lesage-Meessen et al., 1999). One step further, the principle of co-cultivation of different species depends on an internal regulation of the bioprocess, but has been applied with few problems to cheese and other traditional fermented foods (Martin et al., 1999; Midje et al, 2000). Novel developments in enzyme technology are surface-coated lipases (Huang et al, 1998), lipases imprinted by pre-incubation with an ester and a surfactant before freezing the conformation by lyophilisation (Gonzalez-Navarro and Braco, 1998), or the use of a fluidised bed reactor (Laboret and Perraud, 1999). More than 100 g of terpenyl ester per day were produced in a 2-litre bioreactor. No specific preference for a certain type of bioreactor for flavour biotechnology can be recognised. Many authors rely on classical stirred tanks or variants thereof, and examples of a direct comparison of the same reaction proceeding in two different reactor systems are quite rare (Carvalho et al, 2000). Solid-state fermentation was applied to bacteria (Besson et al, 1997) as well as to many fungi (Pandey et al, 2000b). Not much attention was paid to the development of suitable techniques of in situ recovery of flavours. Standard extraction solvents denature or kill most of the biocatalysts; distillation at ambient pressure or under vacuum will produce the same result. A simple and sometimes efficient means is the direct addition of inert resins to the cell suspension (Lomascolo et al, 1999b). The lipophilic, macroporous polystyrenes offer good mechanical stability and a large adsorptive surface of several hundreds of m2 per gram. Zeolites share with polystyrenes the disadvantage of being nonselective (Treffenfeldt et al., 1999), but are chemically perfectly inert and easily reused. First attempts to create more selective polystyrenes have been undertaken (Gehrke et al, 2000). In view of so many parameters that might eventually affect a bioprocess, more emphasis should be placed on factorially designed experiments and statistical evaluation of results. One of the few examples refers to ester formation by Kluyveromyces marxianus (Medeiros et al, 2000), another one to raspberry compound formation by Nidula niveo-tomentosa (Boker et al., 2001). One frequently encountered problem of enzyme catalysis is the occurrence of multienzyme arrangements. Activated substrates are passed from one active site to the next, and no thermodynamically adverse accumulation of intermediates will be observed. In the field of nonvolatile flavours, some progress in constructing enzyme cascades was made. In a four-step-one-pot reaction, glycerol was transformed into 5-deoxy-5-ethylD-xylulose (Schoevaart et al, 2000). Phosphorylation/dephosphorylation steps were controlled by changes of pH to which the respective enzymes responded differentially. One could imagine a comparable arrangement for the synthesis of volatile flavours in aldolase-based or other reactions. 3.8.2 Process developments of plant catalysts Strategies towards improved productivity are key to any commercialisation of plant cell cultures (Goldstein, 1999). Progress was achieved by the selection

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of high-yielding cell lines, variation of the chemical and physical environment, supplementation of precursors and elicitors, in situ product removal, and immobilisation techniques. Simultaneous application of several different strategies may result in a synergistic response and a pronounced secondary metabolite formation (Pedersen etal., 1999; Berger, 1995b; Shuler, 1999: Verpoorte et al.. 1999). The phases applied for in situ extraction procedures can be either waterimmiscible liquids, such as n-hexadecane or Miglyol™, or solid adsorbents, such as hydrophobic polystyrene-divinylbenzene resins, or zeolites (Treffenfeldt et al., 1999). General advantages result from the provision of such an external accumulation site: the danger of evaporative loss of product is minimised, and further catabolism of products or accumulation of cytotoxic product levels is prevented (Gehrke et al., 2000). Current proposals to improve bioreactor designs for suspension cultures and for organised cultures were presented by Scragg (1997). Oxygen transfer rates in large-scale root culture reactors were discussed by Tescione et al. (1999). The immobilisation of cells or enzymes will inevitably decrease oxygen (and nutrient) transfer, but this disadvantage appears to be overcompensated by improved longevity of the catalyst, simplified recovery of biocatalyst and product, and sometimes increased productivity (Fu. 1999). Immobilisation of cell cultures of Solarium aviculare and Dioscorea deltoidea using agents such as polyurethane foam, carrageenan, alginate, pectate or polyphenylene oxide did not affect the ally lie oxidation of (—)-limonene, but changed product ratios (Vanek et al., 1999). Tomato flavour enzymes were harnessed as a crude enzyme preparation in a commercial ultrafiltration unit operated as a hollow-fibre reactor to produce hexanal from linoleic acid (Cass et al., 2000). The reactor proved to be Table 3.3 Recent flavour books containing chapters on biotechnology Year

Editors

Title/source

1997

K.A.D. Swift

1997

R.G. Berger

1999

K.A.D. Swift

1999

R. Teranishi, E.L. Wick. I. Hornstein S.R. Risch. C.-T. Ho P. Schieberle, K.-H. Engel

Flavours and Fragrances/The Proceedings of the RCS/SCI International Conference on Flavours and Fragrances held on 30.04 to 02.05.1997. Warwick, UK Biotechnology of A roma Compounds! Advances in Biochemical Engineering Biotechnology. 55: T. Scheper (managing editor) Current Topics in Flavours and Fragrances: Towards a New Millennium of Discover,-. Kluwer Academic. Dordrecht Flavor Chemistry: Thirty Years of Progress/Proceedings of an ACS Symposium held on 23. to 27.08.1998. Boston. Massachusetts Flavor Chemistry: Industrial and Academic Research/ ACS Symposium Series 756 Frontiers of Flavour Science/The Proceedings of the Ninth Weurman Flavour Research Symposium, held on 22. to 25.06. 1999. Freising. Germany

2000 2000

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stable over an operation period of 5 days, indicating that flavour production with immobilised membrane associated enzymes in a hollow-fibre reactor is a promising technique. It allowed retention of the enzyme system and substrate with concomitant formation and removal of the product, as monitored by solidphase microextraction. Water-soluble crude protein preparations from green pea, soybean or buckwheat were immobilised in calcium alginate gels (Nagaoka and Kayahara, 2000). This simple catalyst produced optically pure l-(4-substituted phenyl)ethanol by selective oxidation of one enantiomer of a racemic mixture. After three consecutive reactions, no decrease of yield or optical purity was observed. 3.9

Conclusion

The global flavour and fragrance market was estimated to exceed $10 billion in 2000, and is expected to grow by 4-5% per year to exceed $12 billion in 2005, with a share of > 10% for biotechnology-derived compounds (Shamel and Udis-Kessler, 2000). In the relatively mature flavour industry, biotechnology is an emerging alternative to generate natural compounds (Sime, 2000), and all of the leading companies in the flavour market are now operating industrialscale production processes. About 80-100 flavours from biotechnology were estimated to be on the market, either as constituents of composed flavours or as pure compounds. The continuing scientific progress is also documented by numerous chapters in recent flavour books authored by scientists from both academia and industry (Table 3.3). Only a few cost considerations have been published (Krings and Berger, 1998). As a rule of thumb, a concentration of one gram of flavour per litre is necessary to render a process economically interesting. Evidently, more progress in the field is required before biocatalytical systems will seriously compete on a broad industrial scale with the conventional sources.

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Pandey, A.. Soccol, C.R., Nigam, P., Soccol, V.T., Vandenberghe, L.P.S. and Mohan. R. (2000a) Biotechnological potential of agro-industrial residues. II: cassava bagasse. Biores. Technol..1481-81. Pandey, A.. Soccol, C.R. and Mitchell, D. (2000b) New developments in solid state fermentation: I. Bioprocesses and products. Process Biochem., 35 1153-1169. Park, O.J.. Holland, H.L., Khan. J.A. and Vulfson. E.N. (2000) Production of flavour ketones in aqueousorganic two-phase systems by using free and microencapsulated fungal spores as biocatalysts. Enzyme Micmbiol. Technol., 26 235-242. Park. S.-H.. Kim. K.-S., Suzuki, Y. and Kim. S.-U. (1997) Metabolism of isopiperitones in cell suspension culture of Mentha piperita. Phytochemistry, 44 623-626. Pedersen, H., Chin, C.-K. and Dutta, A. (1999) Yield improvement in plant cell cultures by in situ extraction, in Plant Cell and Tissue Culture for the Production of Food Ingredients (eds T.-J. Fu et al), Kluwer Academic/Plenum, New York, pp 129-138. Pichersky. E. (1997) Use of linalool synthase in genetic engineering of scent production. World Patent 9742333. Prema. B.R. and Bhattacharyya. P.K. (1962) Microbiological transformation of terpenes II. Transformations of a-pinene. Appl. Micmbiol., 10 524—528. Prestage, S.. Linforth. T. Rob. S., Taylor. A.J.. Lee. E., Speirs. J. and Schuch. W. (1999) Volatile production in tomato fruit with modified alcohol dehydrogenase activity. J. Sci. Food Agric.. 79 131-136. Pretorius, I.S. (2000) Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast, 16 675-729. Pridmore, R.D.. Crouzillat. D.. Walker. C. Foley. S.. Zink, R., Zwahlen. M.C.. Brussow. H.. Petiard. V. and Mollet. B. (2000) Genomics. molecular genetics and the food industry. J. Biotechnoi. 78 251-258. Prince. C.L.. Shuler, M.L. and Yamada, Y. (1997) Altering flavor profiles in onion (Allium cepa L.) root cultures through directed biosynthesis. Biotechnoi Prog., 13 506-510. Pu. F., Jiang. M.. Zhang, Z.J.. Zhang, J.S. and Hao. F.H. (1998) Study on Vanilla curing enzyme treatment method. Acta Botanica Yunnanica. 20 355-361. Rabenhorst, J. (2000) Biotechnological production of natural aroma chemicals by fermentation processes. in Biotechnology. Vol. 8b: Biotransfonnations II (ed. D.R. Kelly) Wiley-VCH. Weinheim. pp 333-350. Ramachandra Rao. S. and Ravishankar. G.A. (1999) Biotransformation of isoeugenol to vanilla flavour metabolites and capsaicin in suspended and immobilized cell cultures of Capsicum frutescens: study of the influence of p-cyclodextrin and fungal elicitor. Process Biochem.. 35 341-348. Ramachandra Rao. S. and Ravishankar. G.A. (2000) Vanilla flavour: production by conventional and biotechnological routes. J. Sci. Food Agric.. 80 289-304. Randez-Gil. F. Pascual. S. and Prieto. J.A. (1999) Engineering baker's yeast: room for improvement. Trends Biotechnoi.. 17 237-244. Rehbock. B. and Berger. R.G. (1998) Covalent immobilization of a hydroperoxide lyase from mung beans (Phaseolus radiatus L.). Biotechnoi. Tech.. 12 539-544. Rehbock. B., Gansser. D. and Berger. R.G. (1998) Efficient generation of 2-( £)-hexenal by a hydroperoxide lyase from mung bean seedlings. Food Chem.. 63 161-165. Reil. G. and Berger, R.G. (1996a) Accumulation of chlorophyll and essential oils in photomixotrophtc cell cultures of Citrus sp. Z. Naturforsch., Sic 657-666. Reil. G. and Berger. R.G. (1996b) Elicitation of volatile compounds in photomixotrophic cell culture of Petroselinum crispum. Plant Cell, Tissue Organ Cult. .46 131 -136. Reil. G. and Berger. R.G. (1997) Variation of chlorophyll and essential oils in photomixotrophic cell cultures of Coleonema album (Thunb.). J. Plant Physiol.. 150 160-166. Rijnen. L.. Bonneau. S. and Yvon. M. (1999) Genetic characterization of the mayor lactococcal aromatic aminotransferase and its involvement in conversion of amino acids to aroma compounds. Appl. Environ. Micmbiol.. 65 4873-4880.

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Rijnen. L., Courtin, P., Gripon, J.C. and Yvon, M. (2000) Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds. Appl. Environ. Microbiol., 66 1354-1359. Risch, S.J. and Ho, C.-T. (eds) (2000) Flavor Chemistry, ACS, Washington, DC. Ronse, A.C., De Footer, H., Van De Vyver, A. and De Proft, M.P. (1998) Otacanthus species: in vitro culture, plant propagation, and the production of essential oil, in Biotechnology in Agriculture and Forestry (ed. Y.P.S. Bajaj), Springer, Berlin, pp 305-319. Rosecke, J. and Konig, W.A. (2000) Odorous compounds from the fungus Gleophyllum odoratum. Flavour Fragrance J., 15315-319. Roy, A, (1999) Chemoenzymatic synthesis of homochiral (/?)- and (S)-karahanaenol from (/?)-limonene. J. Agric Food Chem., 47 5209-5210. Sakho, M., Chassagne, D., Jaus, A., Chiarazzo, E. and Crouzet, J. (1998) Enzymatic maceration: effects on volatile components of mango pulp. /. Food Sci., 63 975-978. Santos, P.M., Figueiredo, A.C., Oliveira, M.M., Barosso, J.G., Pedro, L.G., Deans, S.G., Younus, A.K.M. and Scheffer, J.J.C. (1998) Essential oils from hairy root cultures and from fruits and roots of Pimpinella anisum. Phytochernistry, 48 455-^460. Santos, P.M., Figueiredo, A.C., Oliveira, M.M., Barosso, J.G., Pedro, L.G., Deans, S.G., Younus, A.K.M. and Scheffer, J.J.C. (1999) Morphological stability of Pimpinella anisum hairy root cultures and time-course study of their essential oils. Biotechnol. Lett., 21 859-864. Sarhy-Bagnon, V., Lozano, P., Saucedo-Castaneda, G. and Roussos, S. (2000) Production of 6-pentyla-pyrone by Trichoderma harzianum in liquid and solid state cultures. Process Biochem.. 36 103-109. Saxena, R.K., Ghosh, P.K., Gupta, R., Davidson, W.S., Bradoo, S. and Ruchi, R. (1999) Microbial lipases: potential biocatalysts for the future industry. Curr. Sci., 77 101-115. Schoevaart, R., Van Rantwijk, F. and Sheldon, R.A. (2000) A four step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol. J. Org. Chem., 65 6940-6943. Schieberle, P., Engel, K.-H. (eds) (2000) Frontiers of Flavour Science, Deutsche Forschungsanstalt fur Lebensmittelchernie, Garching. Schreier, P. (1997) Enzymes and flavour biotechnology, in Advances in Biochemical Engineering (ed. R.G. Berger), Springer, Berlin, pp 51-72. Schumacher, K., Asche, S., Heil, M., Mosandl, A., Engel-Kristen, K. and Rauhut, D. (1998) Mircobiological oxidation of 2-methylbutanol of differing enantiomeric ratios by Gluconobacter species. Z. Lebensm.-Unters, Forsch. A, 207 74-76. Schwab, W., Williams, D.C. and Croteau, R. (2000) On the mechanism of monoteipene synthases: stereochemical aspects of (—)-4S-limonene synthase, (+)-bornyl diphosphate synthase and (—)-pinene synthase, in Frontiers of Flavour Science (eds P. Schieberle and K.-H. Engel), Deutsche Forschungsanstalt fur Lebensmittelchernie, Garching, pp 445-451. Scragg, A.H. (1997) The production of aromas by plant cell cultures, in Biotechnology of Aroma Compounds (ed. R.G. Berger), Springer, Berlin, pp 239-263. Serrano-Carreon, L., Flores, C. and Galindo, E. (1997) y-Decalactone production by Trichoderma harzinanum in stirred bioreactors. Biotechnol. Progr., 13 205-208. Shaniel, R. and Udis-Kessler, A. (2000) The sweet taste of success. Chem. Ind., 10 27-28. Shervington,A., Darwish, R. andAfifi, F. (1997) Volatile oil produced by microbial infected Rosmarinus officinal is callus and suspension cultures. Alexandria J. Pharm. Sci., 11 81-84. Shimada, Y., Hirota, Y., Baba, T, Kato, S., Sugihara, A., Moriyama, S., Tominaga, Y. and Terai, T. (1999) Enzymatic synthesis of L-menthyl ester in organic solvent-free system../. Am. Oil Chem. Soc.. 76 1139-1142. Shimoni, E., Ravid, U. and Shoham, Y. (2000) Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin. J. Biotechnol., 78 1-9. Shin, S.-W. (1995) Biotransformation of exogenous monoterpenoids by plant cell culture. Saengyak Hakhoechi (Korean J. Pharmacol), 26 227-238.

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Shuler, M.L. (1999) Overview of yield improvement strategies for secondary metabolite production in plant cell culture, in Plant Cell and Tissue Culture for the Production of Food Ingredients (eds T.-J. Fu et al.) Kluwer Academic/Plenum. New York, pp 75-83. Sime. J. (2000) Biotransformation versus extraction technology. Int. Food Ingredients. ( 1 ) 31-32. Simmonds, J. and Robinson, G.K. (1998) Formation of benzaldehyde by Pseudomonas putida ATCC 12633. Appl. Microbiol. BiotechnoL, 50 353-358. Smit. G.. Verheul, A., Van Kranenburg, R.. Ayad, E.. Siezen. R. and Engels, W. (2000) Cheese flavour development by enzymatic conversions of peptides and amino acids. Food Res. Int.. 33 153-160. Spagna, G., Romagnoli. D., Angelo. M. and Bianchi. G.P.P.G. (1998) A simple method for purifying glycosidases: a-L-arabinofuranosidase and fi-D-glucopyranosidase from Aspergillus niger to increase the aroma of wine. Part I. Enzyme Microbiol. Techno!., 22 298-304 Speelmans, G., Bijlsma, A. and Eggink, G. (1998) Limonene bioconversion to high concentrations of a single and stable product, perillic acid, by a solvent resistant Pseudomonas putida strain. Appl. Microbiol. BiotechnoL. 50 538-544. Speirs. J.. Lee. E.. Holt. K., Yong-Duk. K.. Scott. N.S.. Loveys. B. and Schuch. W. (1998) Genetic manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the balance of some flavor aldehydes and alcohols. Plant Physiol.. 117 1047-1058. Steinbiichel. A.. Priefert. H. and Rabenhorst. J. (1997) Syntheseenzyme fur die Herstellung von Coniferylalkohol, Coniferylaldehyd. Ferulasaure. Vanillin und Vanillinsaure und deren Verwendung. European Patent 0 845532. Steinhart, H.. Stephan. A. and Bucking. M. (2000) Advances in flavor research. J. High Kesolut. Chmmatogr.. 23 489-496. Stentelaire. C.. Lesage-Meessen. L.. Oddou. J.. Bernard. O.. Bastin. G.. Ceccaldi. B.C. and Asther. M. (2000) Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus. J. Biosci. Bioeng.. 89 223-230. Suezawa, Y. Yoshioka. N. and Mori, H. (1998) Bioconversions of ferulic acid and /j-coumaric acid to volatile phenols by Aspergillus spp. and bacteria found in soy sauce koji and mashes. Nippon Nogei Kagaku Kaishi. 72 43-49. Sugawara, E. and Sakurai.Y (1999) Effect of media constituents on the formation by halophilic yeast of the 2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3(2//)-furanone aroma component specific to miso. Biosci. BiotechnoL Biochem.. 63 749-752. Suurmeijer, C.N.S.P.. Perez-Gilabert. M.. Van Unen. D.-J., Van der Hijden. H.T.W.M.. Veldink. G.A. and Vliegenthart. J.F.G. (2000) Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase. Photochemistry, 53 177-185. Svoboda. K.P.. Finch. R.P.. Cariou, E. and Deans. S.G. (1995) Production of volatile oils in tissue culture oi Origanum vulgare and Tanacenim vulgare. Acta Hortic.. 390 147-151. Swift. K.A.D. (ed.) (1999). Current Topics in Flavours and Fragrances. Kluwer Academic. Dordecht. Takeoka. G. (1999) Flavor chemistry of vegetables, in Flavor Chemistry (eds R. Teranishi. E.L. Wick and I. Hornstein). Kluwer Academic/Plenum. New York, pp 287-304. Tan. Q. and Day. D.F. (1998) Bioconversion of (/?)-(+Himonene by immobilized Penicillium digitatum (NRRL 1202). Appl. Microbiol. BiotechnoL. 49 96-101. Tan. Q.. Day, D.F. and Cadwallader. K.R. (1998) Bioconversion of -limonene by P. digitatum (NRRL 1202). Pmc. Biochem.. 33 29-37. Tawfik. A.A.. Read, P.E. and Cuppett. S.L. (1998) Rosmarinus officinalis L. (Rosemary): In vitro culture, regeneration of plants, and the level of essential oil and monoterpenoid constituents, in Biotechnology in Agriculture and Forestry (ed. Y.P.S. Bajaj). Springer. Berlin, pp 349-365. Teranishi. R.. Wick. E.L. and Hornsteim. I. (eds) (1999) Flavor Chemistry. Kluwer Academic/Plenum. New York. Tescione, L.. Asplund. P. and Curtis. W.R. (1999) Reactor design for root culture: oxygen mass transfer limitations, in Plant Cell and Tissue Culture for the Production of Food Ingredients (eds T.-J. Fu et al.) Kluwer Academic/Plenum. New York, pp 139-156.

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4

Plant-derived natural sources of flavours Peter S.J. Cheetham

4.1

Introduction

This chapter attempts to provide examples of the biochemical basis for the wide variety of flavours obtained from plant sources. These include some of the more important sources of the flavour materials themselves and their precursors, which can be converted into flavours by subsequent cooking, enzymatic or microbial steps. There are many excellent articles on particular aspects of natural flavours—such as essential oils, citrus or fruit flavours—but this chapter tries to show the relationships between the source materials and processing methods, and the chemical compositions of the resulting flavours. The flavour characteristics of the resulting flavour extracts and the way they dictate the end uses and social and economic values of the flavours are discussed. To illustrate these effects, examples are included from a wide range of sources, from the wide variety of flavour chemicals contained in these sources and from the very varied organoleptic characteristics produced when they are consumed. It is hoped that this chapter will be of interest to those involved in formulating natural flavour chemicals and natural extracts into complete flavours, to scientists interested in understanding the elusive relationship between the structures of flavour chemicals and their organoleptic sensations, and to those involved in developing new methods of making natural flavour chemicals or extracts to replace the traditional sources. The important principles and concepts are presented, along with some representative examples—such as for extraction technology (citrus), commercial background (vanilla) and isomeric effects on flavour character and thresholds of detection (menthols). Some of the key problem areas are described and some promising opportunities for the future are identified. In preparing this chapter, I have to acknowledge many sources of useful and interesting information, all of which are cited in the references. 4.2

Properties of flavour molecules

The chemical nature of a given flavour molecule determines its properties and how it behaves in a wide range of environments. Flavour perception is one example (Section 4.2.1), as is the effect of isomerism on perceived flavour quality (Section 4.2.2). Extraction of compounds from biological sources (Section 4.2.3)

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also utilises the properties of the molecules for the design of suitable extraction processes. Chemical properties determine the safety in use of flavour chemicals (Sections 4.2.4) as well as the ease with which they can be prepared and shipped. thus affecting the price and the economics of the whole industry (Section 4.2.4). 4.2.1 Flavour perception 4.2.1.1 Receptors Tastants are molecules that interact with receptors in the taste buds of the mouth to produce sensations that are broadly classified as sweet, bitter, salt and sour, although now umami flavour (due specifically to monosodium glutamate) is recognised as a fifth taste sensation. Other sensations in the mouth (and to some extent in the nose) are derived from interaction of chemicals with the trigeminal receptors, epitomised by the hot sensation of chili. Aromas are volatile molecules that are perceived by receptors in the nose, so that their effects are dependent on their vapour pressures, temperature, etc. Taste and smell have probably evolved as a way to assess the quality and safety of foods. Unlike sight, hearing and touch, the senses of taste and smell have not yet been properly explained. Aroma and taste receptors have been isolated and analysed. They have common structural features, belonging to the transmembrane G-protein family. Taste receptors interact with well-defined properties of the molecules eliciting the response. For example, sweet taste is due to the arrangement of certain functional groups on the molecule that form the glycophore (Shallenberger, 1996). Malnic el al. (1999), provided experimental evidence to support a combinatorial mechanism for aroma receptors, whereby each type of receptor can detect a range of different molecules, probably to differing extents. Similarly, any one aroma molecule can bind to more than one type of receptor, probably with different affinities. This means that these signals probably undergo much processing on the way to the brain. This could explain the ways in which smells and tastes can be learned and offers explanations of how molecules as dissimilar as hydrogen cyanide and benzaldehyde can both be perceived as having an almond-like character, whereas isopropylbenzaldehyde smells of cumin, quite unlike almonds. 4.2.1.2 Thresholds To be active as a tastant or as an aroma, the concentration of the molecule has to be above its taste or aroma threshold, which varies greatly from molecule to molecule (Table 4.1). The only exception is when mixtures of high-threshold materials are present; but in such cases the overall intensity is less than expected from the intensities of the individual materials. Odour thresholds can vary widely depending on comparatively minor changes in chemical structure. Thus, whereas vanillin has a good vanilla-like flavour, isovanillin is almost tasteless. Similarly, 2-methyl-3-ethylypyrazine and 3-butyl-3-methoxypyrazine have

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thresholds of 130 and 0.001 ug/1 respectively, a 130000 fold difference. Likewise the intensity of a flavour is roughly related to its concentration in the food or beverage divided by its threshold for perception. Up to 10 000 different flavour molecules have been found in foods and beverages, of which about 10% contain sulfur. A particular food or beverage will usually contain a very large number of flavour and aroma chemicals. This may approach 1000 compounds, especially for materials like coffee, chocolate or bread in which a range of molecules have been generated by fermentation, and then that number has been amplified by their degradation and recombination during thermal processing. Because the taste and aroma thresholds for some molecules are so low, even very minor chemical constituents of foods and beverages can make significant contributions to the flavour quality and character. For instance, total flavour molecules are commonly present at about 20g/t of food, i.e. at 20mg/kg. Assuming that there are a minimum of 20 individual flavour chemicals present, then any one will be present, on average, at a concentration of 1 mg/kg. If it is also assumed that any individual flavour molecule needs to be present at about ten times its threshold of perception to have a significant flavouring effect, then the flavour molecule has to have a threshold of perception of less than 100 ug/1. Flavours also vary in character and intensity depending on which isomer of the flavour chemical is used. Common examples are D- and L-carvones, which taste

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and smell of caraway and spearmint respectively. Again, this effect suggests that interaction with receptors with three-dimensional binding sites must be involved (see Sell, 2001, for the latest views on receptor theories). In several cases, the quality and character of a flavour changes with its concentration, owing to different molecules having different affinities for the flavour receptors. This contrasts with many other biological effects, in which activity varies linearly depending on concentration. However, it must be always be remembered that actual perception of flavour quality, character and intensity is very subjective and very dependent on prior consumer experience, so that large regional, cultural and age differences exist, even for some quite basic flavour sensations. Flavours with quite different taste sensations can (surprisingly) complement each other—for example gammon and pineapple, strawberries and cream, and so on. Also, molecules with quite different chemical structures can have very similar taste sensations, such as the sesquiterpene (/?)-nootkatone and the thioterpenoid l-/?-menthene-8-thiol, which both taste of grapefruit. 4.2.1.3 Range of flavour compounds Some 2000-3000 different flavour chemicals are in use commercially. Typically complete, well-rounded commercial flavours, made to flavour foods and beverages, contain at least 20-50 different flavour chemicals. Many more will often be present if plant extracts are used, as when essential oils are included in the formulation. Many flavour chemicals, including most fruit flavours and many vegetable flavours, are manufactured as pure single chemicals, indeed often as single isomers. Others, especially savoury flavour chemicals, such as reaction flavours, are made as mixtures, often referred to as flavour 'blocks'. Flavours can also vary a great deal in price, from cheap yeast extracts to scarce and expensive specialities such as truffles. It is the flavourist's role to formulate the various flavour chemicals, extracts, blocks, etc. to produce compounded flavours that reproduce the authentic flavour of the traditional products (see Chapter 1). Many flavour chemicals rely on one or more functional groups for their activity. However, the presence of a functional group is not essential, as hydrocarbons such as limonene have flavour. In many cases, particular chemicals are essential flavour constituents and, without them, a good flavour of the particular fruit or vegetable cannot be achieved. In some foods, the flavour quality is predominantly given by a single flavour chemical, the so called 'character impact' compounds, such as benzaldehyde for cherry flavours and vanillin for vanilla flavours. When used alone, these give the characteristic flavour of the product, although invariably the complete flavour as extracted from the source material—as cherry fruit or vanilla beans in these cases—is chemically and organoleptically more complex. Other flavour chemicals are used in combination with the character impact compounds to create an overall impression. Flavour chemicals such as the Cfi alcohols and aldehydes are fairly ubiquitous, contributing a 'green* fresh

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

1 09

character to many fruit and vegetable flavours. In many situations, particular flavour chemicals can be used effectively to create flavours in which they do not naturally occur. A good example is hydroxydimethylfuranone, which is widely used in both fruit and savoury flavours but is only found in a restricted number of sources such as strawberries and pineapples. These differences also hold for flavour and aroma extracts such as essential oils. Mint oil is chiefly used as a source of L-menthol, which has a prized combination of mint taste and cooling sensation, but other plant extracts are used only to supplement flavours, as in the use of buchu oil to supplement blackcurrant. This is analogous to the traditional use of herbs and spices to enhance and/or modify the taste of another food or beverage. It is also illustrative of the general approaches to making and using natural flavouring materials (Figure 4.1). 4.2.1.4 Consumption Indices The proportion of any flavour consumed as present in a natural source divided by that present from deliberate addition to the food or beverage is termed the consumption ratio. Thus vanillin, which is mostly consumed as chemically synthesised material, rather than as present in vanilla bean extracts, has a consumption ratio of 0.02; whereas methyl 2-pyrrolyl ketone, which is almost exclusively consumed as a flavour molecule in roast beef, has a consumption ratio of 2807 and so is called a food predominant flavour chemical. The various isomers of methoxy isopropylpyrazine are consumed fairly evenly in the forms of tomatoes and added flavour, and so has a consumption ratio of 3.5 (Stofberg, 1983). Raw material

Citrus peel (containing flavanone glycosides)

I Flavour precursor

Rhamnose

I

i

Flavour chemical

2,5-Dimethyl-4-hydroxy-3(H)-furanone

I Formulated flavour

I Flavoured consumer product

Strawberry flavour

I Strawberry-flavoured ice cream

Figure 4.1 General approach and specific example for production of flavoured products.

110

FOOD FLAVOUR TECHNOLOGY

4.2.1.5 Flavour quality Flavour quality is comparatively easy to assess, although the detection and identification of individual flavour molecules present at very low concentrations can be challenging. In most cases, the concentration of any single flavour chemical in the end food or beverage product rarely exceeds 10-50ppm (that is 0.01-0.05 g/1). The flavour may be supplied diluted in a food-acceptable solvent such as propylene glycol or ethanol; with a bulking agent, such as maltodextrins; or even microencapsulated, such as in cyclodextrins. Flavour molecules can also have some other functional effects, contributing desirable colour or antimicrobial, or antioxidant activities such as possessed by rosemary extracts, as well as some uses as fragrances. The perceived quality and intensity of a flavour depends on the environment in which it is presented, for instance its temperature or pH. Table 4.2 illustrates the way in which the aroma and taste thresholds for a particular molecule vary depending on whether it is presented to the nose or mouth dissolved in water, or in vegetable or mineral oil, or in air. These data show how the perception thresholds of various Cf, flavour molecules differ depending on the matrix in which they are presented. The effect of protein solutions on flavour release is illustrated by Figure 4.2, which shows that as the protein concentration increases, the headspace concentrations of both allyl isothiocyanate and diacetyl decrease; and that different proteins have different effects, presumably because they bind the flavour molecules to different extents. A particularly big influence comes from the fat content of the food, because of the ability of flavours to partition between the water and fat phases to different extents depending on their lipophilicity/hydrophilicity. requiring rebalancing of the flavour formulations to give the taste required by consumers. Table 4.3 shows this effect for vanilla icecream flavour, with the flavour reformulation factor (FRFF) being that required to produce the same initial flavour intensity in 0% fat ice-cream as in \57c fat ice-cream. 4.2.1.6 Changes on storage Changes in the chemical composition (and thus in taste and smell quality) frequently occur on storage. Sometimes these changes are desirable, as in the Table 4.2 Comparison of the perception threshold values of C& flavour aldehydes in different media Aldehyde

Air (mg/m 3 )

Water (ppm)

Vegetable oil (ppm)

Mineral oil (ppm)

Water (ppm)

Milk (ppm)

Hexanal fra/7j-2-Hexenal fra/i5-2-frani-4-Hexadienal fraHs-3-Hexenal c/5-3-Hexenal frart.v-4-Hexenal

0.053 0.220 0.0018 0.0022 0.0026

0.0045 0.017 0.01

0.12 -

0.32 10.0 0.27 1.2 0.11

0.076 0.45 -

0.05 0.067 -

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

CD O 03 Q.

20

£ 40 o

CD Q

60

80

0.5

1

2

3

4

5

Percentage added protein Figure 4.2 The influence of native proteins on the headspace concentrations of allyl isothiocyanate (solid curves) and diacetyl (dashed lines) in water. A, egg white; •, bovine serum albumin; • casein. From Land (1994), with permission. Table 4.3 Comparison of high- and low-fat ice-cream vanilla formulations Ingredient Vanillin Phenol p-Cresol 4-Ethylguaiacol Eugenol Ethyl benzoate Methyl cinnamate Anethole

Reformulation factor

logP

0.75 0.50 0.30 0.13 0.08 0.03 0.02 0.01

0.40 0.89 1.28 1.74 1.94 2.64 2.79 3.33

maturation of fine wines and spirits, but they can often be negative (Sinki et a/., 1997). Changes can be due to interactions between components, such as esterification, aldol condensation, acetyl formation, oxidation or reduction. For instance, a frequent occurrence is the lipoxygenase-mediated breakdown of unsaturated fatty acid into aldehyde and alcohol fragments with undesirable flavours. Changes can also be due to the processing conditions or to irradiation, microbial contamination or air oxidation. 4.2.1.7 Historical development of food flavourings Originally, the only flavourings available were essential oils and other natural extracts, which were quite variable depending on their source and processing

112

FOOD FLAVOUR TECHNOLOGY

variations. Supply of these commodities was obviously limited in terms of both availability and quality, especially as the size of markets increased in the nineteenth century with the growth of modern consumerism. Then, as a result of advances in chemical analysis and synthetic organic chemistry, came an increasing number of nature-identical and synthetic flavour chemicals, which allowed improved fidelity to the original materials and greater flavour intensity, stability and reproducibility. This is reflected in a comparison of modern and traditional flavour formulations for cherry (Tyrell, 1995) (Table 4.4). Around 1985, a range of natural flavour chemicals produced by enzymic, microbial or mild chemical processes became available, so that good-quality flavour formulations can be made whose compositions closely resemble the analysis of natural extracts of the fruit. A big advantage of this approach is that a range of new raw materials can be utilised that may be cheaper and more available than the traditional raw materials used to manufacture flavours, in the same way that corn starch has become a cheap and large-scale raw material for glucose and fructose sweeteners. The main challenge in this approach remains the development of high-yielding and cost-effective processes. This is because, despite the variety of microbially produced flavours that occur in foods and beverages, these generally only form quite low concentrations of product, otherwise the product would be inedible. However, these flavour-producing strains are only rarely productive enough for use in the manufacturing of concentrated flavours.

Image Not Available

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

13

4.2.2 Differences in sensory character and intensity between isomers Marked differences in aroma-flavour characteristics and intensity (as assessed by threshold values) are common for isomers (Koppenhoefer et al., 1994). For instance, L- and D-carvones have spearmint and caraway flavours respectively, and out of the eight menthol isomers (due to its having three chiral esters) and analogues, such as menthones and menthyl acetates, only one has the prized combination of good mint flavour plus cooling sensations (Table 4.5). Clark (1998) has reviewed menthol flavour, while Benn (1998) has given details of the sniff-GC and aroma extract dilution analysis of mint oils. That isomers really do have quite different flavour characteristics was irrefutably demonstrated by interconverting the (/?)-(+) and (S)-(—) isomers of limonene and showing that the characteristic flavours of each isomer were the same, irrespective of whether they had been isolated or synthesised de novo. Boelens et al. (1993) thoroughly reviewed the information and concluded that three main categories of enantiomeric difference can be identified: • • •

when sensory properties of the two enantiomers differ slightly in intensity or in quality (e.g. terpenoid hydrocarbons); when the enantiomers have the same main character but differ in secondary notes and intensity (such as for aliphatic and monoterpene alcohols); when the odours of the enantiomers differ in both quality and intensity (e.g. carvones, nootkatones).

Image Not Available

5

114

FOOD FLAVOUR TECHNOLOGY

The effects of enantiomer type on odour threshold and odour character are shown in Table 4.1. These differences are quite common. For instance the enantiomers of carvone show the well known caraway and spearmint character while the enantiomers of damascone exhibit both fruity and woody notes with one enantiomer more fruity than woody while the other shows the reverse (Table 4.1) 4.2.3 Extraction of flavours from plant material Figures 4.3 and 4.4 show the general approaches adopted. For plant sources, the tissue must usually be disrupted by mechanical, thermal or enzymic methods to allow the extraction of flavour and aroma materials in good yields. In some cases the required molecules are evenly distributed throughout the plant material, while in others they are present in specialised structures, such as the oil sacs that hold mint oils, present on the underside of the mint leaves. Plant cell walls are L\ ihree layers, the middle lamella and the primary and secondary cell walls. The middle lamella binds between cells and is mostly composed of pectin. The primary cell wall consists of cellulose fibres, together with pectins, hemicelluloses and proteins. The secondary cell wall contains lignin and pectin. It is these structures that must be disrupted to release flavour and aroma chemicals contained with the cells. Typical overall compositions of the cell walls are shown in Table 4.6. Enzymes commonly used in the manufacture of plant extracts include polygalacturonidase, pectin and pectate lyases and esterases, cellulases and hemicellulases. There are four main ways of producing flavour materials. 1.

2.

By direct extraction from the natural source, very often by steam distillation. Other methods are preferred for some materials; for instance, cold expression is the method of choice for obtaining citrus oils. Depending on the method used, various terms are used for the extract, including essential oil, absolute, extract, resinoid and oleoresins. Examples include coffee, cocoa and hop extracts whereas benzoin and chical gum are resinoids; oleoresins include very many spices, such as pepper, capsicum, ginger or paprika. These spice oleoresins are particularly useful because they contain many of the nonvolatile components not present in the corresponding essential oils. Classification of a flavouring material is not always straightforward; for instance, vanilla extract is. technically speaking, really an oleoresin. Compounded flavours are usually complex mixtures of chemically or naturally synthesised flavour molecules, often also incorporating extracts as produced above, and with the chemically or biochemically synthesised chemicals usually based on naturally occurring flavour chemicals discovered by careful chemical analysis. There is also a category

PLANT RAW MATERIAL

Modern extraction methods, e.g. liquid CO2 extraction, use of enzymes

Traditional extraction methods, e.g.grinding, steam distillation

Extracts

Concentration, addition of other materials/formulation*, e.g. grinding, steam distillation, etc.

Heat treatment, and formation of reaction flavours*, e.g. coffee, tea

Fermentation and/or enzyme treatment* e.g. wine, beer, soy sauce

* These steps can be used in combinations, e.g. fermentation followed by heat treatment Figure 4.3 General approaches to production of plant extract ingredients and products.

PLANT MATERIALS

Exudate

i Solvent extraction

i

Nonpolar extraction, e.g. hexane

I

Enflurage1

4 2

Concrete

i

Pomade

Ethanol extraction

1 Tincture

Ethanol extraction and filtration

4 Absolute Figure 4.4 Traditional methods for the production of various aroma-flavour products (underlined) hy the extraction of plant materials. 'Absorption into fat. -Concretes contain the volatile aroma chemicals, hut also waxes. Concretes are usually used as intermediates.

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

1i 7

Table 4.6 Major components of the cell walls of some important fruit sources of flavour Per cent Fresh weight of cell

Cherries Pineapple Mango Apple Pear

3.

4.

Pectin

Hemicellulose

Cellulose

Glycoprotein

0.51 0.21 1.02 0.54 0.42

0.06 0.35 0.23 0.34 0.22

0.17 0.27 0.59 0.70 0.40

0.32 0.12 0.32 0.15 0.12

of semi-compounded flavours in which flavour chemicals are formulated with fruit juices and/or sugar syrups for beverages or dairy products, or with spices, HVP (hydrolysed vegetable protein), MSG (monosodium glutamate) or salt for savoury foods. Reaction flavours are produced by compounding appropriate precursor molecules, usually various sugars, amino acid sources and sulfurcontaining compounds, and then heating or cooking to accelerate chemical reactions, such as the Maillard reaction, that form the required mixture of flavour materials. Enzyme and/or fermentation reactions can be used to produce flavours. These could be traditional, such as the conversion of wine or other sources of ethanol into vinegar, which is principally acetic acid. Modern processes tend to focus on the formulation of individual flavour chemicals such as decalactones, vanillin, etc., but can also produce flavour blocks such as enzyme-modified cheese flavours. These can be extremely expensive when first introduced into the market, but prices usually soon fall as competing suppliers emerge (Figure 4.5).

Irrespective of the methods used, the prime goal is taste quality, followed by considerations of cost, health, nutrition, naturalness and convenience, such as for microwaved foods; and considerations of novelty, as in flavours for exotic foods. New sources of aroma and flavour compounds are consistently being sought. One highly innovative approach is to search for them in flowers in the canopy of rainforests (McGee and Purzycki, 1999). Quite recently, a new factor affecting the ease of extraction of flavours from plant sources has been appreciated, and its exploitation has allowed very good improvements in yields. Many flavour molecules are present in their plant material of origin as glycosides. For instance, damascenone has been shown to be present as its |3-glucoside in Lycium halimolium Mil. (Solanaceae) (Naf et al., 1990). Similarly, tea flavour chemicals have been shown to be present in fresh tea leaf in glycosidically bound forms (Wang et al., 2000); these include hexenol, benzylalcohol, 2-phenylethanol, methylsalicylate, geraniol, linalool

118

FOOD FLAVOUR TECHNOLOGY 12000

Image Not Available

1986

1988

1989

1991

1993

1994

1995

1996

Figure 4.5 Changes in the market price of natural y-decalactone. From Hausler and Munch (1998).

and four isomers of linalool oxide. These tend to be released by the action of endogenous glycosidases during tea processing, as well as by enzymes breaking up the plant matrix to make the glycosides more accessible and to facilitate subsequent extraction of the liberated flavour molecules. A comprehensive review of the occurrence of glycosidically bound plant Cn norisoprenoids as flavour precursors has been made by Winterhalter and Schreier (1994). In particular, the use of enzymes to release glycosidically bound flavours of passion fruit has been described by Chassagne et al (1995). Changes that take place in a plant material following harvesting also affect extraction methods and efficiency, owing to the biochemical processes initiated by the trauma of harvesting. Either increases or decreases in the desired material(s) can result. Table 4.7 shows these effects for y-decalactone in strawberry and peach. Whereas most fruit flavours occur preformed in the plant tissue, most vegetable flavour molecules are formed by enzymic action once the plant tissue has been disrupted, and in addition many flavours are formed by decomposition of precursors, especially by heating. For instance, heating sugars causes

Table 4.7 Comparative analysis of y-decalactone in the headspace volatiles of living and picked fruits Fruit Ripe New Jersey strawberry Ripe New Jersey cling peach

Living fruit, percent y-decalactone

Picked fruit, percent y-decalactone

9.5 2.5

0.3 39.2

Adapted from Mookherjee and Wilson (1990). with permission.

Picked as percent of livine 3 1570

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

1 19

isomerisation and dehydration reactions to form flavour molecules such as hydroxymethylfurfural, acrolein (propenal) and 4-hydroxy-2,5-dimethylfuran-

CH2OH

V

° CHO

Hydroxymethylfurfural

3-one. Polymerisation of such sugar degradation products forms the brown colours characteristic of caramel, beer, bread and toast. 4.2.4 Commercial, economic and safety aspects The worldwide mercantile sales of flavours are substantial, estimated at around $4.5 billion in 1994 (Hartmann, 1995), which excludes the considerable internal production and captive use of flavours by many companies. Sales were growing at 6-7% per annum, with 90% of sales in the big developed areas and 40% of sales in Europe. Sales of flavours are greatest for use in beverages, followed by flavours for savoury products (35% and 26% of total sales respectively). The major consumer trends include convenience, freshness and ethnic and slimming foods or beverages. The largest producers of pure or semi-pure flavour chemicals are the specialised flavour and fragrance companies such as IFF, Givaudan, Quest, Firmenich, Haarmann & Reimer, Takasago and Dragoco. Their competitive advantages lie in their proprietary know-how in manufacturing and formulating flavour materials to produce more complex, addedvalue flavours. Over the last 20 years there has been a strong business trend towards globalisation, which has resulted in the consolidation of the flavour and fragrance industry, with the acquisition of many companies and the broadening of the range of products each large company can provide. Globalisation and consolidation further down the supply chain has been considerable among food manufacturers, who are the customers for flavours and aroma chemicals. To take just one example: the Pepsico/Frito-Lay group (which includes for instance, Walkers Crisps in the UK) now has a 40% share of the world sales of savoury snack products. Next is Procter and Gamble (including the Pringles brand), which holds just 5% of the world market. This makes Pepsi/Frito-Lay the only truly global company in this market, and relegates all the others to somewhat regional and/or niche market positions. The biggest producers of more complex flavour extracts such as essential oils are usually traditional companies, with the biggest concentration present in the Grasse region of Southern France and many others located in tropical countries

120

FOOD FLAVOUR TECHNOLOGY

such as India and Indonesia. Their competitive advantages are in specialised cultivation, extraction and blending techniques. Safety is an important aspect of flavour manufacture. In one sense flavours offer a comparatively low risk because they are used at such low concentrations in most cases, and, if too high a concentration were to be used, the resulting food or beverage would be inedibly strongly flavoured. Thus the use of flavours is, in practice, self-limiting. One new approach to ensuring the safety of foods is through the control and monitoring of the production processes using Hazard Analysis by Critical Control Point (HACCP) (Ropkins and Beck, 2000). In addition, there are the solvents, bulking agents and carriers that usually do not contribute to flavour but serve to stabilise the flavour or make it easier to use. The legal framework surrounding flavour usage is discussed in Chapter 10 and the article by Somogyi (1996) contains further, relevant information.

4.3 Dairy flavours 4.3.1 Background Dairy flavours are particularly interesting because of the wide range of products in which they are found, such as beverages, yoghurts, butters, spreads, cheeses, etc., and because of the wide range of flavour chemicals involved. Raw milk has a very mild but highly complex flavour due to the presence of hundreds of different flavour chemicals, as well as its characteristic mouth feel due to fat globules suspended as a colloidal suspension. The chemicals that make the biggest contribution to the flavour of raw and gently pasteurised milk are dimethyl sulfide formed from methionine, diacetyl made from citric acid, 2-methylbutanol. 4c/5-heptenal, 3-butenyl isothiocyanate, 2-rra/is-nonenal, ethanol, 2- and 3-methylbutanals, 4-pentenenitrile, 2-hexanone, hexanal, ethyl butyrate, 2,4dithiapentane, heptanal, benzonitrile, l-octen-3-one, l-octen-3-ol, nonanal. /7-cresol, 2-trans, 4-frans-nonadienal and 8-decalactones and dodecalactones. in rough order of contribution to the flavour. Sweetness is conferred by lactose, acidity by organic acids such as citrate; milk salts also have an effect. Lowtemperature pasteurisation has little effect on milk flavour. The use of higher temperatures introduces some sulfur flavours, such as hydrogen sulfide produced by the decomposition of milk proteins, and also some methylketones produced by thermal decarboxylation of p-keto acids and y- and S-lactones (formed from hydroxy-fatty acids originally present as glycerides in the raw milk and released by heating). The effects of thermal processing are very marked in UHT milk, which has a distinct cooked flavour. This is due to disulfide exchange reactions between the milk proteins. Attempts have been made to reverse this reaction using the enzyme disulfide isomerase. Other off-flavours can be due to the photooxidation of methionine to methional, and by the action of microbial contaminants, such as

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

121

the conversion of phenylalanine into phenylacetaldehyde and 2-phenylethanol by Streptomyces lactis var. maltigenes. The most important contributors to flavour are 2-heptanone, 2-nonanone, dimethyl sulfide, diacetyl, 4-cw-heptenal, S-dodecalactone, hydrogen sulfide, 3-methylbutanal, dimethyl disulfide, hexanal, 2,3,4-trithlopentane, 2-rra/tsc-nonenal, 2-undecanone, 8-decalactone and y-decalactone. The most extreme example of heating on milk flavour is in sterilised rnilk and condensed milk products, where temperatures have been high enough for Maillard flavours to be formed, such as by reaction between lactose and amino acids, to produce maltol. 4.3.2 Cream and butter Milk-derived products also offer a wide range of flavours. Cream and butter flavour depends greatly on free fatty acids and S-lactones. For instance, 6-cis6-dodecene-y-lactone is an important flavour component of butter. It is formed from linolenic acid by ^-oxidation followed by hydration, hydrolysis of the coenzyme A ester, and subsequent lactonisation. Sour milk products such as yoghurts depend on metabolites of lactic acid bacteria, such as diacetyl and butanediol, lactic acid and ethanol, that are formed by the metabolism of the citric acid originally present in raw milk. The composition of flavour chemicals in a sweet cream butter is given in Table 4.8.

Image Not Available

122

FOOD FLAVOUR TECHNOLOGY

Dairy products are often used as ingredients in other foods to modify the flavour or texture of a food or beverage. One example that has been studied is the addition of cream to heated raspberries, which resulted in a marked decrease in the flavour impact of the four most intense flavour chemicals present in the raspberries, fi-damascenone, vanillin, sotolone and l-nonen-3-one and hydroxyphenylbutanone (raspberry ketone). 4.3.3 Cheese Although derived from milk, cheese flavour is principally determined by complex and individual processing methods (Eaton, 1994). Although hundreds of different cheeses are sold, it has been estimated that there are only 18 basic cheese types. The most important factor is the flavour production by microorganisms present in and/or on the cheese. A good example is the formation of the 8-lactone flavours, shown below. Milk glycerides

Lipase

5-Keto acids

Microbial reduction

5-Hydroxy acid

Ring closure (spontaneous?)

5-Lactone

Free fatty acids make a great contribution to cheese flavours, especially in Parmesan and Romano cheeses. Whereas long-chain fatty acids make little contribution, short-chain fatty acids (such as valeric acid) are important despite their high flavour thresholds. In particular, unsaturated fatty acids, such as trans2-butenoic and hexenoic acids, give a sharp tangy flavour, and thioesters, such as methyl thiobutyrate and methyl (2-methyl)thiobutyrate give a ripe flavour.

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

1 23

,OH

Methyl thiobutyrate

Methyl (2-methyl)thiobutyrate

Intermediate-sized fatty acids have a soap-like flavour and so, in cheese preparation, only lipases that have a substrate specificity for glycerides containing short-chain fatty acids are used (e.g. pancreatic lipase). This use of enzymes as food additives has been employed to its greatest extent in the manufacture of enzyme-modified cheese flavours. This uses carefully selected esterases, lipases and proteases to develop cheese flavours some 20-30-fold stronger than those of conventional cheeses. A particularly useful processing strategy is to use the enzymes sequentially in a 'cascade' to maximise their effectiveness. In addition, some more specialist approaches have been tested, such as the use of methioninase to form methanethiol from methionine (Lindsay and Rippe, 1986). As well as lipids, carbohydrates are very important for cheese flavours. Initially, milk lactose is metabolised into lactic acid by lactic acid bacteria. Lactic acid is the only product formed by streptococci and some lactobacilli, whereas Leuconostoc strains produce lactic and acetic acids and carbon dioxide in equimolar amounts. This results in a more open structure to the cheese, forming gas holes in some cases, or allowing aeration that can lead to the formation of blue-veined cheese varieties. In addition to these general processes, cheese flavour involves a great variety of microorganisms producing a range of chemically quite different flavour chemicals (Bakker and Law, 1994). For instance Streptomyces diaactylactus forms diacetyl, which is the basis of cottage cheese flavour, and Propionibacterium shermanii converts lactic acid into propionic acid, which is characteristic of Swiss cheeses. Other examples include pyrazines that occur in cheese and are produced by strains such as Pseudomonas perolens and Ps. taetrolens and Corynebacterium glutamicum. Diacetyl (2,3-butanedione) contributes to milk and other dairy flavours and is produced by Lactobacillus lactis. It is formed from citric acid via acetolactate and its formation is stimulated by the addition of citric acid and by acidic pH, required by the citrate transport system. Methanethiol and its esters

124

FOOD FLAVOUR TECHNOLOGY

are also important contributors to some cheese flavours, such as Limburger, and are made by bacteria such as Brevibacterium linens and P. freudenreichii. Methylthioacetate is found in cheeses, such as Gruyere and Emmentaler, that are produced by propionic acid bacterial fermentations. Other important cheese flavour chemicals include butyric acid, propionic acid, various y- and S-lactones and also the methyl ketones produced by fungal Penicillium species, which are the characteristic flavour chemicals of blue cheeses such as Roquefort. The initial cheese fermentation is carried out by lactobacilli such as L. casei. The subsequent ripening of the cheeses is much more idiosyncratic. Additional surface ripening by bacteria or fungi occurs in many cases, adding much of the flavour characteristics of particular cheeses. Quite often a mixed population of strains is present with successive growths of different microbial strains. For instance, with Roquefort, Camembert, Brie and others, the initial growth on the surface is of yeasts such as Kluyveromyces lactis or K. fragilis, S. cerevisiae or Debaryomyces hansenii, which deaminate amino acids and oxidise lactic acid, causing an increase in pH. This increased pH encourages the growth of Penicillium roqueforti that gives the cheese its blue colour, especially when holes are bored into the cheese to allow the penicillium to colonise the interior of the cheese. Penicillium roqueforti produces Upases that liberate free fatty acids that not only contribute to the flavour of the cheese but are also the substrates for the production of methyl ketones and also of S-lactones. This lipase activity ratelimits methyl ketone formation, rather than the conversion of the free fatty acids by ^-oxidation. 2-Heptanone is the predominant methylketone of blue cheese, and 2-nonanone that of soft cheese. In addition, Penicillium roqueforti has a good proteolytic activity, so that extensive hydrolysis of the cheese proteins can occur, producing large amounts of complex mixtures of flavour peptides (and amino acids), which also contribute to flavour and can be further converted into other flavour molecules by decarboxylation, deamination and transamination. A most important factor has been to select proteolytic enzymes that minimise the bitterness of the hydrolysates they make. This involves using enzymes with a substrate specificity such that they tend not to produce peptides containing hydrophobic amino acids at the end of the peptide chains. These cheese proteins also have important influences on cheese texture, which is as important as flavour for consumer appreciation. Similar processes occur with different cheese varieties. Camembert is surfaceripened by P. camemberti, and Brie is ripened by a mixture of P. camemberti and the bacterium Brevibacterium linens. Brevibacterium linens acting alone is responsible for the surface ripening of Limberger and similar cheese varieties. It acts only after salt-tolerant yeasts have raised the pH to about 6. In contrast to Penicillium roqueforti, Brevibacterium linens does not produce Upases but does make proteases, metabolising the resulting amino acids into a range of flavour chemicals such as 3-methylbutanol, 2-phenylethanol, methanethiol and many others. Flavour defects can also occur; for instance, reduction of

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

125

diacetyl to acetoin by diacetyl reductase, which is produced by some cheese starter culture strains, is deleterious because the acetoin is much less highly flavoured. The extent of reduction is determined by the redox potential of the cheese. The variety of cheese flavours can be illustrated by Camembert cheese, which is characterised by a mushroom note given by l-octen-3-ol. Camembert's flowery note is due to 2-phenylethanol and its acetate; the hazelnut note is given by 1,3-dimethoxybenzene and methylcinnamate; and the garlic note by molecules such as 2,4-dithiapentane, 2,4,5-triathiahexane and 3-methylthiol-2, 4-dithiapentane. 4.4

Fermented products

4.4.1 Hydrolysed vegetable proteins Hydrolysed vegetable proteins (HVPs) have been used since before scientific times. A good example is soy sauce, which relies on the activities of naturally occurring 'contaminant' strains of Aspergillus, Lactobacillus and Saccharomyces. HVPs are remarkable as they enable meat-like savoury tastes to be produced from entirely vegetable raw materials that can be used to improve the palatability of quite cheap and abundant cereal foods much more cost effectively than if meat-derived flavours were to be used. This effect is achieved not only by the presence of flavours generated during processing but also by flavour enhancers such as MSG, S'-IMP and S'-GMP. HVPs are produced from wheat gluten, defatted soy, peanut or cotton seed flours and other such materials, especially from wheat gluten, which has a high glutamic acid content and so yields high concentrations of MSG in the final product, Hydrolysis is at low pH and around 90°C for several hours, requiring acid-resistant pressure vessels. Under these conditions proteins are broken down into amino acids, and these can undergo Maillard reactions with sugars, also produced from the vegetable starting materials. Then the hydrolysate is filtered, decolorised and concentrated or spray dried (Swaine, 1993). The resulting HVP has a high salt content, derived from the acid used to carry out the hydrolysis, but unfortunately also contains low concentration of mono- and dichloropropanols. Because these are known carcinogens, they have to be maintained at low levels, for instance below 1 and 50 ppb respectively. HVP bouillon type flavours are complex and diffuse meat flavours that cannot easily be described in terms of a few flavour chemicals and that have a 'warm', salty and spicy flavour character. 5-Methyl-4-thiotetrahydrofuran-3one and 2-memyltetrahydrothiophene-3-thiol are top notes. Other flavour chemicals include 5-emyl-4-methyl-3-hydroxy-2(5//)-furanone, methional, sulfurol and 3-hydroxy-4,5-dimethyl-2(5//)-furanone, which gives a meaty, savoury character.

126

FOOD FLAVOUR TECHNOLOGY ,SH

,OH

5-Methyl-4-thio tetrahydrofuran-3-one

2-Methyltetrahydrothiophene-3-thiol

5-Ethyl-3-hydroxy-4-methyl2(5/-/)-furanone

The role of sulfurol is particularly interesting, as it has a relatively high flavour threshold of 10mg/l, but the sulfurol note often increases in intensity with storage. The related molecule 2-methyltetrahydrofuran-3-thiol may contribute to this, as it has a much lower flavour threshold, and may be formed from sulfurol during cooking. ,SH

~S'

v

Sulfurol

4.4.2

*SH

2-Methyltetrahydrofuran-3-thiol

Chocolate

Some of the flavour compounds characteristic of chocolate and cocoa are formed during the fermentation process (Baigrie, 1994) that the beans undergo after harvesting (Table 4.9). During the fermentation, first sugars are metabolised by

Image Not Available

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

! 27

yeasts to produce ethanol, then lactic acid bacteria convert citric acid from the bean extracts into ethanol, and then acetic acid bacteria metabolise this ethanol into acetic acid (Rombaults, 1952). Some other flavours are produced by the fermentation, such as ethyl-2-methylbutanoate, tetramethylpyrazine and some other pyrazines. The bitter taste notes are provided by theobromine and caffeine carried over from the original beans, together with diketopiperizines formed from the thermal decomposition of proteins during the roasting step. Other amino acids released during the fermentation are the precursors for other flavour such as 3-methylbutanol, phenylacetaldehyde, 2-methyl-3-(methyldithio)furan, 2-ethyl-3,5-dimethyl- and 2,3-diethyl-5-methylpyrazine. 2-Acetyl-l-pyrroline is formed by B. cereus acting in the later stages of the fermentation, and also during the drying of the pulp, and so at least some of the acetyl-1-pyrroline of cocoa appears to be formed microbially, and not just by thermally induced reaction during the later stages of cocoa processing (roasting, etc.). This carryover of flavours from fermentation appears to be a general phenomenon, as tetramethylpyrazine has been shown to be made by B. subtilis during the processing of the Japanese fermented soybean product called natto, which has an odour very characteristic of pyrazines. Following fermentation of the bean extract, it is dried, during which polyphenol oxidase activity continues, giving rise to new flavour molecules. Overall, the cocoa flavour is very dependent on the precise conditions and duration of harvesting, fermentation, drying and roasting stages. On further processing to chocolate, the sugar added is the main addition to the flavour, together with cocoa butter and some added flavouring materials, plus special ingredients such as nuts, coffee paste, etc. (Table 4.10).

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4.4.3 Tea

Tea flavour and aroma are complex and depend greatly on the particular type of tea leaf used and the processing conditions. For instance, fully fermented black tea has a very different character from that of partially fermented Oolong tea. Initially the leaves of Camellia sinensis are allowed to wither, allowing a loss of water, then the leaves are macerated so that phenolics and other phytochemicals especially flavanols, such as epi-gallocatechin gallate, are allowed to react with endogenous enzymes. Then the macerated leaves are allowed to 'ferment' at an elevated temperature so that extensive enzyme reactions take place, followed by 'firing' at higher temperatures, which cuts short the enzyme activity but allows chemical reactions, often involving the products of the enzyme reactions, to proceed at a high rate. Some of the important black tea aroma and flavour chemicals are shown in Table 4.11 (Belitz and Grosch, 1999). Others include diacetyl, methylpropanal and 2- and 3-methylbutanals. Many of these chemicals are produced by degradation of carotenoids and unsaturated fatty acids originally present in the tea leaf by the action of endogenous enzymes. Subsequently further chemicals are produced during the firing of the tea, for instance by Strecker reactions. Chemicals produced by fatty acid degradation, such as cis-1,5-octadien-3-one, m-3-hexenal and 3-methyl-2,4-nonanedione. are especially present in green tea and give it its green fresh flavour character. In addition, teas, and especially black teas, have a very marked astringent character. This is due to the presence of thearubigins, which are complex phenols formed by the dimerisation of flavonols by endogenous polyphenol oxidases to form theaflavins, which give tea its red colour, and then further polymerisation to produce the thearubigins.

OR

OH

Theaflavin (R=H or gallic acid)

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

129

Image Not Available

4.4.4

Coffee

Hundreds of flavour and aroma chemicals are present in roasted coffee. Many result from the thermal decomposition of carbohydrates and phenols, especially chlorogenic acids, that are present at significant concentrations in green beans during roasting. In addition, there are marked differences in flavour character and flavour chemical compositions between different coffees. This is due to the different varieties of coffee plant, different mixtures of beans and, of course, different ways of roasting. Only a minority of the compounds present actually contribute to coffee aroma. Furfurylthiol can be detected at 0.005 ug/1 and smells of roast coffee at 0.01-0.5 ug/1, and 5-methylfurfurylthiol is detected at 0.05 ug/1 and gives a stale coffee aroma when present at concentrations of 1-10 ug/1 (Tressl and Silwar, 1981). It is also reported that 2-furfurylthio, 3-methyl-2-butenethio and 3-thio-3-methylbutylformate give the roasted coffee-sulfur flavour note. The phenol-smoky note is given by guaiacol and 4-vinylguaiacol, and the earthy roast and caramel odours are given by pyrazines and furanones respectively, with volatile acids such as formic and acetic acids also modifying the overall flavour. More recently 3-methyl-2butene-1-thiol and 3-thio-3-methylbutanol and its formate have been found in roasted coffee (Holscher, 1992; Blank et ai, 1992). Table 4.12 shows the concentrations and aroma values of many of the more important aroma and flavour chemicals of arabica and robusta coffees, which show significant differences in composition, thus accounting for the quite different tastes and aromas of different varieties of coffee, even when roasted and brewed in the same ways. In addition to the molecules described in Table 4.12, Semmelroch and Grolsch (1996) also include the following chemicals as contributing to coffee flavour and aroma: acetaldehyde, propanal, methylpropanal, 2- and 3-methylbutanals, 2-methyl-3furanthiol, methanethiol, dimethyl trisulfide and 2-ethenyl-3,5-dimethyl- and

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Table 4.12 Concentrations and odour activity values of potent odorants of brews prepared from arabica and robusta coffees

Odorant 2-Furfurylthiol 2-Ethyl-3,5-dimethylpyrazine 2,3-Diethyl-5-methylpyrazine (E)-p-damascenone Methional 3-Mercapto-3-methylbutyl formate Guaiacol 4-Vinylguaiacol 4-Ethylguaiacol Vanillin 4-Hydroxy-2,5-dimethy 1-3(2 //)-furanone 3-Hydroxy-4,5-dimethyl-2(5//)-furanone 5-Ethyl-4-hydroxy-5-methyl-3(2//)-furanone 2-Ethyl-4-hydroxy-5-methyl-3(2//)-furanone 2,3-Butanedione 2.3-Pentanedione 2-Isobutyl-3-methoxypyrazine Propanal Methylpropanal 2-Methylbutanal 3-Methylbutanal Methanethiol

Concentration (mg/kg)

Odour activity value3

Arabica

Arabica

1.7 0.33 0.095 0.195 5.7 5.5 170 1640 51 220 4510 77 8.7 840 2750 1570 1.0 435 800 650 550 210

Robusta 1.7 0.94 0.31 0.205 2.8 4.3 1230 5380 635 740 2480 31 4.4 670 2400 750 0.17 435 1380 1300 925 600

1.7 2.1 1.1 2.6

Robusta 5

x 10 x 103 x 103 x 105 29 1570 68 82 1 9 450 257 1 42 183 52 200 44 1140 500 1570 1050

x 105 x 103 x 103 x 10? 14 1230 490 270 13 30 250 103 <1 29 160 25 34 44 1970 1000 2640 3000

1.7 5.1 3.4 2.7

Data taken from Semmelroch and Grosch (1996), with permission from the American Chemical Society. The odour activity values were calculated by dividing the concentration by the odour threshold value in water.

a

2-ethenyl-3-ethyl-5-methylpyrazines, which goes some way to explaining the complexity and individual variations of coffee flavours. The other major factor affecting coffee quality is the length of storage, as volatile aroma compounds are lost, especially from ground coffee, with methanethiol and 2,3-pentanedione being used as indicators of coffee freshness because they are rapidly lost on storage, especially the 2,3-pentanedione /., 1990). 4.4.5

Beer

Beer flavour is made up of flavour chemicals contributed by the original ingredients, including added materials such as hops, together with yeast metabolites, and also as a result of changes that take place during maturation. For a review, see Verhagen, (1994). One important variable is the type of cereal used and the degree of roasting. For instance, the more extensive roasting used prior to the brewing of dark beers creates methyldihydroxyfuranone. which gives these

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

13 1

beers their caramel note. Many of the flavour chemicals characteristics of beer are produced by yeast enzymes acting on amino acids present in the malt. For example, leucine can undergo transamination and decarboxylation to produce 3-methylbutanal. NH2 JX. J\ ^ ^^ COOH

*~

O J>s. J\ ^ ^^ COOH

|—**"

1 T C02

j>x. /CHO "^ ^^

Other flavour compounds include 2-phenylethanol produced from phenylalanine, which contributes a floral note, dimethyl sulfide and 3-methylbutylformate, 4-vinylguaiacol produced from ferulic acid, which gives a smoky/woody flavour note, and esters such as ethylhexanoate and ethyl butyrate, as well as a number of organic acids such as acetic, lactic, citric, malic and propionic acids, plus amino acids derived from the malt. The individual yeast strains used to brew some speciality beers produce different flavour compounds. For instance, concentrations of vinylguaiacol of over 3 mg/1 are found in German top-fermented wheat beer, well above its flavour threshold of 1 mg/1, whereas vinylguaiacol is well known as an off-flavour when present in many bottom-fermented beers. One important change that occurs in the maturation of lagers is a reduction in the concentration of diacetyl, which gives an undesirable buttery flavour note. Diacetyl is metabolised very slowly by yeast during the long lautering period, but now the enzyme diacetyl reductase can be added to accelerate this process and reduce maturation costs. 3-Methyl-3-butene-l-thiol has been identified as the off-flavour caused in beer by exposure to UV light, the so-called 'sunstruck' off-note.

3-Methyl-3-butene-1 -thiol

It is produced from the hop component humulone, and its flavour threshold has been estimated at 0.2 to 0.3 ng/L (Holscher, 1992). Similarity, trans-2-nonena\ is a major source of stale flavours of beer. Use of hops is very important in flavouring most beers. Hops are an extract of Humulus lupulis that contain humulones (a-acids) such as humulone, cohumulin and adhumalone, and lupulones (j3-acids) such as lupulone, colupulone and adlupulone, as well as terpene flavour chemicals such as myrcene, humulene and caryophyllene. The concentrations, relative amounts and types of the various bitterness conferring acids are all important contributors to beer flavour quality. During boiling of the wort, the humulones are isomerised into isohumulones which are more soluble and more bitter tasting, and then can be further converted

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FOOD FLAVOUR TECHNOLOGY

Myrcene

Humulene

Caryophyllene

into humulinic acids which are less bitter. Lupulones are converted by boiling into hulupones and luputriones which are less bitter than the lupulones. Since the isohumulones are soluble and much more bitter than the hulupones and luputriones, it is the humulones of the hops that make the greatest contribution to flavour, and so the humulone content of the different hop varieties is important both as regards quality control and as a breeding trait. Hops are a good example of a multifunctional ingredient. This is because, in addition to their flavouring properties, hops act as a beer clarifier by precipitating proteins, they have a preservative effect, and the pectins present helps to stabilise beer foam.

Humulones

cis and trans Isomers of humulinic acids R1 = OH in a-acids and

R 2 = OC

' In (3-acids

In humulone and lupulone

OC'

In cohumulone and colupulone

In adhumulone and adlupalone

OC

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

4.4.6

1 33

Wine

The flavour chemicals responsible for the flavour and aroma of wines are either derived from the original grapes, as metabolites of the yeast fermentation, or developed during the maturation of the wine. Thousands of cultivars of grapevines (Vitis vinifera) have been developed worldwide, and very many variations on the wine-producing process, so that enormous variations in the aroma, taste and chemical compositions of different wines occur. Fermentation has relatively little effect on wine flavour. This is mostly determined by the flavour molecules extracted from the grape, so that the length of time that the skins of the grape remain in contact with the fermenting grape skin is important, whereas less commonly the wine flavour is modified by the practice of leaving the wine in contact with the yeast lees for an extended period after fermentation has ceased. Wine flavour is especially reliant on ethyl esters (ethylacetate, propanoate, pentanoate, hexanoate, octanoate, decanoate) as well as the hexyl, 2-phenylethyl, 3-rnethylbutyl and ethyl acetates, plus a number of type-specific flavour chemicals. Methylanthranilate is the character impact compound of Concord and Lambrusco wines.

COOCH3

Methylanthranilate

Ethyl-3-thio-propanoate is also a characteristic flavour chemical of Concord grapes, having a flavour threshold of 2 x 103 ug/1. It has a flavour of fruit and grape at low concentrations, but gives an animalic note when present at higher concentrations. (Kolor, 1983). Perhaps the most striking examples of these grape variety-specific flavour chemicals are the character impact compounds of Sauvignon varieties. A good example is 4-thio-4-methyl-2-pentanone (cat ketone), a key flavour component of Sauvignon grapes and wine, with a flavour threshold of 3 mg/1. 3-Methoxy-3-isobutylpyrazine contributes to the aroma of Sauvignon wine (and also of bell peppers). Furthermore 3-thiohexanol and its acetate occur both in Sauvignon wine and in passion fruit, explaining why some wine tasters describe Sauvignon wines, especially Sauvignon blanc, as having a passion fruit character.

o Ethyl-3-thio-propanoate

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FOOD FLAVOUR TECHNOLOGY

Many of the flavour compounds present in wine occur as glycoside precursors in the grape juice and are released during the wine-making process. Muscat grapes have been shown to contain concentrations of free linalool, geraniol, nerol and a-terpineol of 100, <5, <5 and <5ug/kg fruit respectively, so that only the linalool contributes to taste, as linalool has a flavour threshold of only 6ug/l. But bound concentrations of these molecules of 390, 330, 170 and 40 ug/kg are present, so that release of the bound fractions by treatment with the appropriate glycosidases increase the concentrations of the other three flavour molecules closer to their thresholds. Changes in flavour during wine maturation can be desirable or adverse. For instance, 'musty' off-flavours can be due to the formation of geosmin or 1 -octen-3-one, often caused by unwanted microbial or enzyme action. Desirable improvements in quality are also common during the maturation of wines. For instance, during the maturation of sherry the amounts of acetals, esters and sotolone increase relative to the concentrations of ethanol and volatile acids. Botrytis is common microbial contaminant that infects grapes while still on the vine. It can produce l-octen-3-ol (mushroom flavour) and sotolone, which has a honey-like flavour that can be perceived as either an off-note or as a desirable flavour note as in the case of Tokai and Sauterne wines. A secondary fermentation that is often prized is the malolactate fermentation carried out by certain Lactobacillns or Leuconostoc strains. Country wines, as well as fine vintage wines, also have their characteristic flavour chemicals, which determine their individual flavours. For instance. Jorgensen et al. (2000) analysed elderflower wine by the GC-sniffing technique. They found that cw-rose oxide, nerol oxide, hotrienol, and nonanal contributed to the special elderflower aroma, whereas linalool, a-terpineol. 4-methyl-3penten-2-one and m-^-ocimene contributed floral notes.

c/s-p-Ocimene

Fruity odours were due to pentanal, heptanal, and p-damascenone, and fresh notes to hexenal, hexanol, and c/s-3-hexenol. 4.5

Cereal products

Background information about cereal flavour was published by Eriksson (1994). The flavour and aroma of bread crust is due to a number of compounds either arising from the yeast fermentation or formed owing to the higher temperatures and lower water environment the crust experiences during baking as compared to the

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

1 35

bread crumb. 4-Hydroxy-2,5-dimethyl-3-furanone and 2- and 3-methylbutanals are responsible for the roasted, malty, caramel impression. Methylfurfuryl disulfide has been reported to give the 'golden brown crust aroma' (Mulders 1976). Other aroma chemicals present include 6-acetyltetrahydropyridine, 2-methyl3-ethylpyrazine, 5-methyl-5//-cyclopentapyrazine, 2-methylfurfuryl disulfide, 2-acetylthiazole and acetylpyrazine, as well as 2-acetyl-l-pyrroline that is especially produced from the yeast metabolites ornithine, proline and 2-ketopropanal in the bread crust. r— N

/ / V

o

^

s o

2-Methylfurfuryl disulfide

2-Acetylthiazole

2-Acetyl-1-pyrroline has a very low aroma threshold, but concentrations decrease rapidly as the bread ages, due to air oxidation. Other common bread flavour molecules present in the crumb include methylpropanal, 2-decanal, 2nonenal, diacetyl, methional and l-octen-3-one; plus 2-phenylethanol if the bread has been fermented for a Jong time. Fatty acid decomposition, followed by the condensation of the aldehyde products to form molecules such as 2-butyl2-octenal and 2-butyl-2-heptenal are responsible for rancid off-notes in cereals. Similarily 2-methylisoborneol and geosmin have been found to be responsible for musty off-odours in cereals, and are thought to be microbially produced. 4.6 Vegetable souces of flavour 4.6.1

Vanilla

Vanilla flavour has always been one of the most popular of flavours. Vanilla beans are the fruit of the epiphytic orchids Vanilla planifolia and to a lesser extent Vanilla tahitiensis. Some 20001 per year of vanillin beans are produced, with Madagascar being the largest producer by far. North America is the largest market, using about 14001 per year, and Europe and Japan use 450 and 701 per year respectively (Todd, 1998). CHO

136

FOOD FLAVOUR TECHNOLOGY

Vanilla extracts are generally made by extracting the beans with ethanolwater. The vanillin and other flavours are formed during the curing process by glycosidase action on glycoside precursors present in the green beans. The concentration of vanillin and other flavour chemicals in the extract depends on the proportions of bean and extracting solvent used: this is commonly referred to as the 'fold' of the extract. The flavour obtained varies considerably with source, depending especially on the variety of vanilla grown, climate, the soil type and cultivation conditions, and the methods of harvesting and processing, especially the method of curing. The vanilla flavour is composed of a very wide range of aldehydes, ketones, alcohols, esters, ethers, hydrocarbons, oils, waxes and resins (Adedeji et al, 1993). Of these, vanillin constitutes the 'heart' of the flavour. In a detailed analysis of 10 different vanilla extracts, only 19 out of total 194 organic compounds detected were present in all 10 extracts. These extracts were Bourbon (2 types), Tahitian, Bali (2 types), Java, Mexican, Tonga, Costa Rican and Jamaican. Some of the aroma chemicals are definitive for particular types of vanilla extract. For instance, anise acid and anisaldehyde are only found in concentrations sufficient to influence the flavour in Tahitian vanilla extract. On the other hand, some of the organic compounds detected will contribute little or nothing to the flavour and aroma, because they have only a low flavour impact or/and because they are only present in low concentrations. Most vanilla beans are sold to North America. This is because Americans prefer sweetness in foods and beverages, so that relatively high concentrations of vanilla extract are formulated, and because Americans consume a lot of icecream, which is the single biggest use for vanilla extracts. Vanilla continues to be the biggest selling sweet flavour and the identity of vanilla flavour is strictly regulated in the USA; which defines three categories of vanilla ice-cream: all natural, natural supplemented with artificial, and artificial (categories 1, 2. and 3 respectively). Furthermore, Americans prefer the relatively harsh, phenolic flavour notes of Indonesian vanilla which has a quite smoky aroma. Indonesian vanilla extracts have been cheaper than Madagascan vanilla, and so are costeffective for use in Type 2 vanilla flavour ice-creams supplemented with artificial vanillin. Much vanillin is produced by chemical processes for supplementation of vanilla extract, but now genuine naturally produced vanillin is beginning to be available from microbiological processes. 4.6.2 Spice flavours Spices encompass a huge range of taste and odour sensations, achieved using an equally wide range of chemicals. These include capsaicin and dihydrocapsaicin (peppers), sotolone (fenugreek - curry), zingerone (ginger), trans-2dodecenal (coriander), eugenol (cloves and cinnamon), 1,8-cymene (rosemary, cardomon, allspice and sage), methylchavicol (basil), fenchone and anethole

137

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

(fennel), anthranilic acid ester (mandarin), L-menthol and L-menthone (peppermint) and a-phellandrene (dill). OH

o Sotolone

OCH3

Trans-2-Dodecenal

Zingerone

O

OCH3 1,8-Cineol

p-Cymene

OCH3 Methylchavicol (Estragole)

Fenchone a-Phellandrene

Flavour molecules that have a pungent flavour are found in peppers, ginger and pepper. They show a particular structural similarity. Piperine is the active pungent constituent of peppers. o II

N—C—CH =:CH — C Piperine

13 8

FOOD FLAVOUR TECHNOLOGY

Ginger contains gingerols and shogoals, which vary in proportions depending on the type of ginger used and storage conditions. As can be seen, gingerols are simply a hydrated form of the corresponding shogoals. Ginger flavour is particularly interesting as changes can occur on storage, with gingerol dehydrating into shogoal, and in turn shogoal can be cleaved into zingerone and hexanal, which adds to taste complexity. CH 3 —(CH 2 )n—CH—CH 2 —C —(CH2)2 OH

O

Gingerols

CH3 —(CH2)n—CH2=CH—C—(CH2)2 O

Shogoals

4.6.3 Garlic, onion and related flavours Garlic flavours are based on (di)allyl disulfide (di-(2-propenyl) disulfide), which constitutes 90% of the active flavour of garlic oil. It is produced from the precursor alliin (S-allyl-L-cysteine sulfoxide), via the intermediate allicin. by alliinase enzyme, which is only released when the vegetable tissue is crushed. 1-Propenyl disulfide is also present in garlic oils, but so far no manufacturing process has been developed to make it more readily available for formulation into flavours.

S

Di-(2-propenyl) disulfide Alliin

Allicin

(allyl disulfide)

As well as the allyl disulfide, the trisulfide, mixed disulfide, mercaptan and other products are produced from allyl sulfide by disproportionation and rearrangement reactions. These increase the complexity of the flavour formed. In particular, the savoury flavour note increases with the sulfur content of the flavour molecule.

Trisulfide

Mixed disulfide

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

139

Flavour complexity is also increased because some 5-methyl- and 5propylcysteine sulfoxide precursors are also present in garlic, and give rise to the corresponding flavour chemicals. In addition, chemical interactions with other food components occur, and the overall flavour can be modified by the presence of other flavour chemicals. For instance, in the presence of 3-hydroxy2,5-dimethyl-4(//)-furanone a sweet, roasted character is produced. Onion flavours are based on propyl derivatives, rather than the allyl derivatives found in garlic, and so the flavour chemicals formed tend to be saturated rather than unsaturated. 5-Methyl and 5-propyl precursors also contribute flavour chemicals. Alkyl alkanethiosulfonates, especially propyl methanethiosulfonate and propyl propanethiosulfonates and alkyl thiosulfonates are characteristic of the flavour of raw onion, and have odour thresholds of 1.7 and 1.5 ug/1 respectively (Boelens et al., 1993).

o Propyl propanethiosulfonate

Cooked onion flavour is given by dipropyl disulfide, and cis- and trans2-propenylpropyl disulfides, which have odour thresholds of 3.2 and 2.0 ug/1 respectively, and also by various other sulfides such as trisulfides.

di propenyldisulfide

Dipropyl disulfide

frans-2-Propenylpropyl

disulfide

In fried onions the characteristic flavour chemicals formed are 2-(propyldithio) dimethylthiophenes, which have odour thresholds of 0.01-0.05 ug/1 (Kuo and Ho, 1982).

s 2-(Propyldithio)dimethylthiophene

140

FOOD FLAVOUR TECHNOLOGY

Other vegetables of the allium group contain similar flavour chemicals; for instance, leeks contain methylpropyl di- and trisulfides and dipropyl trisulfide. 4.6.4 Brassica flavours, including mustard and horseradish Isothiocyanates are the characteristic pungent flavouring components in Brassica plants, including mustard, horseradish, broccoli, cabbage and cress. The particular form of isothiocyanate present varies from species to species (Table 4.13), with each species being characterised by the possession of a particular isothiocyanate, such as /?-hydroxybenzyl isothiocyanate in white mustard as compared to isophenyl isothiocyanate in horseradish. The isothiocyanates are formed from glucosylinolate precursors, via unstable thiohydroxamate-0sulfate intermediates, by the action of myrosinase (thioglucosidase) that is liberated when the plant tissue is disrupted, such as by mastication or maceration during processing. The main precursors are allyl isothiocyanates, but methyl, ethyl and isopropyl isothiocyanates are also present, and products of their reaction with myrosinase also contribute to the flavour. In addition to the formation of isothiocyanates, lesser amounts of nitrile and thiocyanate products are also formed from the unstable intermediates produced by myrosinase. Allyl isothiocyanate Black mustard and horseradish

p-Hydroxybenzyl isothiocyanate White mustard

CH2—CH2—N—C=S

N=C=S

2-Phenylethyl isothiocyanate Watercress

Indoylmethyl isothiocyanate Cabbage

Allyl isothiocyanate has a rather particular use, namely as a denaturant to give a repellent bitter taste to many household cleaning products, so as to deter children from accidentally poisoning themselves by drinking these often attractively labelled products. 4.6.5

'Fresh/green/grassy'

This flavour note is conferred by Cf, alcohols and aldehydes, typically trans-2hexenal (leaf aldehyde) and m-3-hexenol (leaf alcohol), as well as others such

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

Image Not Available

as rra/«-2-hexenol and m-3-hexenyl butyrate (for a review see Fabre and Goma, 1999). The former compound is produced by isomerisation of the unstable cis-3hexenal, which is the original product formed by the action of lipoxygenase, and then by lyase enzyme, on linoleic acid. m-3-Hexenal isomerisation is catalysed by both acid and base, so it can only be used effectively at its optimal pH stability point, This is a considerable limitation as ds-3-hexenal has a very low aroma threshold of 0.25 ug/1, whereas frans^-hexenal and m-3-hexenal are less intense, having thresholds of 17 and 70 ug/1 respectively (that is about 64-fold and 280-fold higher).

c/s-3-Hexenal

frans-2-Hexenal

c/s-3-Hexenol

frans-2-Hexenol

The nuances of flavour between the various C& flavour molecules have been compared by Whitehead et al. (1995). Their descriptions were: hexanal, fatty, green and fruity; fraw^-2-hexenal, leafy green and fruity; m-3-hexenal (unstable); hexan-1-ol, oily green; frans-2-hexen-l-ol, sharp, green, fruity; cis-3hexen-1-ol, grassy green, fresh. These Cg flavours are also responsible for the off-flavours that develop in some foods on prolonged storage owing to the action of lipoxygenase on unsaturated fatty acids. This has been combated by blanching to inactivate the enzymes, and irradiation can also stabilise vegetables against the formation of off-flavours. The fresh green flavour sensation extends from €5 molecules, through C? and Cg to Cg nonadienols which have green vegetable-cucumber-melon characters.

142

FOOD FLAVOUR TECHNOLOGY

Image Not Available

especially 3,6-nonadien-l-ol (violet leaf alcohol or cucumber alcohol), which is characteristic of cucumbers. This was the first nonadienol to be isolated and studied. Nonadienols are also formed by lipoxygenase action on linoleic acid and occur in melon and some grapes as well as in cucumbers. Nonadienals give a good illustration of the relationships between structure, flavour character and flavour price (Table 4.14). A further flavour variation is the formation of Cn hydrocarbons, such as l,3-frans-5-c/.s-undecatriene and \3-trans-5,S-cisundecatetraene found in various fruits and vegetables, by ^-oxidation of linoleic acid and then lipoxygenase activity, nonenzymic oxidation and decarboxylation. 4.7 4.7.1

Fruit Apples

Esters are the main contributors. For instance, ethyl 2-methylbutyrate is a key contributor of apple flavour, with an odour threshold of 0.1 ug/1, together with a range of other acids, aldehydes, alcohols and esters such as c/s-3-hexenol, trans2-hexenal, ^-damascenone, ethylbutyrate, c/s-3-hexenylbutyrate and hexyl2-methylbutyrate.

Ethyl 2-methylburyrate

c/s-3-Hexenyl buryrate

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

143

A detailed examination comparing three different quantitative structureactivity relationship approaches between the various ester molecules with a fruity aroma has been carried out by Rossiter (1996). An interesting study of the supply of exogenous n-butanol vapour to apples as a substrate for flavour ester formation shows that, whereas butanol penetrates deeply into the apple, flavour esters predominate close to the surface of the apple, strongly indicating that the enzymes responsible for flavour formation are located in, or close to, the skin of the apple. The great increase in ester concentrations formed from the supplied precursor—up to 200 times the levels normally present—proves that effective concentrations of the enzymes are present. Probably these esters are formed via coenzyme A ester intermediates. The characteristic flavours of particular varieties of fruit can often be accounted for by the presence of one variety-specific flavour chemical or a small number of such chemicals. For instance the characteristic aniseed flavour note of the apple variety 'Ellisons Orange' has been identified as being due to the presence of 1-methoxy-4-propeny Ibenzene (estragol). Much more basic flavour characteristics are variety dependent, as with acid apple varieties (Cox) as compared with sweeter varieties of lower acidity (Jonagold). The actual situation is more subtle as acidity tends to reduce during storage, unless the fruit is stored under a controlled low-oxygen atmosphere, and can result in depletion of important flavour compounds such as butyl and hexyl acetates. This behaviour has led to selective breeding studies to produce new hybrid varieties with improved flavour storage characteristics. The 2-methylbutyl esters predominate not only in apples but also in strawberries, papaya, blackcurrant, pineapple and other fruits. But in cider, which is also produced from apple juice, considerable amounts of 3-methylbutyl ester flavour chemicals occur. Other fruit esters frequently present, but without conferring a flavour character as does ethyl-2-methylbutyrate, are ethyl butyrate, and ethyl hexanoate, which have odour thresholds of 1, 0.1 and l-2ug/l respectively. The complexity of the various flavours possible is clearly illustrated by sensory mapping of apple drinks, which shows clear variations in perception resulting from the differences in types of apple, processing methods, and so on. 4.7.2 Pears Pear flavour is chiefly given by esters of unsaturated fatty acids, such as ethyl 2,4-decadienoate, ethyl-2-octenoate, hexylacetate, ethyl-4-decenoate, butyl acetate and ethylbutyrate. A particularly important flavour component of pears is 2-m2«s-4-cw-decadienoic acid ethyl ester. It is believed to be produced in the fruit from linoleic acid by ^-oxidation, isomerisation of a double bond and further ^-oxidation, followed by desaturation and esterification with ethanol.

144

4.7.3

FOOD FLAVOUR TECHNOLOGY

Grapefruit

A very good example of character impact compounds is provided by grapefruit. For some time it has been recognised that the sesquiterpene (/?)-nootkatone has a potent grapefruit flavour character with a quite low odour threshold of 1 ug/1. More recently, it has been discovered that a quite different chemical, (/?)-(+)-/?1 -menthene-8-thiol (grapefruit mercaptan) also gives grapefruit character and has a remarkably low threshold of 0.000 02 ug/1. Indeed, it is so potent that it only exhibits the grapefruit character when diluted to below 10 ug/1; at higher concentrations it only has a nondescript rubbery odour. By contrast the (S)-(—)p-1 -menthene-8-thiol isomer is reported to have an unpleasant sulfur flavour, and a quite different threshold of detection.

SH

Nootkatone

1 -p-Menthene-8-thiol

4.7.4 Blackcurrant Various sulfur-containing flavour chemicals give the characteristic flavour of blackcurrant. Examples are cat ketone (4-thio-4-methylpentan-2-one) blackcurrant mercaptan (4-methoxy-2-methyl-2-butanethiol) and particularly 8-thio/?-menthan-3-one. Blackcurrant mercaptan is also found in olive oil and has a flavour threshold of 0.2 ug/1.8-Thio-/?-menthan-3-one is found in Buchu leaf oil and is used to boost the flavour of blackcurrant extracts. Only the 1S, 4/? isomer out of the four possible isomers was described as blackcurrant-like (Kopke andMosandl, 1992) l-Methoxy-3-methyl-3-butanethiol also has a blackcurrant flavour and a threshold of 0.08-3 ug/1, and has been detected in virgin olive oils (Guth and Grosch, 1991). The composition of blackcurrant leaf oil from different cultivators extracted by steam distillation and low-temperature solvent extraction has been reported by Marriott (1988).

SH

4-Thio-2-methyi-2-butanethiol

8-thio-p-methan-3-one

4-thio-4-methylpentan-2-one

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

145

4.7.5 Raspberry l-/?-Hydroxyphenylbutan-3-one (raspberry ketone), which has a flavour threshold of 5 ug/kg, gives raspberries its characteristic flavour.

Hydroxyphenylbutanone

Other flavour chemicals that make a big contribution to raspberry flavour include p-damascenone, vanillin, 4-hydroxy-2,5-dimethyl-3-furanone, sotolone, l-nonen-3-one, as-3-hexenal, a- and |3-ionones and the ethyl esters of 5-hydroxyoctanoic acid and 5-hydroxydecanoic acid, with these esters capable of hydrolysing and cyclising to form lactones during cooking. 4.7.6 Strawberry 4-Hydroxy-2,5-dimethylfuran-3-one and m-3-hexenol are important in determining strawberry flavour. Also contributing are methylbutanoate, ethyl 2-methylbutanoate, methyl-2-methylbutanoate, acetic acid, 2,3-butanedione and methyl and ethyl cinnamates. Significant changes occur on cooking or freezing. On freezing, concentrations of hydroxydimethylfuranone increase 6-fold and cw-3-hexenol concentrations fall 60-fold. On heating, ds-3-hexenol concentrations also fall and the concentrations of hydroxydimethylfuranone, p-damascenone, 2,4-decadienol and guaiacol increase markedly. COOCH3

°H

4-Hydroxy-2,5-dimethylfuran-3-one

Methyl cinnamate

For comparison it is interesting to note that early strawberry flavours used the synthetic compounds ethylphenylglycidate and ethylmethylphenylglycidate, the merits of which are discussed in Chapter 1 (see page 8 for more details).

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FOOD FLAVOUR TECHNOLOGY

Ethyl-3-methylphenylglycidate

4.7.7 Apricot and peach Various flavour chemicals contribute to apricot flavour. These include myrcene, limonene, />-cymene, terpinolene, a-terpineol, geraniol and geranial, linalool, acetic and 2-methylbutyric acids, fraw.s-2-hexenol, and the lactones y-caprolactone, y-octalactone, y-dodecalactone, S-octalactone and 5-decalactone. In peaches, the main flavour character is provided by lactones, especially y-decalactones, and also other C& to C\2 y-lactones and QO and C\2 8-lactones. Other contributors include benzyl aldehyde, benzyl alcohol, ethyl cinnamate, isopentyl acetate, linalool, a-terpineol, a- and p-ionones, 6-pentyl-a-pyrone. and hexanal, c/s-3-hexenal and fran.s-2-hexenal. 4.7.8 Citrus and citrus processing Orange flavour is contributed by minor constituents of the orange oil such as Cg-Cn saturated aldehydes (octanal, decanal), undecanal terpenes such as p-sinensal, esters such as ethylbutanoate, ethyl-2-methylbutanoate and ethyl isobutyrate, and unsaturated aldehydes such as m-3-hexenal, and especially fra/75-2-decenal. By contrast, mandarin flavour contains a-sinensal.

CHO

frans-2-Decenal

a-Sinensal

Citral, which is a mixture of the two stereoisomers geranial and neral, is a characteristic flavour of lemons. Linalool, myrcene and limonene, which are the major components of citrus oils, also make a contribution to flavour. Traces of hydrogen sulfide and dimethyl sulfide are also present in all citrus fruits and contribute to the overall flavour.

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

CHO

147

n

CHO

Geranial

Neral

Unfortunately, the excellent flavour and aroma of fresh, cold-pressed citrus oils is unstable. This is because the unsaturated hydrocarbon terpenes such as limonene, which comprise 80-95% of the oil but have very little aroma, are rapidly oxidised to produce molecules that cause strong off-tastes. Therefore, various techniques have been developed to reduce or remove the hydrocarbon constituents of oils before they can become oxidised. Unlike with some sources of flavours, considerable efforts have been made in the plant breeding of citrus for improved flavour characteristics. 4.7.8.1 Citrus processing The orange processing industry worldwide has a turnover of over $2 billion per year. Over 400-500 million trees are under cultivation, mainly in Brazil and Florida, which have around 150 and 60 million trees respectively, and some 75 0001 per year of D-limonene and orange oil are produced from the fruit. These figures give an impressive indication of the size of the industry (Bovill, 1996), especially when it is considered that the yield of D-limonene from the fruit is only 0.27%, compared with a yield of 53% for the juice. Oranges are squeezed to extract the juice, which is concentrated by evaporation about 6-5-fold before being shipped. The remaining orange peel is then subject to pressing or rasping to extract the so-called cold-pressed oil, which is the source of many useful flavour and fragrance molecules. These mainly oxygenated terpenes are a minority of the oil, which is mostly composed of limonene and some other 'hydrocarbon' terpenes. Since oxidation of the terpenes can cause off-flavour, a major objective of citrus oil processing is to reduce its hydrocarbon content. The traditional method is by fractional distillation followed by washing, or by 'folding'. This involves taking a manyfold concentrate and directly dissolving it in a water-ethanol solution. Although distillation is cheap, some volatile flavour materials are lost, some thermal degradation occurs, and the sesquiterpene hydrocarbons are not removed and so are still available for oxidation. Therefore, a molecular still (thin-film evaporator) has to be used to reduce residence times and to minimise thermal degradation. An alternative method is to wash the citrus oil with ethanolic solutions, as the valuable oxygenated terpenes are soluble in ethanol whereas the hydrocarbon terpenes are insoluble. The hydrocarbon extract is called washed citrus oil,

148

FOOD FLAVOUR TECHNOLOGY

and the alcohol solution containing concentrated oxygenated terpenes is called 'washed extracts'. Although effective, this countercurrent process is difficult to operate because of the difficulty in efficiently contacting the two phases without emulsifying them so finely that their subsequent separation is difficult. Countercurrent extraction with liquid carbon dioxide, which has a polarity as an extracting solvent close to that of hexane, has been used. However, this method suffers from high capital costs. Absorption column chromatography using silica gel as the absorbent and ethyl acetate or hexane as the eliciting solvent has been developed (Moyler and Stephens, 1992). The newest method for removing the hydrocarbon terpenes is the so-called poroplast (PTFE) extraction method. This involves the aqueous phase passing through a column containing a low-polarity stationary phase on to which the

Image Not Available

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

149

hydrocarbons absorb, with the oxygenated hydrocarbons being carried through and out of the column by a flow of aqueous ethanol. Once extraction is complete, a fresh charge of oil is applied to the column and the separation procedure is repeated (Fleisher, 1994). The compositions of conventional and poroplastextracted hydrocarbon orange oil are compared in Table 4.15. New solvents have been tested for extraction of essential oils; for instance, 1,1,2-2-tetrafluoroethane (hydrocarbon-134a) (Wilde and McClory, 1994). Although this is a poor solvent, the product is made in the form of a clear, mobile oil with only a very low solvent residue, and with no degradation of the solutes occurring. In addition, the solvent can be reused without further processing, and the product can be used without additional processing, in contrast to conventional extraction processes that produce a concrete that must be further refined by treatment with ethanol. One critical factor, whichever extraction method is used, is the influence of harvesting time on the chemical composition of the extract produced (Chalchat et al., 1997), particularly if an enantiomeric analysis is required (Dugo et al., 2001). This is one of the factors that makes the standardisation of the chemical composition of extracts difficult and meeting tight product specifications challenging (Moyler and Moss, 1998). Another important factor is the presence of any pesticide residues (Dugo et al., 1997). The methods developed for some of the more traditional flavour products have also provided a basis for recovery and purification processes for new bioprocesses; for instance, see the review of Fabre et al (1996) on the processing of 2-phenylethanol.

4.8

Other flavour characteristics

Sweetness is very important in flavour perception and sucrose is a plant extract produced by hot water extraction of sugar cane or sugar beet. Similarly, glucose and fructose sweeteners are produced by the action of a-amylase and glucoamylase acting on starch extracted from cereals followed by glucose isomerase to produce fructose. In addition, there are the more recently introduced sweeteners, such as Aspartame™ and Sucralose™. Acidity and sourness in foods and beverages is achieved using organic acids such as citric, malic and tartaric acids. Originally these were made by extraction from citrus fruits, apples and grapes respectively. Now production of citric and malic acids is mostly by fermentation, although tartaric acid is still extracted from grapes and is an important component of wines. The type of acid used affects the intensity and quality of the flavour. Some less common acids are also consumed; for instance, isocitric acid is the major organic acid in blackberries. Bittering materials can also be derived from plant extracts; in particular hops used to bitter beer, contain humulone, cohumulone and adhumulone. Other

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FOOD FLAVOUR TECHNOLOGY

bittering materials include quinine used to bitter soft drinks, and naringin and limonin that make oranges and grapefruit bitter.

HO H3CO

4.9

Conclusion

This chapter has explored the range of flavour chemicals available from natural resources and the variety of organoleptic effects that they produce, together with an indication of how they can be used to flavour foods and beverages and their commercial value. It illustrates a little of what is already known but indicates how much still needs to be discovered. An indication of the great variety and range of natural flavour chemicals has been presented. Besides chemical diversity, there are also enormous variations in the intensities of aroma/flavour chemicals, as measured by their detection thresholds, which span a range of 16 orders of magnitude. Even within a single 'class' of flavour chemicals, there is a wide variation in flavour/aroma characters. Despite this, some good structure-function relationships have been identified for both taste and aroma molecules. Flavour creation is still something of an art, and much research is still required to put flavours on a truly rational and predictive basis. References Adedeji, J., Hartman, T.G. and Ho, C.T. (1993) Perfum. Flavour. 18 24-33. Alston, F.H. (1992) in Bioformation of Flavours (eds Patterson. R.L.S. etal.). Royal Society of Chemistry. Cambridge, pp 33^tl. Aoki, T. and Uchida. K. (1991) Agric. Biol. Chem., 55 2113-2117. Baigrie, B.D. (1994) in Understanding Natural Flavours (eds Piggott. J.R. and Paterson. A.). Chapman and Hall, pp 268-282. Bakker, J. and Law. B.A. (1994) in Understanding Natural Flavours (eds Piggott. J.R. and Paterson. A. i. Chapman and Hall, London, pp 283-295. Belitz, H.D. and Grosch. W. (1999) Food Chemistry. 2nd edn. Springer-Verlag. Berlin. Benn, S. (1998) Perfum. Flavour.. 23 5-18. Berger. R.G. (1990) Perfum. Flavour.. 15 33-39. Blank, I. (1992) Z. Lebemm.-Unters. Forsch., 195 239-245.

PLANT-DERIVED NATURAL SOURCES OF FLAVOURS

15 1

Boelens, M.H. and Van Gemert, LJ. (1993) Perfum. Flavour., 18 29-39. Boelens, M.H., Boelens, H. and Van Gemert, L.J, (1993) Perfum. Flavour., 18 1-18. Bovill, H. (1996) Perfum. Flavour., 21 9. Chalchat, J.C., Michel, A. and Pasquier, B. (1997) Perfum. Flavour., 22 15. Chassagne, D., Bayonove, C, Crouzet, J. and Baurnes, R. (1995) Bioflavour 95, Dijon, INRA Paris 1995, (Les Colloques No. 75) pp 217-222. Clark, G.S. (1992) Perfum. Flavour., 17 107-109. Clark, G.S. (1998) Perfum. Flavour., 23 33-48. Clark, G. (1999) Perfum. Flavour., 24 25-30. Dugo, G., Saitta, ML, DiBella, G. and Dugo, P. (1997) Perfum. Flavour., 22 33^4. Dugo, G., Mondello, L., Cotroneo, A., Bonaccorsi, I. and Larnonica, G. (2001) Perfum. Flavour., 26 20-36. Eaton, D.C. (1994) in Bioprocess Production of Flavour, Fragrance and Color Ingredients (ed. Gabelman, A.), Wiley Interscience, New York, pp 169-204. Eriksson, C. (1994) in Understanding Natural Flavours (eds Piggott, J.R. and Paterson, A.), Chapman and Hall, London, pp 283-297. Fabre, C. and Goma, G. (1999) Perfum. Flavour., 24 1-8. Fabre, C.E., Blanc, P.J., Marty, A., Goma, G., Sauchon, I. and Voilley, G.A. (1996) Perfum. Flavour., 21 27-39. Fleisher, A. (1994) Perfum. Flavour., 19 11-15. Guth, H. and Grosch (1991) Fat. Sci. Technoi., 93 335-339. Hartmann, H. (1995) Perfum. Flavour., 20 35-42. Hausler A. and Munch, T. (1998) ASM News, 63 551-559. Holscher, W. (1992) /. Agric. Food. Chern., 40 655-658. Holscher, W. and Steinhart, H. (1992) Zeit schrift fur lebensmittel - unter suchung und - forschung, 195 (1)33-38. Jorgenson, U., Hansen, M., Christensen, L.P Jensen, K. and Kaack. K. (2000) J. Agric. Food. Chem., 48 2376-2383. Kolor, M.G. (1983) J. Agric. Food Chem., 31 1127-1129. Kopke, T. and Mosandl, A. (1992) Z. Lebensm.-Unters. Forsch., 194 327-376. Koppenhoefer, B., Behnisch, R., Epperlein, U. and Holzschuh, H. (1994) Perfum. Flavour., 19 1-10. Kuo, M.C. and Ho, C.T. (1992) J. Agric Food Chem., 40 111-117, 1906-1910. Land, D.G. (1994) in Understanding Natural Flavours (eds Piggott, J.R. and Paterson, A.), Chapman and Hall, London, pp 298-306. Lindsay, R.C. and Rippe, J.K. (1998) in Biogeneration of Aromas (eds Croteau, R. and Parliament, T.). Symposium Series 317, American Chemical Society, Washington, DC, pp 286-308. McGee, T. and Purzycki, K. (1999) Perfum. Flavour., 24 1-10. Malnic, B., Hirono, J., Sato, T. and Buck, L.B. (1999) Cell 96 713-716. Marriott, R.J. (1988) in Flavours and Fragrances, A World Perspective, Proc. 10th Int. Congress of Essential Oils (eds Lawrence, B.M. et al.), Elsevier Science, Amsterdam, pp 387-392. Mookherjee, B. and Wilson, A. (1990) Perfum. Flavour., 15 27-49. Moyler, D. and Moss, N. (1998) Perfum. Flavour., 23 37-39. Moyler, D.A. and Stephens, M.A. (1992) Perfum. Flavour., 23 37-39. Mulders, E.J. (1976) Chem. Ind. (London), pp 613-614. Mulheim, A., Hausler, A., Schilling, B. and Lerch, K. (1998) Perfum. Flavour., 23 21-27. Naf, R. and Velluz, A. (2000) Flavour Fragrance J., 15 329-334. Naf, R., Vellez, A. and Thommen, W. (1990) Tetrahedron Lett., 31 6521-6522. Rombaults, J.E. (1952) Proc. Soc. Appl. BacteriL, 15 103-111. Ropkins, K. and Beck, AJ. (2000) Trends Food Sci. Technoi., 11 10-21. Rossiter, K. (1996) Perfum. Flavour., 21 33-39. Rowe, D.J. and Tangel, B. (1999) Perfum. Flavour., 24 36-44.

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Sell, C. (2001) Perfum. and Flavour., 26 2-7. Semmelroch, P. and Grosch, W. (1996) J. Agric. Food Chem.. 44 537-543. Shallenberger, R.S. (1996) Food Chem., 56 209-214. Sinki. G., Assaf, R. and Lombardo, J. (1997) Perfum. Flavour.. 22 23-31. Somogyi, L. (1996) Chem. Ind., March 170-173. Stofberg. J. (1983) Perfum. Flavour.. 8 61-64. Swaine, R.L. (1993) Perfum. Flavour, 18 35-38. Todd, H. (1998) Perfum. Flavour.. 23 23-25. Tressl, R. and Silwar, R. (1981) J. Agric Food Chem.. 29 1078-1082. Tyrell, M. (1995) Perfum. Flavour., 20 13-22. Verhagen, L.C. (1994) in Understanding Natural Flavours (eds Piggott, J.R. and Paterson. A.). Chapman and Hall, London, pp 283-297. Wang, D., Yoshimura, T.. Kubota, K. and Kobayashi, A. (2000) / Agric. Food Chem., 48 5411-5418. Whitehead, I.M., Muller, B.L. and Dean. C. (1995) Cereal Foods World. 40 193-197. Wilde, P.P. and McClory. P.O. (1994) Perfum. Flavour.. 19 25-26. Winterhalter, P. and Schreier, P. (1994) Flavour Fragrances, 9 281-297. Wright. J. (1995) in Food Flavourings, 2nd edn (ed. Ashurst, PR.). Blackie Academic and Professional/ Chapman & Hall, Glasgow and London, pp 25-57.

Flavour encapsulation using polymer-based delivery systems Daniel Benczedi

5.1

Introduction

The encapsulation of flavours was triggered by the need to protect volatile food components from evaporation and oxidation by locking them in a solid carrier able to dissolve rapidly in water (Schultz, 1955). Nowadays more emphasis is laid on slowing down aqueous dissolution and on release systems activated by heat and mechanical stress: the controlled release of flavours is taken as an opportunity to provide new solutions to the food industry and to promote the development of innovative products (Roberts and Taylor, 2000). Flavour encapsulation is now used to modulate perception by generating flavour impact and persistence where needed. The edible ingredients used to design flavour delivery systems include polysaccharides, proteins, fats and waxes, sometimes used in combination to reduce the migration of oxygen, flavours or moisture (Kester and Fennema, 1986; Miller and Krochta, 1997). As we shall see, flavours are generally wellretained and protected from oxygen by hydrophilic polymers, whereas hydrophobic polymers are used to protect delivery systems from moisture. To be commercially successful, the selected encapsulation process has to be costeffective and adapted to the phase behaviour of the selected barrier material. Flavours are traditionally encapsulated by spray-drying an aqueous polymer solution, and the resulting glassy powders have a fine granulometry, favouring quick aqueous dissolution. The selective retention of volatile molecules during spray drying is reviewed elsewhere (Risch and Reineccius 1995; Coumans et al., 1994; King, 1995; Rosenberg etal., 1990). When extrusion or fluid bed coating is used, a much coarser granulometry is possible, with the advantages of improving oxidation stability and of slowing down aqueous dissolution kinetics by varying the surface-to-volume ratio. Starch hydrolysates are excellent oxygen and flavour barriers, frequently used in combination with octenyl-succinylated starches or gum arabic to provide some lipophilic character to the carrier (Buffo and Reineccius, 2000). To improve oxidative stability of orange oil, starch hydrolysates are used in combination with lower molecular weight plasticisers such as amorphous sucrose or corn starch syrups (Anandaraman and Reineccius, 1986). This is counterintuitive because, above the glass transition temperature (7"g), plasticisation is

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FOOD FLAVOUR TECHNOLOGY

generally coupled to a density reduction followed by an increase in permeability. The opposite is observed below 7g, in which case the addition of a plasticiser leads to a densification of the polymer, as observed at low water concentrations in starch (Benczedi, 2001). Cross-linked polymers are used to slow down aqueous dissolution or to provide systems that release upon exposure to heat, chemical or mechanical stress. Gelatin and starch build thermally reversible gels, while alginates and pectins can be cross-linked by calcium ions (Harris, 1990). The use of glutaraldehyde to cross-link gelatin irreversibly is restricted to specific food applications by the Food and Drug Administration. The manufacture of delivery systems by polymer extrusion, phase separation (simple or complex coacervation), gelation (nozzle extrusion) and coating (Wiirster) is reviewed elsewhere (Kondo, 1979; Bakan. 1986; Jackson and Lee, 1991; Shahidi and Han, 1993; Risch and Reineccius. 1995; Gibbs etal., 1999; Qi and Xu, 1999; Benczedi and Blake, 1999). The semi-empirical principles of chemical engineering presented here are particularly useful in addressing the performance and limitations of controlledrelease systems (Prausnitz, 1999). Hildebrand's solubility parameter is used as a solvent scale to estimate the polarity of flavours and the cohesive energy density of polymers and to assess their mutual compatibility (Hildebrand el a/.. 1970: Barton, 1991). The Flory model of polymer solutions is used to highlight the limiting phase behaviour of glassy polymers at low partial pressures of water and of hydrogels at saturation vapour pressure of water (Flory, 1953: de Gennes 1979; Tanaka, 1979). A power law is used to illustrate how mass transport in polymers is affected by polymer cross-linking or plasticisation. as induced by temperature or partial pressure of water. Pick's laws of diffusion are used to distinguish between diffusion-controlled kinetics, scaling with the square root of time, and zero-order kinetics observed if mass transport is polymer relaxation-controlled (Atkins, 1994). 5.2

Compatibility and cohesion

The second law of thermodynamics states that the mixing of molecules is spontaneous when the contributions of the heat of mixing A// and of the entropy of mixing AS lead to a negative Gibbs free energy of mixing AG. AG = A// - 7AS < 0

(5.1)

In the absence of any heat of mixing, the sole driving force is the configurational entropy gain, which is proportional to the number of molecules involved and is thus less when the mixture contains a polymeric component. The sign and absolute value of A H are thus critical for spontaneous mixing, more particularly when polymers are involved.

POLYMER-BASED DELIVERY SYSTEMS

1 55

Following Hildebrand et al. (1970), the heat of mixing is approximated as A// = (8, -& 2 ) 2 Vic!>2

(5.2)

where V\ is the molar volume of the solvent, 4>2 is the volume fraction of polymer and 5 is the solubility parameter defined as the square root of the cohesive energy density, (c.e.d). 8 - (c.e.d.)

=

y]/2

—

(5.3)

where Vis the molar volume, R is the universal gas constant, T is the temperature and A// v is the enthalpy of vaporization. To be miscible, the difference in & of chemical species indexed 1 and 2 must be small. If this is not the case, it can be minimised with a co-solvent with intermediate & value. Originally formulated for regular solutions, Hildebrand's approach has been extended to include associative attractions (hydrogen bonding) in addition to nonpolar and polar attractions. As a consequence of the specific nature of such forces, materials with weak cohesive forces cannot make the associations needed to dissolve materials with strong attractive forces (Barton, 1991). In the solvent scale presented in Table 5.1, heptane represents a molecule held together by apolar attractions, and water represents one held together by apolar, polar and associative attractions. A value estimated for gaseous diatomic oxygen is included to emphasise its apolar character. It can be seen that the range of hydrophobicity characteristics of essential oils spans from limonene to eugenol, although some flavour ingredients such as Furaneol show a hydrophilicity closer to that of ethanol and of sucrose, a widespread carbohydrate flavour carrier ingredient. The proximity of solubility parameters of sucrose and starch (see Table 5.2) suggests that the former is a good plasticiser for the latter. If the Table 5.1 Molecular weight M, boiling point 7},, and solubility parameter S, of selected chemicals at 25 D C Diatomic oxygen n-Heptane D-Limonenea D-Carvonea n-Octanol Acetaldehyde Eugenol" Ethanol Furaneolh Sucrose Water a

M (Da)

7b(°C)

8(MPa) ) / 2 t

32 100 136 150 130 44 164 46 128 342 18

gaseous at 25°C 98 176 230 194 21 255 79 crystalline at 25' C crystalline at 25 C 100

11.7 15.1 16.6 19.0 21.1 21.1 23.3 26.4 26.8 45.1 48.0

Barton (1991); Peppas and Am Ende (1997). Furaneol is a Firmenich trade name of 4-hydroxy-2,5-dimethyl-3(2//)-furanone. C 5 calculated with group contributions compiled by Beerbower and listed in Barton (1991). h

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FOOD FLAVOUR TECHNOLOGY

Table 5.2 Solubility parameter &, density p, difference between experimental temperature (25'C) and glass transition temperature (T — Tg). and rounded oxygen and water permeability of polymers P(O 2 ) and P(H2O) Polyethylene Poly(vinyl acetate) Poly(vinyl alcohol) Starch

8 (MPa) l/2

p (g/cm3)

T - rg ro

/MO2)a

P (H 2 O) a

16 19 30 35b

1.0 1.2 1.3 1.5b

>0

10-'

102 104 dissolves dissolves

%o
2

10~ 10~6 n.a.c

a

Permeability is expressed in cm3 nm/(m 2 day kPa). Miller and Krochta (1997); Benczedi et al. (1998); Brandrup et al. (1999). c n.a.. not available. b

vaporisation enthalpy is not available experimentally, as in the case of polymers, indirect methods are used to estimate o experimentally and group contribution methods are available to calculate 8 for a molecule of known chemical structure (van Krevelen, 1976; Barton, 1991; Hu et al., 1997; Tse et al., 1999). Table 5.2 shows the solubility parameters of selected polymers in which polyethylene and starch represent the lipophilic and hydrophilic ends of the solvent scale respectively. The increase of polarity corresponds to an increase of density and glass transition temperature. It is also apparent from Table 5.2 that the hydrophilicity of a polymer increases the water permeability coefficients while the oxygen permeability coefficient is decreased. The glass transition temperature, Tg, of polymers increases with their molecular weight, their stiffness and their polarity. The addition of a soluble low molecular weight diluent decreases Fg by plasticisation. Flavours are thus usually poor plasticisers for the hydrophilic polymers used to encapsulate them. On the other hand, water is a powerful plasticiser for hydrophilic flavour carriers, as is apparent from the similarity of their solubility parameters. The Tg of hydrophilic polymers is thus sensitive to the partial vapour pressure of water (Benczedi et al., 1998). In contrast, the lack of compatibility between lipophilic flavours and hydrophilic carrier material justifies the success of hydrophilic polymers as encapsulation carriers. Hildebrand's approach is thus useful for predicting polymer-solvent compatibility, ranking polymers according to their hydrophilicity and, more generally, predicting the impact of formulation ingredients on the morphology and performance of delivery systems (Michaels etal, 1975; Vaughan. 1985; Rowe. 1988; Sakellariou and Rowe, 1991; Moldenhauer and Nairn. 1992;Archer. 1992: Peppas and Am Ende, 1997; Rodriguez et al., 2000). The densification process of polymer glasses by solvent uptake mentioned in the introduction is known as anti-plasticisation because it affects the mechanical and barrier properties of polymer glasses in ways opposite to those expected of plasticisation (Fischer et al., 1985; Guo, 1994). Its occurrence thus cannot be predicted by Hildebrand's solvent scale. Anti-plasticisation is usually described

POLYMER-BASED DELIVERY SYSTEMS

1 57

with free volume models assuming that the specific volume of a molecular liquid always contains a fraction of space left unoccupied by the molecules. The permanent redistribution of this free volume by random thermal agitation causes density fluctuations by which molecular transport is assumed to take place in liquids. In polymer glasses there is an excess free volume component, which is not redistributed by thermal agitation. As we shall see, the occurrence of an excess free volume can be detected by the particular vapour and gas sorption phenomenology of glassy polymers. The latter suggests that sorption takes place in fixed pre-existing sites composing the excess free volume, in the attempt of glassy materials to recover their equilibrium state (Meares, 1954; Cohen and Turnbull, 1959; Berens, 1975; Fan and Singh, 1989; Debenedetti, 1995; Benczedi, 2001). 5.3

Sorption and swelling

The solubility of volatile components is characterised in sorption experiments by measuring their equilibrium volume fraction, ${ , sorbed by a polymer at any given solvent activity, a\ = pi/p®. The deviation from ideal solution property is defined by the activity coefficient, yi = a\l§\, an excess thermodynamic function relating the actual free energy change upon mixing to the free energy change observed in the ideal case, AG E = AG uctual - AG ideal . I n y i = (AGE/RT) = (&HE/RT) - (&SE/R)

(5.4)

Thus in the ideal case, In yi is zero (yi = 1) over the entire concentration range and the distribution of molecules is random, as predicted by Raoult's law for noninteracting and equal-sized molecules. Endothermal and exothermal deviations from ideal mixing are then characterised by positive and negative deviations of yi from 1, as predicted by Guggenheim's quasi-chemical approximation (Guggenheim, 1952). If y is moreover invariant with concentration at infinite dilution of the volatile species, the mole fraction-based limiting activity coefficient (Deshpande et al., 1974) is related to the Henry's law constant, //, which reflects its volatility in a given physical-chemical environment.

The configurational entropy of mixing is less favourable when polymers are involved and the activity coefficient of solvents in rubbery polymer solutions is larger than predicted by Raoult's law even when athermal polymer-solvent mixing occurs. Endothermal mixing is then treated semi-empirically with a concentration-independent Flory interaction parameter, x • I n y , ^(4) 2 + x 4>2) 2

(5.6)

158

FOOD FLAVOUR TECHNOLOGY

In the athermal case, the Flory interaction parameter / is zero. At infinite dilution of the solvent (4>2 = 1), Flory's expression predicts that In y^ = 1 in the athermal case (y™ = 2.1...). x varies between 0 and 1/2 for rubbery polymersolvent mixtures and phase separation is predicted to occur at / values above 1/2. Solvent uptake by rubbery polymers is thus usually endothermal over the entire concentration range and x shows only weak composition dependence (Guggenheim, 1952; Flory, 1953). An extension of the model to more than two components is used to describe polymer phase separation, as is practised in coacervation (Tompa, 1956; Hsu and Prausnitz, 1973). In contrast, the water sorption isotherm of polar biopolymers is sigmoidal (type IV sorption) because their configurational entropy of mixing is frozen at low solvent concentrations where they are in a glassy state (Benson and Seehof. 1951; McLaren and Rowen, 1951). As a consequence, such systems exhibit an exothermal deviation from Flory's athermal case and the limiting behaviour of the solvent activity coefficient at infinite dilution is thus specified by In y^ < 1. At higher water concentrations these solid systems are brought above their glass transition point by water and the Flory phenomenology is recovered when (T — Tg) becomes positive. As a further consequence, the activity coefficient is no longer independent of concentration, as postulated in the original Flory treatment. To match sigmoidal sorption phenomenology, Flory's model is used in combination with a generalised Freundlich adsorption isotherm taking into account the adsorption occurring at low solvent concentrations (Sips, 1948, 1952; Benczedi el al., 1998). The exothermal solvent uptake characterised by In yi > 1 at low solvent concentrations suggests that the adsorption process takes place in preexisting sites of the excess free volume of polymer glasses mentioned above. The same phenomenology is reported for gas adsorption by glassy polymers with a sorption following Henry's law above To, whereas below To, the gas sorption first follows Langmuir adsorption phenomenology before recovering to follow the Henry mode at higher partial pressures (Paul, 1985; Ganesh et al., 1992). The high x values (above 1/2) characterising water uptake at higher activity are observed in systems in which the poor solubility of solvent in rubbery polymers constrains the solvent molecules to swell the polymer by forming clusters composed of several solvent molecules (Zimm and Lundberg. 1956; Berens, 1975; Stanett et al., 1980; Benczedi et al., 1998). A swollen hydrogel is obtained when the dissolution of a polymer is still hindered at the saturation vapour pressure of water or in bulk water by thermally reversible or irreversible polymer-polymer cross-links. The case of finite polymer swelling without dissolution is illustrated in Figure 5.1 by setting x > 1 in Flory's approximation. At equilibrium, the osmotic pressure is zero and the activity of the solvent inside and outside the gel is identical. The Flory treatment assumes that the total osmotic pressure of a nonionic gel is the result of a swelling force, driven

159

POLYMER-BASED DELIVERY SYSTEMS

0)

E

J3 O

X=1

Solvent activity a, Figure 5.1 Sorption isotherms showing the volume fraction (j>i of solvent in a polymer as a function of the solvent activity a\. The dashed diagonal line represents Raoult's law and the activity coefficient yi = a\/§\, is thus smaller than 1 above the line (exothermal mixing) and greater than 1 below it (endothermal mixing). The phase behaviour predicted with Flory approximation is shown by setting X = 0 (upper dotted line), x = 1/2 (intermediate dotted line) and x = 1 (lower dotted line); the latter illustrates finite polymer swelling ((() | (a] = 1) < 1) in the phase domain characterised by solvent clustering, i.e. / > 1 /2. The sigmoidal (solid line) is typical for water sorption by a hydrophilic polymer frozen in a glassy state at low water activity (McLaren and Rowen, 1951; Benczedi et al, 1998). The corresponding gas adsorption phenomenology in polymer glasses is shown in the insert where c is the ratio of gas to polymer volume at standard temperature, plotted as a function of the gas pressure,/? (Paul, 1985;Ganeshe-ra/., 1992).

by polymer-solvent affinity, opposed by a contracting (shrinking) force due to the rubber-like elasticity of the three-dimensional polymeric network. The swelling ratio, Q, defined as volume ratio of swollen to dry gel, decreases when the density of cross-links increases in a polymer as the polymer chain length between them is reduced (Flory, 1953; Tanaka, 1979). The solubility of polymers is usually increased by temperature unless they exhibit a lower critical solution temperature, as in the case of some cellulose derivatives in water (Doelker, 1993). A third contribution needs to be considered with ionic networks to account for ion-solvent interactions responsible for the pH sensitivity of polyelectrolyte gels. Gels containing acidic (electrophilic) functional groups such as carboxyl groups are ionised at pH > pK,d, whereas gels containing basic (nucleophilic) functional groups such as amino groups are ionised at pH < pK^. The swelling is then driven by the repulsion between the fixed ionised functional groups of polymers until they are neutralised by

160

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hydration following Donnan equilibrium, i.e. electroneutrality in each phase and phase equilibrium for each mobile ion (Prausnitz, 1995; Khare and Peppas. 1995). 5.4 Diffusion and release The transport of volatile molecules such as gases, vapours or liquids through amorphous polymeric barriers is characterised by a permeability coefficient. P. The permeation of volatile molecules takes place in the amorphous domains of polymers, while the crystalline domains are impermeable to molecular transport (Paul, 1985; Avranitoyannis etal, 1994). The thermodynamic component of the permeability coefficient P, describing how many volatile molecules are soluble in a given rubbery polymer, is the solubility coefficient 5, the reciprocal of the Henry law constant, H = S~l. The kinetic component of P is the diffusion coefficient D, describing how rapidly molecules translate in a given polymer. At infinite dilution of the permeating molecule, the permeability coefficient is the product of solubility and diffusion coefficients. P = SD

(5.7)

Permeation data are then interpreted using either an activated process approach or the free volume approach. In an activated process, the energy for diffusion is postulated to arise from the need to separate polymer segments sufficiently to allow the permeating molecule to make a unit diffusion jump. In the free volume approach, the permeating molecule is postulated to move from one place to another only when local free volume around this molecule exceeds a certain critical value (Paul, 1985). Under stationary conditions (steady-state equilibrium), Pick's first law of diffusion defines the rate of mass transfer across an isotropic material as proportional to the concentration gradient measured perpendicularly to the section. Beyond stationary conditions, Pick's second law relates the rate of change of concentration at a point to the spatial variation of concentration at that point (Atkins, 1994). The following power law is used to illustrate how Pick's laws of diffusion can be used to analyse the release of functional molecules (flavour, fragrance, drug, etc.) from a polymer or the uptake of solvent (water) by a polymer. -^- - U"

(5.8)

M, is the amount of volatile component released at time /, Mx is the equilibrium value at large / (sum of active component released or of solvent absorbed), k is a rate constant, and n is a scaling exponent. If a linear release curve is observed on plotting M,/MX as a function of / 1 / 2 , the release or uptake is diffusioncontrolled. A scaling exponent n of 1/2 is thus the fingerprint of the Brownian

POLYMER-BASED DELIVERY SYSTEMS

motion of molecules diffusing in a stationary concentration gradient as predicted by classical Pick's diffusion (case I transport) (Fan and Singh, 1989). If a linear sorption curve is obtained by plotting Mt/M^ as a function of t, the amount released is constant in time (n = 1) and the release kinetics are controlled by the relaxation of the polymer-solvent system (case II transport: zero-order kinetics). For values of the exponent n between 1/2 and 1, the release reflects both diffusion and relaxation of the polymer-solvent system (anomalous transport). The scaling exponent n is thus larger than 1/2 whenever it reflects the time dependence of polymer relaxation around the glass transition point of the polymer-solvent system (Fujita, 1961; Crank and Park, 1968). This is illustrated in Figure 5.2 with a dimensionless Deborah number De, used to distinguish the limiting cases of mass transport in polymers by the ratio of two characteristic times, a characteristic relaxation time A for the polymer-solvent system, and a characteristic diffusion time, 0 (Vrentas and Duda, 1975).

(5.9)

De =

T>T

0) 0.

E

De»1 n = 0.5 T
0.10.5 D = D(a1;0

Solvent activity a,

Figure 5.2 Schematic temperature-solvent activity (T-a\) diagram of mass transport in polymersolvent systems. The solid line is the glass transition temperature, Ts, of the system. The dashed lines delimit domains of mass transport characterised by a Deborah number, De, a scaling exponent in equation (5.7) and a diffusion coefficient that may be independent of concentration and time, D°, dependent on concentration but independent of time, D(a\), or dependent on both concentration and time, D(a\; t). Adapted from Hopfenberg and Frisch (1969).

162

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In the liquid state characterised by De <£ 1, diffusion occurs within a viscous medium, while in the solid state characterised by De ^> 1, diffusion occurs within an elastic medium. As shown in Figure 5.2, the diffusion coefficient is time dependent for 0.1 <$C De <£ 10 in the viscoelastic regime around the glass transition points where anomalous mass transport is observed because A and 0 are of the same order of magnitude (Ferry, 1980; Frisch. 1980; Fan and Singh, 1989). As shown in Table 5.2, hydrophilic polymers are at the same time good oxygen barriers (S = 11.7MPa 1/2 ) but poor water barriers (8 = 48MPa' 2 ) and oxygen permeability, P(O2) in polyethylene (S = 16 MPa l/2 ) is five orders of magnitude larger than in poly(vinyl alcohol) (8 = 30MPa l/2 ) (Salame and Steingiser, 1977; Miller and Krochta, 1997). As the size of the permeating molecule increases, P is expected to decrease because a linear increase in S following an elevation of the boiling point is more than compensated by an exponential drop in D (Naylor, 1989). Diffusion is related to the mobility of polymer chains and thus to the temperature of the system relative to its glass transition temperature. As the temperature is lowered and approaches 7g, the free volume available for diffusion decreases. Diffusion coefficients exhibit either a continuous or a discontinuous change at rg. The change is continuous if the size of the diffusing molecule is smaller than the average void size and diffusion occurs by localised activated jumps from one pre-existing cavity to another. As the size of the diffusing molecule grows, the number of polymer segment rearrangements involved in an activated jump increases and the process becomes dependent on the excess (sub-7 g ) free volume of the system, as reviewed elsewhere (Fan and Singh, 1989). An increase of the size of the permeating molecule in fact causes their diffusion coefficient D to decrease by only two orders of magnitude in natural rubber (r > Tg, 8 = 17MPa 1/2 ) prior to reaching an asymptotic minimum value, while it decreases by ten orders of magnitude in glassy poly(vinyl chloride) (T < 7g, 8 = 20MPa 1/2 ). D is reported to increase by up to three orders of magnitude when the permeating molecule is elongated rather than spherical (Naylor, 1989). The effect of temperature on D is of the same order when a packaging material is brought to retort temperature or when a hydrophilic polymer is plasticised by water following exposure to high relative humidity (DeLassus, 1994; De Lassus et al., 1988). The plasticisation of the polymer makes the diffusion coefficient time-dependent as reflected by n > 1 /2 (Yapel etal., 1994; Beck and Tomka, 1997). The permeation of gases and vapours in rubbery polymers is not affected by polymer relaxation and is thus diffusion-controlled (n = 1 /2) if plasticisation or swelling is negligible, which is always the case at infinite dilution (Figure 5.2). This is the case if the chemical affinity of the volatile compound for the polymer is insufficient or if its partial pressure is low enough, and the relevant diffusion coefficient is then invariant with time (D(a\) in Figure 5.2) or even invariant with time and concentration (D° in Figure 5.2). In packaging applications, the

POLYMER-BASED DELIVERY SYSTEMS

163

partial pressure of volatile compounds is often less than 0.2 and insufficient for plasticisation to affect the permeability, as illustrated by the Fickian diffusion of D-limonene in rubbery polyethylene (Hernandez, 1986; DeLassus, 1994). In controlled-release applications, the partial pressure of flavours is much higher and polar polymers of appropriate polarity and molecular weight are needed to prevent a flavour-induced plasticisation and to minimise oxygen permeability (Anandaraman and Reineccius, 1985). Diffusion coefficients can also be adjusted by cross-linking (Lieberrnan and Guilbert, 1973), which reduces polymer segment mobility and is able to change the release kinetics from diffusion-controlled (n — 1/2) at low cross-link density to polymer relaxationcontrolled (n = 1) at high cross-link density, as shown for release of eugenol in ethanol from gels of poly(2-hydroxyethy 1 methacrylate) (Peppas and Am Ende, 1997). The same strategy can thus be applied to provide sustained flavour release in high-moisture conditions if the polymers are selected to limit carrier dissolution above 70-80% relative humidity and in bulk water. The aqueous release of water-soluble drugs by cellulose ethers or methylcellulose exhibits diffusion-controlled release (n ~ 1/2), whereas release from hydroxyethyl-, hydroxypropyl- and hydroxypropylmethylcellulose is characterised by (1/2 < n < 1) (Rodriguez et al., 2000). The poor water solubility of many flavours and perfumes contrasts with the water solubility of drugs and thus favours their sustained release from hydrogels in aqueous environments. The choice of the right delivery system is ultimately a compromise between oxygen and moisture stability on one hand, and cost-efficiency on the other.

5.5

Summary

Semi-empirical principles are used to review the phase behaviour of hydrophilic polymers such as those applied to encapsulate flavours and fragrances. A solvent scale obtained with Hildebrand's solubility parameter is used to outline how the polarity of a polymer increases its cohesive energy density, its density and its glass transition temperature, and thus ultimately reduces its permeability to apolar species such as gaseous oxygen and most flavouring ingredients. The Flory model of polymer solutions is used to highlight the limiting phase behaviour of glassy polymers at low partial pressures of water and of hydrogels at saturation vapour pressure of water. It is suggested that the sigmoidal phenomenology characterising water sorption by glassy polymers is the macroscopic fingerprint of an excess free volume in glassy polymers, which needs to be minimised to keep the diffusion of oxygen and other volatile species as low as possible. A power law is used to illustrate how mass transport in polymers is affected by polymer cross-linking or plasticisation, as induced by temperature or partial pressure of water. Pick's laws of diffusion are used to distinguish between

164

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diffusion-controlled kinetics, scaling with the square root of time, and zeroorder kinetics observed when mass transport is polymer relaxation-controlled. References Anandaraman, S. and Reineccius, G. (1986) Stability of encapsulated orange peel oil. Food Techno!.. 40(11)88-93. Archer, W.L. (1992) Hansen solubility parameter for selected cellulose ether derivatives and their use in pharmaceutical industry. Drug. Dev. Ind. Pharm., 18 (5) 599-616. Atkins, P.W. (1994) Physical Chemistry. 5th edn, Oxford University Press. Oxford. Avranitoyannis. I., Kalichevsky, M., Blanshard. J.M.V. and Psomiadou. E. (1994) Study of diffusion and permeation of gases in undrawn and uniaxially drawn films made from potato and rice starch conditioned at different relative humidities. Carbohydr. Polym.. 24 1-15. Bakan. J.A. (1986) Microencapsulation, in The Theory and Practice of Industrial Pharmacy. 3rd edn (eds. L. Lachman, H.A. Lieberman, J.L. Kanig). Lea & Febiger. Philadelphia, pp 412^429. Barton. A.F.M. (1991) Handbook of Solubility Parameters and Other Cohesion Parameters. 2nd edn. CRC Press, Boca Raton. Beck. M.I. and Tomka, I. (1997) Effects of the degree of substitution in ethylcellulose on the clustering of sorbed water. J. Macromol. Sci.-Phys.. B36 (1) 19-39. Benczedi. D. (1999) Estimation of the free volume of starch-water barriers. Trends Food Sci. Technol.. 10 21-24. Benczedi. D. (2001) Plasticisation and antiplasticisation of starch glasses by water. ISOPOW 8. (eds Z. Berk. R.B. Leslie. P.J. Lillford and S. Mizrahi), Technomic Publishing. Lancaster. 273-281. Benczedi. D. and Blake, A. (1999) Encapsulation and the controlled release of flavours. Leatherhead Food RA Food Industry J.,2(\) 36-48. Benczedi. D., Tomka, I. and Escher. F. (1998) Thermodynamics of amorphous starch-water systems. Macromolecules. 31 (9) 3055-3074. Benson, S.W. and Seehof, J.M. (1951) The surface of proteins: IV. Sorption of polar gases. / Am. Cheni. Soc.. 73 5053-5058. Berens. A.R. (1975) Solubility of vinyl chloride in poly(vinyl chloride). Angew. Makmmol. Chem.. 47 97-110. Brandrup. J., Immergut. E.H. and Grulke. E.A. (eds) (1999) Handbook of Polymers. 4th edn. Wiley. New York. Buffo. R. and Reineccius, G.A. (2000) Optimisation of gum acacia-modified starch-maltodextrin blends for the spray drying of flavours. Perfum. Flavor.. 25 (May-June) 37-51. Cohen M.H. and Turnbull. D. (1959) Molecular transport in liquids and glasses. J. Chem. Phys.. 31 (51 1164-1169. Coumans, W.J., Kerkhof, P.J.A.M. and Bruin, S. (1994) Theoretical and practical aspects of aroma retention in spray-drying and freeze-drying. Drying Technol., 12 (1-2) 99-149. Crank. J. and Park. G.S. (eds) (1968) Diffusion in Polymers. Academic Press. New York. Debenedetti. P.G. (1995) Metastable Liquids: Concepts and Principles. Princeton University Press. Princeton. de Gennes. P.G. (ed.) (1979) Scaling Concepts in Polymer Physics. Cornell University Press. Ithaca. DeLassus, P.T. (1994) Permeation of flavors and aroma through glassy polymers. Tappi /. 77 ( 1 ) 109-113. DeLassus, P.T.. Strandburg, G. and Howell, B.A. (1988) Flavor and aroma permeation in barrier film: the effects of high temperature and high humidity. Tappi J.. 71 (11) 177-181. Deshpande. D.D.. Patterson, D. Schreiber. H.P. and Su. C.S. (1974) Thermodynamic interactions in polymer systems by gas-liquid chromatography. Macromolecules. 7 (4) 530-535.

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Doelker, E. (1993) Cellulose derivatives. Advances in Polymer Science, 107, Springer Verlag, Berlin, pp 199-265. Fan, L.T. and Singh, S.K. (1989) Controlled release: a quantitative treatment. Polymer Properties and Applications, 13, Springer Verlag, Berlin. Ferry, J.D. (1980) Viscoelastic Properties of Polymers, 3rd edn, Wiley, New York. Fischer, E.W., Hellmann, G.P., Spiess, H.W., Horth, F.J., Ecarius, U. and Wehrle, M. (1985) Mechanical properties, molecular motion and density fluctuations in polymer-additive mixtures. Makromol. Chem.Suppl.,12 189-214. Flory, P.J. (1953) Principles of Polymer Chemistry. Cornell University Press, Ithaca. Frisch, H.L. (1980) Sorption and transport in glassy polymers, a review. Polym. Eng. ScL, 20 ( 1 ) 2-13. Fujita, H. (1961) Diffusion in polymer-diluent systems. Fortschr. Hochpolym.-Forsch., 3 1-47. Ganesh, K., Nagarajan, R. and Duda, J.L. (1992) Rate of gas transport in glassy polymers: a free volume based predictive model. Ind. Eng. Chem. Res., 31 746-755. Gibbs, B.F., Kermasha, S., Alii, I. and Mulligan, C.N. (1999). Encapsulation in the food industry: a review, int. J. Food. Sci. Nutr, 50 213-224. Guggenheim, E.A. (1952) Mixtures. Clarendon Press, Oxford. Guo, J.-H. (1994) A theoretical and experimental study of additive effects of physical aging and antiplasticisation on the water permeability of polymer film coatings. J. Pharm. ScL, 83 (3) 447-449, Harris, P. (ed.) (1990) Food Gels, Applied Food Science Series, Elsevier Applied Science, London. Hernandez, R.J.. Baner, L.A. and Giacin, J.R. (1986) The evaluation of the aroma barrier properties of polymer films. J. Plast. Films and Sheeting, 2 187-194. Hildebrand, J.H., Prausnitz, J.M. and Scott, R.L. (1970) Regular and Related Solutions, Van NostrandReinhold, Princeton. Hopfenberg, H.B. and Frisch, H.L. (1969) Transport of organic micromolecules in amorphous polymers. J. Polym. Sci. Polym. Lett, B7 405^109. Hsu, C.C. and J.M. Prausnitz (1973) Thermodynamics of polymer compatibility in ternary systems. Macmmolecules, 1 (3) 320-324. Hu, Y, Zhou, H., Liu, H., Wu, D.T. and Prausnitz, J.M. (1997). Estimation of vapor—liquid equilibria for polymer solutions by a group-contribution method. Fluid Phase Equilibria, 134 43-54. Jackson, L.S. and Lee, K. (1991) Microencapsulation and the food industry, Lebemm.-Wiss. u.-Technoi. 24 289-297. Khare, A.R. and Peppas, N.A. (1995) Swelling/deswelling of anionic copolymer gels. Biomateriah, 16 (7)559-567. Kester, JJ. and Fennema, O.R. (1986) Edible coatings: a review. Food TechnoL, (December) 47-59. King, C.J. (1995) Spray drying: retention of volatile compounds revisited. Drying TechnoL, 13 (5-7) 1221-1240. Kondo, A. (1979) Microcapsule processing and technology (ed J.W. van Valkenburg), Marcel Dekker. New York. Lieberman, E.R. andGuilbert, S.G. (1973) Gas permeation of collagen films as affected by cross-linkage. moisture and plasticiser content. J. Polym. Sci. Symp., 41 33-43. McLaren, A.D. and Rowen, J.W. (1951) Sorption of water vapor by proteins and polymers: a review. J. Polym. ScL, 7 (2-3) 289-324. Meares, P. (1954) The diffusion of gases through polyvinylacetate. J. Am. Chem. Soc., 76 3415-3422. Michaels, A.S., Wong, P.S.L., Prather, R. and Gale, R.M. (1975) A thermodynamic method of predicting the transport of steroids in polymeric matrices. A/Che J., 21 1073-1080. Miller, K.S. and Krochta, J.M. (1997) Oxygen and aroma barrier properties of edible films: a review. Trends Food Sci. TechnoL, 8 228-237. Moldenhauer, M.G. and Nairn, J.G. (1992) The control of ethylcellulose microencapsulation using solubility parameters../. Controlled Release, 22 205-218.

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Naylor, T.d.V. (1989) Permeation properties, in Comprehensive Polymer Science. Vol. 2 (eds C. Booth and C. Price) pp 643-668. Paul. D.R. (1985) Transport property of polymers. Applied Polymer Science. ACS Symposium Series 285, American Chemical Society. Washington, DC, pp 253-274. Peppas, N. and Am Ende, D.J. (1997) Controlled release of perfumes from polymers. II. Incorporation and release of essential oils from glassy polymers. J. Appl. Polym. Sci., 66 509-513. Prausnitz, J.M. (1995) Some new frontiers in chemical engineering thermodynamics. Fluid Phase Equilibria. 104 1-20. Prausnitz. J.M. (1999) Thermodynamics and the other chemical engineering sciences: old models for new chemical products and processes. Fluid Phase Equilibria. 158-160 95-111. Qi. Z.H. and Xu, A. (1999) Starch-based ingredients for flavor encapsulation. Cereal Foods World. 44 (7) 460-465. Risch, S.J. and Reineccius, G. A. (eds) (1995) Encapsulation and Controlled Release of Food Ingredients. ACS Symposium Series 590, American Chemical Society. Washington. DC. Roberts, D.D. and Taylor, A.J. (eds) (2000) Flavor Release. ACS Symposium Series 763. American Chemical Society. Washington, DC. Rodriguez. C.F., Bruneau. N.. Barra. J.. Alfonso. D. and Doelker. E. (2000) Hydrophilic cellulose derivatives as drug delivery carriers, in Handbook of Pharmaceutical Controlled Release Technology (ed. D.L. Wise), Dekker. New York, pp 1-30. Rosenberg, M.. Kopelman, I.J. and Talmon. J. (1990) Factors affecting retention in spray-drying microencapsulation of volatile materials. J. Agric. Food. Sci.. 38 1288-1294. Rowe. R.C. (1988) Binder-substrate interactions in tablets: a theoretical approach based on solubility parameters. Acta Phann. Technoi. 34 144-146. Sakellariou. P. and Rowe. R.C. (1991) Phase separation and morphology in ethylcellulose/cellulose acetate phthalate blends. J. Appl. Polym. Sci.. 43 845-855. Salame, M. and Steingiser, S. (1977) Barrier polymers. Polym. Plast. Technoi. Ent>.. 8 (2) 155-175. Schultz. T.H.. Dimick, K.P. and Mackower, B. (1956) Incorporation of natural fruit flavors into fruit juice powders: I. Locking of citrus oil in sucrose and dextrose. Food Technoi.. (January) 57-60. Shahidi. F. and Han. X.-Q. (1993). Encapsulation of food ingredients. Cm. Rev. Food Sci. Nutr.. 33 (6) 501-547. Sips. R. (1948. 1952) On the structure of a catalyst surface. J. Chem. Phys.. I. 16 (5) 490-495: and II. 18(8)1024-1026. Stannett. V, Haider. M.. Koros. W.J. and Hopfenberg. H.B. (1980) Sorption and transport of water vapor in glassy poly(acrylonitrile). Polym. Eng. Sci.. 20 (4) 300-304. Tanaka. T. (1979) Phase transitions in gels and a single polymer. Polymer. 20 1404-1412. Tompa. H. (1956) Polymer Solutions. Butterworth Scientific. London. Tse. G.. Blankstein. D.. Shefer. A. and Shefer. S. (1999) Thermodynamic prediction of active ingredient loading in polymeric microparticles. J. Controlled Release. 60 77-100. van Krevelen, D.W. (1976) Properties of Polvmers: Estimation and Correlation with Chemical Structure. 2nd edn, Elsevier. Amsterdam. Vaughan. C.D. (1985) Using solubility parameters in cosmetic formulations. J. Soc. Co.smet. Chem.. 36 319-333. Vrentas. J.S. and Duda. J.L. (1975) Diffusion in polymer-solvent systems: III. Construction of Deborah number diagrams. J. Polym. Sci.. 15 441-453. Yapel, R.A.. Duda. J.L.. Lin. X. and von Meerwall. E.D. (1994) Mutual and self-diffusion of water in gelatin: experimental measurement and predictive test of free volume theory. Polymer. 35 ( 1 1 ) 2411-2416. Zimm, B.H. and Lundberg. J.L. (1955) Sorption of vapors by high polymers. J. Phys. Chem.. 60 425-428.

Delivery of flavours from food matrices Saskia M. van Ruth and Jacques P. Roozen

6.1

Introduction

It is generally accepted that aroma, taste, texture and mouth feel account for the major stimuli that contribute to the perception of flavour. Stimulation occurs when compounds from the food come into contact with receptor cells in the mucous membranes of nose (odour/aroma) and mouth (taste) or when food structures such as emulsions or rigid cell walls affect the chewing process (texture) or interact with the mouth lining (mouth feel) (Taylor, 1996). Mouth feel responses are concerned with the heat sensation of spices and the cooling sensation of menthol. Taste is concerned with the sensations of sweet, sour, salty, bitter and umami, which are associated with receptors on the tongue. Aroma is a much broader sensation and encompasses an estimated 10 000 or more different odours (Reineccius, 1993). The flavour perceived during eating is not simply an addition of the four basic stimuli but is a complex pattern that has different characteristics for particular foods. When food is eaten, flavour molecules are released from the food into the mouth and the volatile flavour compounds pass back up through the nasopharynx into the nose. A sufficiently high concentration of flavour molecules has to be released from the food to stimulate the olfactory system and elicit a response. Flavour release and delivery depend on the nature and concentration of volatile compounds present in the food, as well on as their availability for perception as a result of interactions between the major components and the aroma compounds in the food (Bakker et al., 1995). Food composition factors and eating behaviour determine the extent of flavour release, delivery and perception (Bakker et al., 1996). With the growing range of new foods available, many with lower fat or lower sugar formulations than the traditional foods, it is becoming increasingly important to understand the factors that affect the perception of flavour, including how flavour is released from food matrices, in order to deliver an acceptable flavour from these foods. Knowledge of the binding behaviour of flavour compounds in relation to various food components and their rates of partitioning between different phases is of great practical importance for the flavouring of foods, in determining the relative retention of flavours during processing or the selective release of specific compounds during processing, storage and mastication (Kinsella, 1988).

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FOOD FLAVOUR TECHNOLOGY

Thermodynamic and kinetic factors control the release of flavour from food products and thus its delivery. The influence of properties of the flavour compounds as such and the thermodynamic aspects and kinetic aspects of flavour release as a function of food composition and oral manipulation (salivation, mastication) will be discussed in the following sections. This chapter mainly involves volatile flavour because most studies in this area deal with volatile compounds.

6.2 Flavour properties Flavour delivery depends on the availability of the flavour compounds in the gas phase and, therefore, on the affinity of the flavour compounds for the food matrix. Various properties of the flavour compounds determine the interactions with food components, e.g. molecular size, functional groups, shape, volatility (Kinsella, 1988). Properties such as molecular weight, vapour pressure, boiling point, octanol-water partition coefficient (log P) have been used to predict the volatility of the compounds under static conditions (Roberts and Acree. 1996: Linforth et a/., 2000; van Ruth et al, 2000).

6.3 Thermodynamic aspects of flavour delivery Interactions between flavour substances and major food components are of two types: attractive and repulsive interactions. Attractive interactions involve fixation of flavour compounds on food components, whereas repulsive interactions concern the release of aroma compounds. The nature of these interactions depends on the physicochemical properties of the compounds and the food matrix (Le Thanh et al., 1992). 6.3.7 Definition of gas/product partition coefficients and activity coefficients The discussion of thermodynamic aspects of flavour release, such as phase partitioning, requires a definition of gas/product partition coefficients and activity coefficients. Flavour release will only take place if the gas/product phase equilibria are disturbed. In other words, nonequilibrium is the driving force for mass transfer. Equilibrium between the gas phase and the product phase exists only if there is no effective transfer at the product-gas interface (de Roos. 2000). The equilibrium partition coefficient can be expressed as

Kl = CC

(6.1)

DELIVERY OF FLAVOURS FROM FOOD MATRICES

1 69

and

#'=y/q

(6.2)

where Kl is the partition coefficient, //' is the Henry's law constant, Cp is the food product concentration (mole fraction), C'g is the gas phase concentration (mole fraction), and pl is the partial pressure in the gas phase of the flavour compound /, all at equilibrium. Partition coefficients can also be expressed using concentrations in the gas and product phases. Gas/product partition coefficients are temperature dependent: the log-transformed gas/liquid partition coefficient is linearly related to the temperature (Kolb et al., 1992). Henry's law explains the behaviour of the flavour compound and holds for a restricted range of conditions (Taylor, 1998). The concentration of the flavour compounds should be such that they can be considered infinitely dilute. Furthermore, molecules must remain unimolecular. Compounds that dissociate, such as organic acids (de Roos and Sarelse, 1996), or that associate, e.g. those that form micelles (Piggott et al., 1996), do not show ideal behaviour. Another important property is the relative volatility of the volatile component with respect to water, since it determines the relative proportions in which the aroma compound and water come off during an equilibrium vaporisation. This relative volatility a'w is defined by the ratio of the partition coefficient of flavour compound i (K*) to the partition coefficient of water (Kw). K w in turn is defined w ae c r"g Cpw . as // c ,^i

f^i ^w

=h _£_ a- = !L /fw /""j /""w ft

l^

L,

Activity coefficients in the liquid phase, y( are defined by reference to the concept of an ideal solution: pi = yc'pPl()

(6.3)

where P'° is the vapour pressure of the pure component i at the temperature in question. The activity coefficient is a measure of the degree of compatibility of i with the liquid phase, the tendency for intermolecular forces to develop between / and the major constituents of the food, in comparison to the strength of intermolecular forces among the major components of the food itself. A similar definition may be made for the activity coefficient of water. Activity coefficients may also be defined for the gas phase, but these tend to be important only at pressures higher than those normally encountered under food conditions. Putting the equations for compound / and water together and recognising that by Dalton's law Y' - —

(6.4)

1 70

FOOD FLAVOUR TECHNOLOGY

where P is the total pressure, gives v' P'° K'^-jr and

v' p'° OLIW = --

(6.5)

(6.6)

For many important flavour compounds, the vapour pressure of the pure substance is not greatly different from that of water. On the other hand, values of y1 tend to be very large, with values of the order of 1 000 being common. These very large activity coefficients stem from the fact that common volatile flavour compounds tend to be relatively nonpolar, and therefore relatively incompatible with a highly polar, aqueous solution in terms of intermolecular forces. Conversely, values of yw tend to be around 1 , since water is a major component of many food systems (King, 1983). 6.3.2

Types of binding

6. 3. 2. 1 Absorption and adsorption Binding of flavour compounds in food systems is synonymous with 'sorption' in its broad sense, including adsorption, absorption, physicochemical binding and chemical binding. Adsorption and absorption are types of binding specific for low-moisture food systems. Dry foodstuffs consist of particles of variable size with an outer surface and, usually, an inner surface made up of fine pores and channels. Volatile compounds can therefore be sorbed onto both the outer and the inner surface: this process is called adsorption. The aroma compound may also 'dissolve' in the material of the particle: this process is called absorption. In crystalline nutrients of low molecular mass, the process is mainly adsorption to the outer surface. Pores play no great role. Sorption of sugars and salts is normally physical, according to molar heat of sorption at low vapour pressures of volatiles. In special cases, even crystalline substances can bind very large amounts of aroma compounds, irreversibly under some conditions. An example is the binding of volatile acids and amines to amino acids (Maier. 1975). Physical and chemical binding of various food components, which are the same for low- and high-moisture food systems, will be discussed in the following section. 6.3.2.2 Physicochemical and chemical binding Flavour binding/complex formation in food systems is the result of specific physicochemical and chemical interactions between major food components and the flavour compounds. It is important to discriminate here between dissolved, bound and total flavour concentration. Only the free dissolved flavour molecules

DELIVERY OF FLAVOURS FROM FOOD MATRICES

17 1

exert a vapour pressure (de Roos, 2000). Fixation of aroma substances in food results from a number of binding processes: (a)

Chemical binding •

(b)

Covalent bonds, which are irreversible and involve the transfer of electrons between two atoms

Physicochemical binding • • • •

Van der Waals forces Hydrogen bonds Hydrophobic interactions Ionic bonds (Solms et al., 1973; Kim and Min, 1988; Voilley et al., 1990)

The composition of the food matrix determines the extent and type of aroma binding. Apart from the aqueous phase present in high-moisture foods, the major food components with respect to binding are lipids, carbohydrates and proteins. 6.3.3 Lipid-flavour interactions Most lipids are hydrophobic, nonpolar materials that exist naturally as liquids (oils) or solids (fats). Lipids may be regarded as material of biological origin consisting of one or more of the following classes: free fatty acids; mono-, diand triglycerides; phospholipids; sterols; plasmalogens; and lipoproteins (Forss, 1969). Of all food components, lipids probably have the strongest impact on gas/product partitioning. In lipid-containing food systems, lipophilic flavour compounds are bound to the lipid molecules by weak, reversible van der Waals forces and unspecific hydrophobic interactions (Plug and Haring, 1993). Lipids act as solvent for lipid-soluble, hydrophobic flavour compounds. Table 6.1 illustrates the effect of the interactions of flavour compounds and the oil phase by comparing the gas/liquid partition coefficients of flavour compounds in sunflower oil, in water and in a mixture of oil and water. The generally hydrophobic nature of flavour compounds results in considerably differences in headspace composition if the lipid phase is removed, as is the case in fat-free foods. In the absence of fat, the food matrix retains lipophilic flavours poorly and the resulting headspace concentrations are high, as indicated in Table 6.1 (Plug and Haring, 1993). Binding to the water phase tends to reduce the volatility of polar compounds in much the same way that oils bind nonpolar flavour compounds (Forss, 1969). Buttery et al. (1971,1973) found that gas-oil partition coefficients of aliphatic aldehydes and ketones decreased with increasing chain length of the flavour molecule. More recently, other authors (Gijs etal, 2000; HaahretaL, 2000) have reported a similar relationship. The effect of chain length can be explained by the

172

FOOD FLAVOUR TECHNOLOGY Table 6.1 Gas/liquid partition coefficients ( K x 1000) of 18 flavour compounds in sunflower oil, water and a 3:2 mixture thereof Oil

Oil/water mix

Water

3.8 1.3 0.6 0.9 0.5 0.3

1.0 0.8 0.6 0.8 0.6 0.3

0.6 0.8 1.2 1.7 2.4 5.6

4.8 4.9 0.5 0.3 0.4

3.9 2.6 0.6 0.4 0.4

4.3 1.9 15.6 21.8 25.5

0.6 0.3 0.2

0.9 0.4 0.3

23.6 35.8 44.1

5.3 2.0 0.5 1.1

5.8 2.7 0.7 1.3

13.0 21.8 22.8 25.0

Alcohols 1 -Propanol 1 -Butanol 3-Methyl-l-butanol 2-Pentanol 1-Hexanol 2-Nonanol Ketones 2-Butanone 2,3-Butanedione 2-Heptanone 2-Octanone 2-Decanone Aldehydes Hexanal Heptanal Octanal Esters Ethyl acetate Propyl acetate Butyl acetate Ethyl butyrate

lipophilicity of the flavour compounds, which is an important factor with respect to the affinity of aldehydes, ketones, esters, thioesters, sulfides and disulfides for the lipid phase (Piraprez et al., 1998; Gijs et al., 2000). Fat concentration (Schirle-Keller et al., 1994) and composition (Druaux et al., 1998), pH (van Ruth et al., 1999) and temperature (Hall and Andersson, 1983) determine the extent of interactions between lipids and small molecules. The occurrence of compound-liquid interactions can also be expressed as activity coefficients y'. Interactions cause the value of y' to differ from 1. Compounds like 2,5-dimethylpyrazine in oil have y' values that are lower than 1. This is the result of the size of the flavour molecule, which differs significantly from that of the solvent molecules and results in repulsive forces. For other compounds in oil, y' values larger than 1 indicate attractive forces between flavour compounds and oil (Druaux et al., 1998). 6.3.4

Carbohydrate—flavour interactions

The retention of flavour compounds in systems rich in carbohydrates is more complex than the retention caused by lipids. Simple sugars (e.g. glucose, maltose)

DELIVERY OF FLAVOURS FROM FOOD MATRICES

1 73

produce an increase in vapour pressure for a number of components at low concentrations and a marked decrease for others (Buttery et al, 1971; Nawar, 1971). However, higher concentrations of simple sugars generally result in increased gas/liquid partition coefficients (Nahon et al., 2000; Hansson et al., 2001). A sort of 'salting-out' effect is likely to be the reason for this phenomenon, whereby the sugar interacts with water, increasing the concentration of flavour compounds in the remaining volume of free water (Voilley et al., 1977). This hypothesis was confirmed by Kieckbusch and King (1979), who calculated partition coefficients of some acetates in sucrose solutions on a free-water basis. Although initially a sharp increase in partition coefficients was found with increasing sucrose concentrations, the corrected partition coefficients remained nearly constant. Whereas some studies have used just a few, similar flavour molecules to investigate the effect of sugars on partition, Friel et al. (2000) used 40 different compounds and sugar concentrations up to 60%. The partition behaviour was expressed as a Quantitative Structure-Property Relationship (QSPR) model and the factors describing behaviour were log P and some topological (molecular shape) factors. Generally speaking, significant changes in headspace were seen only for sugar concentrations above 20%. Binding of flavour compounds to simple sugars is not likely as it can occur only through loose hydrogen bonds, in which case the flavour compounds have to compete with the water molecules. Polysaccharides, such as dextrins and gums, are known to interact with flavour compounds, and are used to stabilise flavours in food preparations (Versic, 1988). Dextrins can reduce the activity coefficients of flavour compounds in water and, accordingly, gas/liquid partition coefficients (Lebert and Richon, 1984). The binding is of hydrogen bond type (Solms etal., 1973), which results in competition of flavour compounds for the binding sites (Goubet et al., 2000). Among enzyme-modified starch derivatives, cyclodextrins are known to entrap flavour compounds of specific geometry and polarity (Szente and Szejtli, 1988) and are used for flavour encapsulation (Hedges et al., 1995). Gums, such as xanthan and guar gum, are generally used as thickeners and also exhibit interactions with flavour compounds. The type of compound affects the extent of binding. As competition between flavour compounds with respect to binding to these gums has been observed, the binding mechanism is likely to be of a more general hydrogen bond nature (Roberts et al., 1996; Yven et al., 1998). The interactions of flavour substances with starch are of special importance, since starch is one of the most commonly found components in food systems. Interactions involve the formation of so-called starch inclusion complexes. Inclusion compounds are not the result of chemical reactions but have been defined as addition compounds in which one entity fits into and is surrounded by the lattice of the other. Starch combines with a variety of substances to form inclusion compounds that are insoluble at room temperature. The inclusion compounds can be formed by the addition of particular substances to

174

FOOD FLAVOUR TECHNOLOGY

a molecularly dispersed solution of starch. Usually the complex is formed from a hot solution by slow cooling in presence of an excess of guest molecules. The formation of a helical arrangement of amylose molecules has been recognised to be responsible for inclusion complex formation. It is also conceivable that single helices are induced; in this case the flavour compound is located in the free space between the helices (Osman-Ismail and Solms, 1973; Escher el al.. 2000). Alcohols, aldehydes, ketones, terpenes and fatty acids have been reported to form inclusion complexes with starch (Osman-Ismail and Solms, 1973; Solms etal., 1973; Niissli, 1998). Starch affects flavour retention not only at molecular level by the complexation of flavour compounds with amylose and amylopectin; it also has an effect at supramolecular level through crystallisation of inclusion complexes, and at colloidal level through formation of dispersions in which aggregation, phase separation and network formation of starch and amylose complexes occur (Escher et ai, 2000). 6.3.5 Protein—flavour interactions Two types of interactions can occur between flavour compounds and proteins: (a) reversible physical adsorption via noncovalent interaction and (b) chemical reaction via covalent linkages. In the first case, the heat released by the reaction is less than 20 kJ/mol. In the second case, the heat released is at least 40 kJ/mol and includes formation of salts, amides and esters formation, and aldehyde condensation with NH2 and SH groups. Flavour compounds, especially aldehydes, can react either with free amino acids or with free amino groups of proteins and reversibly form Schiff bases. However, the process by which flavour compounds bind to proteins through covalent linkages is irreversible, as is seen in the case of interaction between formaldehyde and proteins. Formaldehyde reacts not only with primary amino groups in proteins but also with sulfhydryl groups. Flavour compounds bind to protein only when binding sites are available; that is, if the sites are not engaged in protein-protein or other interactions. Loops projecting into the aqueous phase are especially favourable sites for interaction with flavour compounds (Kim and Min, 1988). The reversible and noncovalent binding of a flavour compound obeys the Scatchard equation: —~±- = K(n - Vbound)

at equilibrium

(6.7)

where Vbound is the number of moles of flavour compound bound per mole of protein, V is the molar concentration of the free volatile compound, K is the association constant and n is the total number of binding sites per mole of protein. As binding proceeds, the protein can undergo conformational changes and more binding sites can become available. Nonpolar flavour compounds can diffuse into the hydrophobic core of the protein, replace intra- or intermolecular proteinprotein hydrophobic interactions and result in a change in the protein solubility.

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175

The amount of flavour adsorbed to proteins increases with the hydrophobicity of the proteins. The amount of irreversibly bound acetone and ethanol, however, increases with the polarity of the proteins (Kim and Min, 1988). As was found for nonspecific binding to carbohydrates, some flavour compounds exhibit competition for binding into the hydrophobic pocket of proteins (Muresan and Leguijt, 1998; Jouenne and Crouzet, 2000). Infrared spectroscopy and fluorescence quenching have indicated that flavour compounds seem to have different binding sites (Dufour and Haertle, 1990; Guichard and Langourieux, 2000). Lactoglobulin, albumin and soy proteins are the most intensively studied proteins with respect to flavour-protein interactions. 6.4

Kinetic aspects of flavour delivery

Flavour release is determined by thermodynamic and kinetic factors; this is illustrated by the differences in flavour release under static and dynamic conditions in Figure 6.1. The factors influencing the equilibrium concentrations have been discussed in preceding paragraphs. Kinetic factors determine the rate at which equilibrium is achieved. Once a food is ingested, it is rapidly coated by a thin film of saliva. It can therefore be assumed that flavour, on release from a solid food, must first pass through the saliva phase before partitioning into the headspace of the oral cavity. In the case of liquid and semi-solid foods, flavour is already in the liquid phase and therefore can be released into the headspace directly. Thus, passage of flavours from the food to the headspace is a three-phase arrangement involving the food, saliva and gas phases. It is unlikely that simple diffusion of flavour molecules in the bulk phases of the food or saliva can determine the rates of release in the mouth, as mastication disturbs diffusion gradients and generates fresh interfaces (Harrison, 2000). 6.4.1 Principles of interfacial mass transfer Under the nonequilibrium conditions that exist during eating, the driving force for transfer of flavour compounds across the interface is the difference in flavour concentration between product and gas phase. The rate of the unidirectional diffusion from the product to the gas phase is determined by the concentration gradients as well as the mass transfer coefficients of the flavour compounds in each of the phases (Pick's law). The rate of mass transfer in product (denoted by subscript p) and gas phase (subscript g) can be described as

di

- = £p[C'Fnt - Cpl

(6.8)

£ — ^gL^g T, \r _ ^a ^' n t lI

r'A Q^ \v.y)

176

FOOD FLAVOUR TECHNOLOGY

(a)

CO

IDU

£ 140 -

8 120 £

100 -

—

2£ 860°-

| 40 -

H

20 -

_J Fl

1

••

mam

i^—

A

(b) Figure 6.1 Flavour release under (a) equilibrium and (b) dynamic conditions from water and oil. D water: • oil.

where M is the total mass of flavour compound diffusing in the two phases and kf and kg are the mass transfer coefficients. Cg Cp and Cmt are the concentrations of the flavour compound in the product phase, the gas phase and at the interface respectively. Figure 6.2 illustrates the concentrations at the interface and in the gas and product phase. Flavour compound diffusion is based on two mechanisms: molecular and eddy diffusion. Molecular or static diffusion is the random movement of the molecules in the stagnant fluid. Typical molecular diffusivities are 10~5 m2/s and 10~9 m2/s in gas and liquid-aqueous phases respectively. The rate of molecular diffusion varies only slightly between flavour compounds. The

177

DFXIVERY OF FLAVOURS FROM FOOD MATRICES

Air flow 0

0

° Gas phase

Product phase

V

Figure 6.2 Flavour concentrations in gas and product phase under nonequilibrium conditions (redrawn from de Roos and Graf, 1995) where Cg and Cp are the concentrations in the gas and product phase, C"11 is the concentration at the interface and Vg and Vp are the gas and product phase volumes respectively.

second mechanism is eddy or convective diffusion, which transports element or eddies of the fluid from one location to another, carrying with them the dissolved flavour compounds. The rate of eddy diffusion is usually much higher than the rate of molecular diffusion and is independent of flavour type (de Roos, 2000). In general, it is assumed that diffusion of flavour compounds in the gas phase is extremely rapid and as a result the concentration gradient in the gas phase is neglected (Harrison et al., 1997; Harrison and Hills, 1997a). Consequently, the concentration of the flavour compound at the product side of the interface determines the concentration in the gas phase (C' = C g /AT'), which allows reformulation of the previous equations as dMp/dt =

(6.10)

The concentration gradient depends on the depletion of the flavour compound at the interface. Depletion is favoured by a high gas pressure and a low mass transfer coefficient of the flavour compound in the product phase. If compounds are completely depleted at the interface (C' ~ 0), the release of these kinetically controlled conditions is similar for all flavour compounds as kp varies only slightly (molecular diffusion) or not at all with the type of flavour compound (eddy diffusion) (de Roos, 2000). Three mathematical models, which differ in the mechanisms of mass transport, have been derived for predicting flavour release under dynamic conditions. (a) Stagnant-film theory. The stagnant-film model assumes that the boundary layers at the interface are stagnant and that mass is transported through these layers as a result of molecular diffusion. The mass transport coefficient k varies with the first power of the diffusion coefficient D and the reciprocal of the effective thickness of the stagnant layer (Hills and Harrison, 1995).

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FOOD FLAVOUR TECHNOLOGY

(b) Penetration theory. The penetration theory takes into account that the boundary layers are often not completely stagnant and that there is also mass transport by eddy diffusion. It is assumed that a volume element of liquid from the bulk comes into contact with the interface layers, and is exposed to the second phase for a definite interval of time. During this time, equilibrium is attained by surface layers through a process of unsteady-state molecular diffusion of flavour into the gas phase, before the volume element is remixed with the bulk liquid. In the penetration model k varies with the square root of the diffusion coefficient (Harrison et al., 1997). (c) Nonequilibrium partition model. The nonequilibrium partition model assumes that mass transfer takes place only by eddy diffusion. The independence of the diffusion constant allows a multiple extraction model. It is assumed that flavour compounds are extracted from the product with infinitesimal volumes of gas. During successive extraction, full equilibrium is achieved only at the gasproduct interface in the infinitesimal volumes of product and gas phase. After each extraction the initial flavour concentrations at the surface of the product are restored by diffusion and turbulence before the next extraction takes place (McNulty and Karel, 1973; de Roos and Graf, 1995). 6.4.2 Liquid food products Release rates of flavour compounds from liquid foods, both directly after consumption and after swallowing will depend on how the flavour compounds interact with other components of the food during dilution. Firstly, the concentrations of free flavour compounds are affected by the thermodynamic interactions described in Section 6.3. The majority of flavour compounds are hydrophobic and therefore preferentially partition into the lipid phase and not into the aqueous or gas phases. Dilution with saliva of a lipid-containing liquid food will therefore shift the aroma partitioning and change the release kinetics (Harrison, 1998; van Ruth et al., 2001). Diffusion of flavour compounds between lipid and aqueous phases is extremely rapid in liquid foods (Harrison et al., 1997). The mass transfer coefficient is influenced by the viscosity of the phases, and thus by the lipid fraction and droplet size. Experimental studies confirm the importance of these factors for flavour release (Charles et al., 2000). Flavour release from lipidcontaining liquid foods, such as emulsions, has been described by the penetration theory (Harrison et al, 1997; Harrison and Hills, 1997a). Furthermore, macromolecules, such as proteins and polysaccharides can bind both reversibly and irreversibly to flavour compounds, as described in section 6.3, thus reducing the free flavour available for release. Diluting the macromolecular concentration with saliva will also shift the binding equilibrium and change the flavour release kinetics. In addition, the presence of these food components will affect the overall viscosity of the saliva, further

DELIVERY OF FLAVOURS FROM FOOD MATRICES

1 79

influencing the rates of release. Dissociation between the bound and unbound states of a flavour-macromolecule complex is extremely fast and therefore will not affect the rate of flavour release. At first sight, this may seem surprising, but it is a consequence of the extremely small amount of flavour released in the headspace compared with the amount of free flavour retained in the aqueous phase. Reversible binding of flavour compound and macromolecule can be described with first-order chemical kinetics. The penetration theory allows modelling of flavour release across the gas-liquid interface of solutions containing macromolecules (Harrison and Hills, 1997b). Interactions between methyl ketones and (3-lactoglobulin (Andriot et al, 2000), and flavour compounds and liquid gelatin resulted in effects on flavour release that are in agreement with the penetration theory (Bakker et al, 1998). Apart from the direct effect of viscosity on flavour release kinetics, the viscosity of saliva will also influence the residual thickness coating the inside of the oral cavity long after the majority of the food has been swallowed, thus potentially influencing the aftertaste. Breath-by-breath mass spectrometry has shown that some flavour compounds persist in the nose-space long after the food has been swallowed (Taylor, 1996). Viscosity is an important parameter of flavour release in liquid food systems. It determines the diffusion coefficient and, as a consequence, the mass transfer coefficient. Food components determining the viscosity of the liquid food-saliva mixture have an effect on flavour release kinetics. A practical example is liquids with high sucrose concentrations, flavour release from which shows penetration model behaviour (Nahon et al., 2000). 6.4.3 Semi-solid food products For semi-solid foods like gels, which possess melting points below the mouth temperature, the driving force for flavour release is the rate at which heat can diffuse into the gel matrix and initiate melting. For harder gels with melting points above mouth temperature, the diffusion of sucrose from the surface of the gel into the adjacent saliva phase is the rate-limiting step for flavour release, because it lowers the melting temperature of the surface. In vitro release from gelatin gels containing sucrose shows good agreement with the stagnant layer model. The model can also be applied to other foods when flavour release is accompanied by a melting transition. Ice-creams and chocolates are expected to behave like soft gels with low sucrose concentrations, the rate-limiting step of which is thermal diffusion (Harrison and Hills, 1996). 6.4.4 Solid food products For simple solid foods, where dissolution of the sugar matrix determines flavour release, e.g. boiled sweets, the driving force for release across the interface

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FOOD FLAVOUR TECHNOLOGY

is the sucrose gradient between the food product and saliva. Flavour release from this matrix shows stagnant layer behaviour. As the matrix dissolves, all flavours are simultaneously released into the surrounding saliva, from where they partition into the headspace of the oral cavity. The mass transfer coefficient of this type of food is determined by oral manipulation. Higher rates of manipulation will reduce the thickness of the stagnant layer, will increase mass transfer rates and, therefore, will increase flavour release (Hills and Harrison. 1995). Flavour release and delivery from nondisintegrating solid foods is more complex. Transfer of flavour compounds to the gaseous phase is a three-phase problem for foods of this type, with saliva as the intermediate medium. In principle, the relationship for mass transfer from saliva to the gas phase is similar to that for liquid foods (Harrison et aL, 1998; de Roos, 2000), described earlier in this section. The rate-limiting step for release from solid foods is the transport of flavour compounds across the food-saliva interface (Harrison et al, 1998; de Roos, 2000; Lian, 2000). Both the particle and the surrounding fluid affect the transfer of the compounds (Lian, 2000). Flavour compounds are transferred from the food into the saliva at different rates determined by the product-saliva partition coefficients. The transfer can be described by the stagnant-layer theory. The physical processes occurring during eating, such as saliva flow, mastication and swallowing, are important factors determining the particle size distribution and thus food product interface. Initial rates of flavour release from solid foods are less sensitive to chewing frequency and saliva flow rate, but are extremely dependent on the fracture mechanics of the food and the mass transfer coefficient. This implies that the structure and composition of a food determine the initial rate of flavour release. With longer eating times, eating behaviour begins to play a role in flavour release rates. Increasing chewing frequency and/or the efficiency of particle selection increases the rate at which new surfaces are created and therefore the rate of flavour release into the saliva phase. Once the food has been swallowed, the flavour-enriched saliva will continue to release flavour compounds into the oral cavity (Harrison et al.. 1998). Flavour delivery has been described in this chapter for liquid food, semi-solid food, very simple solid food and nondisintegrating solid food. For many other foods, however, mechanisms such as dissolution, melting and hydration play a role. Breaking of the food structure might increase viscosity. Furthermore, released flavour compounds may bind to macromolecules and partition into lipids and therefore reduce the free flavour present in the saliva. In some foods, the microenvironment may determine flavour release and delivery rather than the food's gross composition. Those factors further complicate the elucidation of flavour delivery from complex solid foods (Harrison et al.. 1998) and are a challenge for future research.

DELIVERY OF FLAVOURS FROM FOOD MATRICES

6.5

181

Conclusions

Flavour release and delivery are complex owing to the rapidly changing conditions during eating. Many factors affecting flavour delivery have been determined. However, only sporadic attempts have been made to study several factors simultaneously in order to determine the contribution of the individual factors to flavour release and delivery, and validate theoretical models, because of the large-scale experiments required. Nevertheless, with the development of rapid, sensitive methods for measuring flavour release in the mouth (Taylor and Linforth, 2000; Yeretzian et al., 2000), it is expected that more attempts will be made to relate food-flavour-mouth interactions to theoretical predictions of in vivo flavour release in the near future.

References Andriot, I., Harrison, M., Fournier, N. and Guichard, E. (2000) Interactions between methyl ketones and p-lactoglobulin: sensory analysis, headspace analysis, and mathematical modelling. J. Agric. Food Chem., 48 4246-4251. Bakker, J.. Langley, K.R., Elmore, J.S., Brown, W., Martin, A. and Salvador, D. (1995) Release of flavour from food matrices and effect on flavour perception, in Aroma: Perception, Formation, Evaluation (eds M. Rothe and H.-P. Kruse), Deutsche Institut fur Ernahrungsforschung, Potsdam-Rehbriicke, pp 191-207. Bakker, J., Brown, W., Hills, B., Boudard, N., Wilson, C. and Harrison, M. (1996) Effect of the food matrix on flavour release and perception, in Flavour Science, Recent Developments (eds A.J. Taylor and D.S. Mottram), Royal Society of Chemistry, Cambridge, pp 369-374. Bakker, J., Boudaud, N. and Harrison, M. (1998) Dynamic release of diacetyl from liquid gelatin in the headspace. J. Agric. Food Chem., 46 2714-2720. Buttery, R.G., Bomben, J.L., Guadagni, D.G. and Ling, L.C. (1971) Some considerations of the volatilities of organic flavor compounds in foods. J. Agric. Food Chem., 19 1045-1048. Buttery, R.G., Guadagni, D.G. and Ling, L.C. (1973) Flavor compounds: volatilities in vegetable oil and oil-water mixtures. Estimation of odor thresholds. J. Agric. Food Chem., 21 198-201. Charles, M., Rosselin, V., Beck, L., Sauvageot, F. and Guichard, E. (2000) Flavor release from salad dressings: sensory and physicochemical approaches in relation with the structure. J. Agric. Food Chem., 48 1810-1816. de Roos, K.B. (2000) Physiochemical models of flavor release from foods, in Flavor Release (eds D.D. Roberts and A.J. Taylor), American Chemical Society, Washington, DC, pp 126-141. de Roos, K.B. and Graf, E. (1995) Nonequilibrium partition model for predicting flavor retention in microwave and convection heated foods. /. Agric. Food Chem., 43 2204-2211. de Roos, K.B. and Sarelse, J.A. (1996) Volatile acids and nitrogen compounds in prawn powder, in Flavour Science, Recent Developments (eds A.J. Taylor and D.S. Mottram), Royal Society of Chemistry. Cambridge, pp 13-18. Druaux, C., Le Thanh, M., Seuvre, A.-M. and Voilley, A. (1998) Application of headspace analysis to the study of aroma compounds-lipids interactions. J. Am. Oil Chem. Soc., 75 127-130. Dufour, E. and Haertle, T. (1990) Binding affinities of p-ionone and related flavor compounds to p-lactoglobulin: effects of chemical modifications. J. Agric. Food Chem., 38 1691-1695.

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Escher, F.E., Niissli, J. and Conde-Petit (2000) Interactions of flavor compounds with starch in food processing, in Flavor Release (eds D.D. Roberts and A.J. Taylor). American Chemical Society. Washington, DC, pp 230-245. Forss, D.A. (1969) Role of lipids in flavors. 7. Agric. Food Chem.. 17 681-685. Friel, E. N., Linforth, R. S. T, Taylor, A. J., (2000) An empirical model to predict the headspace concentration of volatile compounds above solutions containing sucrose. Food Chem.. 71 309-317. Gijs, L.. Piraprez, G., Perpete, P., Spinnler, E. and Collin. S. (2000) Retention of sulfur flavours by food matrix and determination of sensorial data independent of the medium composition. Food Client.. 69319-330. Goubet, I., Le Quere, Semon, E. and Seuvre. A. (2000) Competition between aroma compounds for the binding on p"-cyclodextrins: study on the nature of interactions, in Flavor Release (eds D.D. Roberts and A.J. Taylor), American Chemical Society, Washington. DC. pp 246-259. Guichard, E. and Langourieux, S. (2000) Interactions between {i-lactoglobulin and flavour compounds. Food Chem., 71 301-308. Haahr. A.-M., Bredie. W.L.P.. Stahnke, L.H., Jensen. B. and Refsgaard. H.H.F. (2000) Flavour release of aldehydes and diacetyl in oil/water systems. Food Chem.. 71 355-362. Hall. G. and Andersson. J. (1983) Volatile fat oxidation products II. Influence of temperature on volatility of saturated, mono- and di-unsaturated aldehydes in liquid media. Lehensm.- Wiss. u-TechnoI.. 16 326-366. Hansson. A.. Andersson, J. and Leufven. A. (2001) The effect of sugars and pectin on flavour release from a soft drink-related model system. Food Chem.. 72 363-368. Harrison, M. (1998) Effect of breathing and saliva flow on flavor release from liquid foods. J. Auric. Food Chem., 46 2727-2735. Harrison. M. (2000) Mathematical models of release and transport of flavours from foods in the mouth to the olfactory epithelium, in Flavor Release (eds D.D. Roberts and A.J. Taylor). American Chemical Society, Washington, DC, pp 179-191. Harrison. M. and Hills. B.P. (1996) A mathematical model to describe flavour release from gelatine gels. Int. J. FoodSci. Technol.. 31 167-176. Harrison, M. and Hills, B.P. (1997a) Effects of air flow-rate on flavour release from liquid emulsions in the mouth. Int. J. FoodSci. Technol.. 32 1-9. Harrison. M. and Hills, B.P. (1997b) Mathematical model of flavor release from liquids containing aroma-binding macromolecules. 7. Agric. Food Chem.. 45 1883-1890. Harrison. M., Hills, B.P. Bakker, J. and Clothier, T. (1997) Mathematical models of flavor release from liquid emulsions. / Food Sci.. 62 653-658, 664. Harrison. M., Campbell, S. and Hills, B.P. (1998) Computer simulation of flavor release from solid foods in the mouth. J. Agric. Food Chem.. 46 2736-2743. Hedges. A.R., Shieh. W.J. and Sikorski. C.T. (1995) Use of cyclodextrins for encapsulation in the use and treatment of food-products, in Encapsulation and controlled release of food ingredients. S. J. Risen, G. A. Reineccius. Washington. DC. American Chemical Society. 590 60-71. Hills. B.P. and Harrison, M. (1995) Two-film theory of flavour release from solids. Int. J. Food Sci. Technol.. 30 425^36. Jouenne. E. and Crouzet. J. (2000) Determination of apparent binding constants for aroma compounds with (3-lactoglobulin by dynamic coupled column liquid chromatography. 7. Agric. Food Chem.. 48 5396-5400. Kieckbusch. T.G. and King. C.J. (1979) Partition coefficients for acetates in food systems. 7. Agric. Food Chem.. 27 504-507. Kim, H. and Min, D.B. (1988) Interaction of flavor compounds with protein, in Flavor Chemistry of Lipid Foods (eds D.B. Min andT.H. Smouse). American Chemical Society. Washington. DC. pp 404-420. King, J. (1983) Physical and chemical properties governing volatilisation of flavor and aroma components, in Physical Properties of Food (eds M. Peleg and E.B. Bagley). AVI. Westport. CT. pp 399^21.

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Kinsella, J.E. (1988) Flavour perception and binding to food components, in Flavor Chemistry ofLipid Foods (eds D.B. Min and T.H. Smouse), American Chemical Society, Washington, DC, pp 376-403. Kolb, B., Welter, C. and Bichler, C. (1992) Determination of partition coefficients by automatic equilibrium headspace gas chromatography by vapor phase calibration. Chromatogmphia. 34 235-240. Lebert, A. and Richon, D. (1984) Infinite dilution activity coefficients of n-alcohols as a function of dextrin concentration in water-dextrin systems. J. Agric. Food Chem., 32 1156-1161. Le Thanh, ML, Thibeaudeau, P., Thibaut, M.A. and Voilley, A. (1992) Interactions between volatile and non-volatile compounds in the presence of water. Food Chem., 43 129-135. Lian, G. (2000) Modeling flavor release from oil-containing gel particles, in Flavor Release (eds D.D. Roberts and A.J. Taylor), American Chemical Society, Washington, DC, pp 201-21 1. Linforth, R.S.T., Friel, E.N. and Taylor, AJ. (2000) Modelling aroma release from food using physicochemical parameters, in Flavor Release (eds D.D. Roberts and A.J. Taylor), American Chemical Society, Washington, DC, pp 166-178. Maier, H.G. (1975) Binding of volatile aroma substances to nutrients and foodstuffs, in Proceedings of the International Symposium on Aroma Research (eds H. Maarse and P.J. Groenen), Pudoc, Wageningen, pp. 143-157. McNulty, P.B. and Karel, M. (1973) Factors affecting flavour release and uptake in O/W emulsions. 1. Release and uptake models. J. Food Technoi., 8 309-318. Muresan, S. and Leguijt, T. (1998) Fluorimetric analysis of interactions between p-lactoglobulin and small ligands, in COST Action 96. Proceedings of the Meeting in Goteborg (ed. A. Zacharoff), European Commission, Luxembourg, pp 45-52. Nahon, D.F., Harrison, M. and Roozen, J.P. (2000) Modelling flavor release from aqueous sucrose solutions, using mass transfer and partition coefficients../. Agric. Food Chem., 48 1278-1284. Nawar. W.W. (1971) Some variables affecting composition of headspace aroma. J. Agric. Food Chem., 19 1057-1059. Niissli, J. (1998) Complexation behavior of amylose with small ligands in aqueous starch systems. PhD thesis No. 12518, Swiss Federal Institute of Technology (ETH), Zurich. Osman-Ismail, F. and Solms, J. (1973) The formation of inclusion compounds of starches with flavor substances. Lebensm.-Wiss. u-Technol., 6 147-150. Piggott, J.R., Gonzalez Vinas, M.A., Conner, J.S., Withers, S.J. and Paterson, A. (1996) Effect of chill filtration on whisky composition and headspace, in Flavour Science, Recent Developments (eds A.J. Taylor and D.S. Mottram), Royal Society of Chemistry, Cambridge, pp 319-324. Piraprez, G., Herent, M.-F. and Collin, S. (1998) Determination of the lipophilicity of aroma compounds by RP-HPLC. Flavour Fragrance J., 13 400-408. Plug, H. and Haring, P. (1993) The influence of flavour-ingredient interactions on flavour perception. Food Quality Preference, 5 95-102. Reinecdus, G. (1993) Biases in analytical flavor profiles introduced by isolation method, in Flavor Measurement (eds C.T. Ho and C.H. Manley), Marcel Dekker, New York, pp 61-76. Roberts, D.D. and Acree, T.E. (1996) Retronasal flavor release in oil and water model systems with an evaluation of volatility predictors, in Flavor-Food Interactions (eds RJ. McGorrin and J.V. Leland). American Chemical Society, Washington, DC, pp 179-187. Roberts, D.D., Elmore, J.S., Langley, K.R. and Bakker, J. (1996) Effect of sucrose, guar gum, and carboxymethylcellulose on the release of volatile flavor compounds under dynamic conditions. J. Agric. Food Chem., 44 1321-1326. Schirle-Keller, J.-P, Reineccius, G.A. and Hatchwell, L.C. (1994) Flavor interaction with fat replacers: effect of oil level. J. Food Sci., 59 813-815, 875. Solms, J., Osman-Ismail, F. and Beyeler, M. (1973) The interaction of volatiles with food components. Can. hist. Food Sci. Technoi. J., 6 A10-16. Szente, L. and Szejtli, J. (1988) Stabilisation of flavours by cyclodextrins, in Flavor Encapsulation (eds S.J. Risch and G.A. Reineccius), American Chemical Society, Washington, DC, pp 148-157.

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Taylor, A.J. (1996) Volatile flavor release from foods during eating. Crit. Rev. Food Science Nutri.. 36 765-784. Taylor, A.J. (1998) Physical chemistry of flavour. Int. J. Food Sci. Techno!.. 33 53-62. Taylor, A.J. and Linforth, R.S.T. (2000) Techniques for measuring volatile release in vivo during consumption of food, in Flavor Release (eds D.D. Roberts and A.J. Taylor). American Chemical Society, Washington, DC, pp 8-21. van Ruth, S.M., Roozen, J.P., Posthumus, M.A. and Jansen. F.J.H.M. (1999) Volatile composition of sunflower oil-in-water emulsions during initial lipid oxidation: influence of pH. J. Agric. Food Chem., 47 4365-4369. van Ruth, S.M., O'Connor, C.H. and Delahunty, C.M. (2000) Relationships between temporal release of aroma compounds in a model mouth system and their physico-chemical characteristics. Food Chem., 71 393-399. van Ruth, S.M., Grossmann, I., Geary, M. and Delahunty. C.M. (2001) Interactions between artificial saliva and twenty aroma compounds in water and oil model systems. J. Agric. Food Chem.. 49(5) 2409-2413. Versic, R.J. (1988) Flavor encapsulation: an overview, in Flavor Encapsulation (eds S.J. Risen and G.A. Reineccius), American Chemical Society, Washington. DC. pp 1-11. Voilley. A., Lamer. C., Dubois. P. and Feuillat. M. (1990) Influence of macromolecules and treatments on the behavior of aroma compounds in a model wine. J. Agric. Food Chem.. 38 248-251. Voilley, A.. Simatos, D. and Loncin, M. (1977) Gas phase concentrations of volatile* in equilibrium with a liquid aqueous phase. Lebensm.-Wisse. u-Technol.. 10 45^49. Yeretzian, C., Jordan, A.. Brevards. H. and Lindinger. W. (2000) Time-resolved headspace analysis by proton-transfer-reaction mass-spectrometry, in Flavor Release (eds D.D. Roberts and A.J. Taylor). American Chemical Society. Washington. DC. pp 58-72. Yven, C., Guichard. E., Giboreau, A. and Roberts. D.D. (1998) Assessment of interactions between hydrocolloids and flavor compounds by sensory, headspace. and binding methodologies. J. Agric. Food Chem.. 46 1510-1514.

7

Modelling flavour release Rob Linforth

7.1

Introduction

Why model flavour release? The main driver behind model production is the generation of an equation or series of equations that describes the behaviour of a system. This can then be used to predict how a given system should function or how the performance of the system will be affected by any changes made to it. In terms of flavour release (or delivery), the ultimate objective is to understand and estimate the intensity and timing of aroma delivery to the consumer. There are two main approaches to the construction of models. The first is essentially a theoretical approach, using the principles of physics and chemistry to describe how a system is likely to behave. Equations are constructed that contain parameters, describing the attributes of the system (the flavour molecules, the matrix and the phases that surround them) and the way these components interact to influence flavour release. Because this is a purely theoretical approach, these models are not initially dependent on experimental data being available; however, experimental data are necessary for model validation. This is one of the major advantages of this approach, since it allows the production of models for systems even if analytical methods are too insensitive to study them. The second approach is data driven. Data (such as headspace volatiles concentrations) are collected for a range of flavour compounds or a range of food matrices. A model is then constructed with sufficient components to describe the variation in the data. These models may contain some of the same parameters used in the theoretical approach described above, or they may contain parameters that just numericise the differences that occur in the ranges of the system studied (e.g. differences in molecular weight). The principal difference between the two approaches is the amount of data required. A theoretical model may only require 5-10 experimental values for it to be tested and validated, whereas empirical models require much larger data sets to increase their accuracy. However, there are limits to the size of data sets that should be generated, because a model that included every compound of interest would have nothing left for it to predict! Models can therefore exhibit many different forms and cover different aspects of a particular system. Key questions that can be asked of any model are; • • •

What information is needed to make a prediction from the model? Which are the most important factors in the model? How reliable are the predictions obtained?

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The last point is perhaps the most important question that can be asked about any model—namely how well does it work? Model validation in its simplest form should be a relatively easy task (assuming data can actually be obtained). Use the model to make predictions about how a system should perform; obtain data on the behaviour of the system under the conditions used for the prediction; and then compare the data obtained experimentally with the predicted values. The quality of the correlation (determined statistically) between the two sets of values is a good indicator of the validity of the model. The system modelled can be considered as belonging to one of three basic types. Those of the first type are the simplest models that describe the equilibrium partitioning of volatile compounds from a matrix into the gas phase: typically these have been developed for solutions containing volatile and nonvolatile solutes or a second phase such as lipid. Similar studies with low water content matrices are less common owing to 'complicating' factors such as the heterogeneity of the system and the difficulty of defining factors such as surface area in such systems. The second type of model is more complex and attempts to model dynamic partitioning where the gas phase above a matrix is disturbed or diluted to simulate the processes that happen in real life. These models now include a temporal dimension. In addition, the exact conditions used (flow rates, volumes, etc.) can result in different degrees of volatiles depletion in the headspace and mixing within the matrix, complicating the comparison of different systems. In contrast, measurements of volatiles partitioning in static systems at equilibrium give the same 'release', providing temperature and pressure are constant. Models of the third type are those that attempt to describe flavour release during eating, where a number of factors (dilution with saliva, temperature changes, bolus breakdown or dissolution, phase inversion, swallowing and breathing) can act in concert, making these models the most complex (the temporal dimension is almost unavoidable). They have also been difficult to validate, principally because of the lack of experimental data available for comparison with their predictions.

7.2 Equilibrium partition models 7.2.1 The air—water partition coefficient The static partition (at equilibrium) has the potential to describe the maximum volatile concentration that may occur above a sample, under defined conditions of temperature and pressure. One of the simplest static partition situations that can be modelled is the equilibrium partitioning of a volatile compound between water and air in a closed vessel. The concentration of a volatile in the air (C a ) above a solution is directly proportional to the concentration in the liquid phase, in this case water (Cw), when in ideal solution. This can be expressed as a single

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187

value, the air-water partition coefficient (A^ uw ): Kaw=<^

(7.1)

C-\v

This ratio does not, however, have the power of prediction, it simply describes the state of equilibrium. However, given the air-water partition coefficient, it is possible to estimate the equilibrium concentration of the volatile in the headspace for any concentration of the volatile in (ideal) solution. Estimates of Kaw can be obtained from values such as the activity coefficient of a compound and its vapour pressure. These values, however, are not known for all compounds. Measuring them experimentally is not an ideal solution; it would be just as easy to measure Kaw itself. The activity coefficient for a compound can be estimated using methods such as ASOG (analytical solution of groups) or UNIFAC (universal functional group activity coefficients). These methods predict the physical and topological properties of compounds using algorithms that calculate the sum of each functional group's contributions (Reid et al, 1987). Since the values for the group contributions have themselves been derived by reduction of experimental data, they too are semi-empirical values. The vapour pressure of a compound can be estimated from tables listing critical properties of compounds, or these too can be estimated using group contribution methods. These methods for estimating K&w can yield estimates close to those obtained experimentally (Marin et al., 1999). It is important to note that there can be substantial differences in the values obtained by different methods of parameter estimation (Sorrentino et al., 1986), which would in turn influence the accuracy of the Kaw estimate. 7.2.2 Estimation of Kaw using quantitative structure—property relationships In addition to the methods described above, Kaw estimates can be obtained using Quantitative Structure-Property Relationships (QSPR) as outlined in Figure 7.1. The QSPR method is effectively an extension and development of the group contribution approach. Group counting is based on the principle that certain aspects of the compound (in this case the number and type of subunits present) influence its behaviour, and a value is ascribed to each one empirically. Such methods are simple and can be performed manually without too much difficulty. With the increasing availability of computer technology, we have the potential to rapidly perform large numbers of complex calculations so as to determine a wide range of parameters to describe aroma compounds (this would be virtually impossible manually). The parameters can be calculated for any compound; first the compound is drawn using the software package, and then the computer calculates the desired parameters on the basis of this structure.

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Measure Property '—K. of volatile /> compounds 1 I/

C •I

Calculate Parameters to ] describe compound

Select (statistically) Parameters that correlate with Properties

A Build model by Model Equation regression analysis Property = <' "Xi-i of Parameters and a*Para 1 + b*Para 2 + c Properties

-J

"=-

Model Failed

^nNC-r^

Predict Properties of |_jx further compounds using Model Equation P"^

>

c>

Correlate with |_ actual Properties of compounds |~

Is correlation good enough? ^JYESL^

Model Validated

Figure 7.1 QSPR modelling of the behaviour of volatiles. The parameters would be molecular descriptors for the compounds; a and b would be regression coefficients for individual parameters (Para 1 and Para 2 respectively), with intercept c.

Calculations can be carried out on the structure under a range of conditions, e.g. as a pure compound or in aqueous solution, substantially increasing the number of individual parameters that can be estimated. Parameters can include simple values, such as group counts and the molecular weight, or more complex terms (describing the whole molecule) such as the solvent-accessible surface area and molecular energies. Once a wide range of estimates for parameters that describe each compound have been obtained, these can then be statistically compared against a data set. such as tables of known values for Kaw. Once the key descriptors of compounds have been found that describe their behaviour, these can be formulated into an equation. This equation can then be used to estimate the Kaw of further compounds based on the values for the key descriptors for those molecules. The quality of the model is determined statistically by the correlation coefficient ( R 2 ) and the cross-validated correlation coefficient (R2y). R2 values approaching 1.0 indicate a high correlation between the values predicted by the model and those of the data set, and, if the value of R2V is close to that of R2. the model should have good predictive power.

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189

QSPR models have been developed to estimate the solubility of compounds in water and their vapour pressures as separate functions, which can be combined to estimate the air-water partition coefficient (Katritzky et al., 1998). QSPR models have also been produced to directly estimate the Kaw of volatile compounds. Katritzky and co-workers (1996) used a data set of 406 structurally diverse compounds to develop a model (equation 7.2) with a final R2 of 0.9407 and an R^ of 0.9386, indicative of good predictive power. Key terms in the model were HDCA(2), related to the hydrogen-bonding ability of the compound; O + 2*N, the number of oxygen and nitrogen atoms present; EHOMO — £LUMO, related to the dispersion energy of a polar solute in solution; PCWTE, which is the most negative partial charge-weighted topographical electronic index; and finally A^ngs. the number of rings present in the compound. All of these terms can be calculated using chemical modelling software and require no experimental data before further predictions can be made. The model for K&w shown in equation (7.2) was generated not with the food industry in mind but for other situations such as the fate of environmental pollutants. Consequently, the 'structurally diverse compounds' also included halogenated organic compounds and hydrocarbons, which are not of major importance to the food industry, except perhaps as contaminants. The opportunity certainly exists for the generation of QSPR models based on data sets of organoleptically significant compounds. logKaw = I/(42 x HOC A (2) + 0.71 x (O + 2*N) — 0.17 x (£HOMO — £LUMO) + 0.13 x PCWTE + 0.79 x A/rings + 2.82)

(7.2)

Therefore, just as the group counting approach estimates the effect of a —CH2OH group on a property, QSPR determines a coefficient expressing the influence of a parameter (which may be associated with part or all of the molecule) on the behaviour of a compound. This has the advantage that QSPR can readily take into account not only the presence but also the position of functional groups, and can incorporate the overall shape of the molecule. For both approaches a data set is required to determine the influence of a group or a QSPR parameter. Further models in addition to those for ATaw can effectively be obtained by either approach to describe how volatile compounds behave in food. 7.2.3 Effect oflipids on volatile partitioning Food systems are rarely simple solutions of volatile compounds in water, and other solutes or phases may influence partitioning substantially. One of the most significant influences on volatile behaviour is the concentration of lipid in the system. The partitioning effect of emulsions can be described using the

1 90

FOOD FLAVOUR TECHNOLOGY

equation of Buttery and co-workers ( 1 973), where the air-oil ( A^o) and air-water (^aw) partition coefficients, are combined with their respective oil and water volume fractions (F0 and Fw) to produce an overall air-emulsion ( Kae ) partition coefficient: Kae = -

(7.3)

This model has been found to work in some cases (Doyen et al., 2001) but not others (Voilley et al, 2000); this may be due to interactions with the emulsifier itself. Equation (7.3) was originally generated from studies of oil and water layers without any added emulsifiers. Consequently it does not include any terms associated with the potential chemical interaction with emulsifiers. or with their presence as a bulk component of the system. The behaviour of some compounds such as the organic acids will be poorly described by equation (7.3) because partition will depend on the degree of dissociation of the acid. Further models have been developed to describe the partitioning of these compounds in lipid-containing systems, including factors such as the pH of the system and the pKa of the acid (de Roos and Sarelse. 1996), shown in a simplified form (assuming equal volumes of gas and liquid phases) in equation (7.4). Based on this equation, a decrease of the pH of the system relative to the pA"a of the acid would increase pA"a/pH, increasing the significance of the term Fw/#aw and therefore decreasing ATae. This would reflect the association of the acid at pH values below its pKa, enhancing its potential to partition into the lipid. A"ae = -

(7-4)

The key parameters for both of these models (equations 7.3 and 7.4) are the air-oil and air-water partition coefficients. Kaw can be estimated for compounds using either thermodynamic parameters or the QSPR approach. We do not have the same options with ATao; therefore, unless a published value for A"ao exists, it is necessary to determine (i.e. measure) Kao before an estimate of Arae can be made. This is the major limitation of many models, in that experimentation has to be performed (or previously published values have to be obtained) before an estimate can be made. However, once the partition coefficients have been determined, it is possible to estimate Kae for any oil fraction. 7.2.4

QSPR estimation of the air—emulsion partition

coefficient

A QSPR model has been developed by Carey et al. (submitted for publication) to describe the concentration of volatile compounds in the headspace above low

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lipid concentration emulsion systems relative to that of water, expressed as the lipid effect: Lipid effect(%) =

Concentration of volatile in headspace above emulsion x 100 Concentration of volatile in headspace above water (7.5)

The key components of the model were (log P)2 (an estimate of the octanolwater partition coefficient, squared); log Sol (a QSPR estimate of solubility in water; Liang and Gallagher, 1997); a dipole vector term squared (DV2) and the oil fraction (F0). The model was based on 92 observations and had an R2 value of 0.83 and an R2V of 0.80. The model was tested with an external test set of 25 compounds (i.e. ones not used in the development of the original model), which showed a correlation coefficient of R2 = 0.82 between the observed and predicted values. The effect of (log P)2 and the oil fraction are shown in Figure 7.2 (for a constant value of log Sol and DV2). The contour lines on the plot link points that would be expected to show the same change in their 16.0

100

40

12.0

O. o> o

60 8.0

80

4.0

0.0

0.00

0.0005

0.001

0.0015

0.002

Oil fraction Figure 7.2 The effect of oil fraction and (log P)2 on the partitioning behaviour of compounds (for log Sol = -2.0, and DV2 = 3).

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headspace volatile concentration based on their (log P)2 and the oil fraction. In Figure 7.2 a compound with a (log P)2 of 6.1, in an emulsion with an oil fraction of 0.0005, would decrease in headspace concentration to the same extent (i.e. 20% decrease, Lipid effect = 80%) as a compound with a log P2 of 1.8 in an emulsion with an oil fraction of 0.0015. Therefore, using the contour plot in Figure 7.2 we can see how the volatile headspace concentration would be affected as changes in (log P)2 and F0 occurred. It is interesting to note that the contour lines in Figure 7.2 are in fact curved, owing to the use of quadratic terms in the QSPR function (7.6) and the interactive term between (log P)2 and oil fraction. This reflects the fact that as (log P) 2 increases there is a general decrease in the relative headspace concentration of volatiles up to a (log P)2 of around 10, after which the headspace concentration increases again (presumably as a result of steric effects restricting partitioning into the emulsion). An estimate of the change in volatile headspace concentration relative to that of water can be made for compounds simply by calculating their log P, log Sol and dipole vector (at a given oil fraction) and substituting these values into equation (7.6). Lipid effect = 107 - 6.3(log P) 2 - 3.2(logSol) + 0.28DV2 + 106F0 + 0.39(log P) 4 - [(2 x 105) x (log P) 2 x F0] + [(7.6x 105) x (logSol) x F0] -[(9.3 x 104) x D V 2 x F0] (7.6) The model does not estimate the absolute headspace concentration of aroma compounds as the lipid concentration increases; it specifically models the relative change in headspace concentration. However, from this and equation (7.3) it is possible to estimate the relative difference between Kao and tfaw; and since we can estimate Kaw either by the thermodynamic properties of a compound or using QSPR, this model also provides a means by which ATao itself can be estimated for a compound. It is important to remember that models such as the emulsion model of Carey are based on data collected using a specific range of experimental factors. In this case the oil fraction used was from 0.0 to 0.002 (0.0-0.2% lipid), and the quality of estimates for higher oil fractions would be less reliable. Equally with any QSPR model, it is important to know the range of compounds used in the development of the model. For example, it might be unwise to try to estimate the behaviour of acetaldehyde (molecular weight 44 Da) using a model based on compounds with molecular weights > 100 Da. 7.3 Dynamic systems Models for dynamic systems can take many forms owing to the potential variation in the systems themselves. Systems may be unstirred, in which case

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stagnant layers may form and the movement of volatiles is then dependent on processes such as diffusion. Alternatively, as agitation increases, factors such as eddy diffusion exert a more significant influence on volatiles release (see Chapter 6 for discussion of flavour release mechanisms). 7.3. 1 Modelling flavour release from a retronasal aroma simulator An empirical model of flavour release from systems containing oil or thickeners at different temperatures was developed by Roberts and Acree (1996). The data set they used was obtained using their Retronasal Aroma Simulator (RAS), which is a headspace sampling system designed to mimic eating conditions inmouth. In this instance, the sampling method results in a single measurement of volatiles release, and the system is dynamic by virtue of the sampling apparatus, through which a stream of air passes. The model was similar to that of Carey in that it focused not on absolute concentrations but on the relative differences between a range of matrices and water. The equation generated to describe the air-product partition coefficient K.dp, (equation 7.7) has three main components. The first relates to the partition between air and water for a compound and its affinity for lipids (the authors would have used the oil-water partition coefficient instead of log P had data been available). This is effectively an alternative form of equation (7.3) in which the numerator and the denominator are divided by Kav/. The second describes the influence of the thickeners on flavour release, due to changes in viscosity of the food phase. The third and final term describes the change in volatile behaviour as temperature varies. Va is the volume of air (Va has little impact in this equation, assuming similar volumes of headspace were analysed, since the model is relative to water), r\ is the viscosity of the matrix (cP), T is its temperature in Kelvin and TO and TJO are standard conditions 298 K and 1 cP respectively. r *awVa (T\Q\™ T- T K dp — — Pexp-—0— (logP)F 0 W 21.5

(7.7)

The model described well the behaviour of aroma compounds used in the development of the model (R2 — 0.90), whereas single factors such as log P, vapour pressure and boiling point showed little correlation with the data. Comparisons of data from the RAS with actual volatile concentrations in the breath during the consumption of a range of food have shown that for some systems it generates similar volatile profiles, although at higher concentrations than observed in vivo (Deibler et ol., 2001). By extrapolation, equation (7.7) may be related to matrix-driven changes in volatile concentration in vivo. It is interesting to note (from equation 7.7) that if identical volumes of headspace are sampled, from equiviscous systems at the same temperature, then it is only the first component of equation (7.7) that has any effect. Since this

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is very similar in form to equation (7.3), we have a situation where a dynamic headspace system is effectively modelled by a model that describes a static equilibrium. 7.3.2 Nonequilibrium partition modelling of volatile loss from matrices de Roos and Wolswinkel (1994) have also developed equations to describe the partitioning of volatile compounds in a range of matrices (relative to water) including thickened solutions, with and without the addition of lipid. The equation (7.8) models the amount of volatile retained in the product phase (X,,), after a series of n extraction steps, relative to the initial volatile concentration (Xo). Their approach is typical of the theoretical approach to model development (Figure 7.3).

v:

^pa r + V*/V* ^ a / vp

+

(7.8)

1-

Va and Vp are the volumes of the air and product phases respectively, and it is assumed that only small proportions of these volumes (V* and V*) are

Generate Model Equation based on theoretical considerations

Use part of data set to estimate values for Constants in model

Collect data from model system

Predict behaviour of remainder of data using Model Equation,Constants and key Variables

Figure 7.3 Theoretical modelling of the behaviour of volatiles. The Constants would be components of the equation that should be the same for all compounds and the key variables are typically compound specific factors, such as partition coefficients.

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actually in equilibrium at the air-product interface (Kpa being the product-air partition coefficient). The ratio between the volumes in equilibrium with each other (V*/V*) is affected by gas flows, mixing in the solution and diffusion, and thereby represents differences in the mass transfer. The relative values of this ratio to Kpa determine whether it is the equilibrium or mass transfer that is the dominant factor affecting losses of volatiles into the gas phase. Values of V*/VP, V*/V* and n can be determined experimentally from the study of volatile retention in different systems (given that Kp3l is known). In practice n can be set to a high number and the two volume ratios can each be determined as single values, following the solution of simultaneous equations. These values will be similar for different compounds (the differences between compounds are expressed by the values for Kpa) if the model is correct. When the model was used to describe the retention of volatile compounds in water, V*/V* was found to be much smaller than Kpa, suggesting that the losses of volatiles were mainly influenced by equilibrium conditions. For solutions of carboxymethylcellulose (CMC) and CMC-foil, however, V*/V* was larger, reflecting changes in mass transport. The values obtained for the components of the equation (Table 7.1) show a decrease in the proportion of the product in equilibrium with the gas phase (V*/VP) as resistance to mass transfer increases (Va*/Vp*). The change in V*/VP was independent of the value for V*/V*, which de Roos and Wolswinkel attributed to changes in mass transport not only in the liquid phase but also in the gas phase as viscosity increased. For the CMC solutions, the changes in V*/V* are not accompanied by changes in Kpa itself, hence the new values for V*IV* a p have a direct effect on volatile retention. The addition of lipid did alter the equilibrium partition coefficient (the Kpa for methyl benzoate was around 400 for water and 900 for 1 % oil), but the change was not as great as that for V*/V*, such that this system was also more dependent on restriction of mass transfer than the equilibrium partition coefficient. The estimates of the amount of volatiles retained using the values in Table 7.1 and equation (7.8) showed a good correlation with the experimental values (Figure 7.4), hence the model has been validated. Once values have been found for the parameters present in equation (7.8) for a given matrix, it should be Table 7.1 Values used for the parameters in equation (7.8) to model the behaviour of volatiles in water and in 1 % CMC with 1% oil added Parameter V /V

p S>

v*/v* n

Water

0.001 30 14000

1 % CMC

0.000 1 35 140 14000

From de Roos and Wolswinkel (1994). CMC = carboxymethylcellulose.

1 % CMC + 1 % oil

0.000068 250 14000

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75"

3

50

CO

O 25-

25

50 Observed

75

100

Figure 7.4 Observed vs calculated values (based on equation (7.8) and the values in Table 7.1) for volatiles retention in water (•). and solutions containing CMC (•) or CMC + oil (D). From de Roos and WolswinkeH 1994).

possible to predict the proportion of any volatile retained in solution, based on values for Kli. 7.3.3 Modelling the gas phase dilution of equilibrium headspace It is also possible to model the changes in volatile headspace concentration with time using on-line analytical techniques. Dilution of the gas phase is easily achieved by introducing a gas flow through the headspace and monitoring volatiles concentration in the outflow. The apparatus used by Marin et al. ( 1 999) consisted of a flask containing an aqueous sample with a headspace volume of about 25 ml. There were two ports for gas flow, one in and one out. After a period of equilibration, the out port was connected to a mass spectrometer (designed to measure the concentration of volatiles in real time). This drew headspace out of the flask (about 70ml/min) causing air to flow in via the second port, thereby diluting the headspace. The first signal detected by the mass spectrometer effectively corresponded to undiluted equilibrium headspace. which served as a reference point. Thereafter compounds were found to vary in the rate at which their headspace concentration decreased with time. The key element driving this was described by equation (7.9), where ko is the overall mass transfer coefficient, and kg and k\ are the mass transfer coefficients in the gas and liquid phases respectively. =

(7.9)

Given that &g and k\ were similar for different volatile compounds, the main factor responsible for differences in ko (which resulted in the different rates

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of headspace depletion for the compounds studied) was the air-water partition coefficient. Compounds with high values for ^Taw (10~ 2 ) were readily depleted from the headspace, whereas compounds with low Kaw values (10~5) were not (Figure 7.5). When compounds have a low A'aw the value of Kaw/ki decreases in significance (trends towards zero) and the main driving factor is then the mass transfer coefficient in the gas phase. Similarly, when Kaw values are high, Kaw/k\ increases in significance and the mass transfer in the liquid phase becomes important, such that surface depletion of volatiles from the solution decreases their maximum potential headspace concentration. This model describes the behaviour of compounds in situations where equilibrium headspace is disturbed by an external gas flow, such as when sniffing a glass of wine. The headspace system the model describes is very specific, but other parameters can be built into the model to describe how other factors such as the air flow rate and surface area of the solution affect the overall mass transfer coefficient (Marin et al., 2000). The two parameters kg and k\ were not determined by solving simultaneous equations but by an iterative process of parameter-fitting using Matlab. The initial values were set to those found in the literature and these were gradually modified until the theoretical change in headspace volatile concentration matched the experimental data. The fact that kg and k\ were similar for different compounds was indicative of the validity of the model. If substantial differences in kg and k\ were needed (for each compound) to fit theoretical curves to experimental data, they would be little more than variable fitting factors.

Time

Figure 7.5 Schematic representation of the differences in the rate of headspace depletion for compound with different air-water partition coefficients as a function of time.

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7.3.4 Modelling the gas phase dilution of equilibrium headspace above emulsions In addition to describing the behaviour of purely aqueous systems, this model has also been applied to the study of emulsion systems. An emulsion can result in a decrease in the volatile headspace concentration and the new equilibrium partition coefficient #ae will be lower than ATaw. If the differences in the two partition coefficients are large enough they may result in differences in behaviour during headspace dilution similar to those shown schematically in Figure 7.5. This, however, will depend on the behaviour of the volatiles in the aqueous phase. If a volatile can readily partition between the lipid and the water, it can partition between the liquid and gaseous phases. However, if the rate of partitioning between the lipid and the water is slow, the volatiles may be effectively trapped in the lipid phase. Substantial differences were observed in the rate of gas phase dilution (relative to the initial gas phase concentrations) of ethyl octanoate in the presence of emulsified lipid (Figure 7.6). It was found (Doyen et al., 2001) that the ethyl octanoate partitioned readily between the lipid and the water phases such that the emulsion appeared to behave as one homogenous phase. These differences were related to the change in the partition coefficient, which altered overall mass transfer as described by equation (7.9). KaVt,/k\ would decrease in significance and the relative headspace concentration would depend more and more on the gas phase mass transfer coefficient as the lipid fraction increased. &o only showed significant change as Kae varied, with the two mass transfer coefficients £g and k\ having similar values to those found for water in this system.

Image Not Available

Figure 7.6 Behaviour of ethyl octanoate in emulsions (oil fractions from 0.0 to 0.02). Relative changes in headspace concentration over time during headspace dilution. Reprinted with permission from Doyen et al. (2001). © 2001 American Chemical Society. • 0.0200:0 0.0100: • 0.0050: D 0.0025: + 0.0000.

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7.3.5 Modelling the rate of volatile equilibration in the headspace above emulsions Harrison et al. (1997) have also developed models to describe the behaviour of emulsion systems. The theoretical system they modelled was the inverse of that modelled by Marin et al. (1999). They modelled the way in which an emulsion would release aroma into the gas phase, from initial conditions in which there were no molecules of the aroma compound in the gas phase up to the point of equilibrium. Their model was based on penetration theory where bulk elements of the emulsion phase come into contact with the interface so that partitioning can take place, rather than a stagnant layer situation. The equation they produced describes the concentration of the volatile in the gas phase over time c g (/):

Cg(/) =

K v iv -4-T r ~ exp

+

~

KaeV/a/V/e+1 I

{7JO)

At first sight this equation looks rather complex. However, Va and Ve are the volumes of air and emulsion phases (respectively), which for simplicity we can assume to be identical and equal to 1. Equation (7.10) can then be written as

1 — exp +

-

1 +

ATae for aroma compounds is typically < 10 2 , therefore £ ae H-l, is effectively 1 and likewise l + l/Kae can be approximated by l/K.de to give equation (7.12). cg(0 - KaeQ(O) ( 1 - exp F-^^1 } I

L

K ae

(7.12)

JJ

This allows us to see more clearly the key elements of the equation. The first term in equation (7.12), /faece(0), is effectively the partition coefficient multiplied by the initial concentration of the compound in the emulsion: this product gives the headspace concentration at equilibrium (when time t trends towards infinity). The second part of the equation, 1— exp[— (/z D A ae ?//r ae )], governs the rate at which the headspace moves from its initial concentration (i.e. containing no volatile) towards equilibrium. The rate is influenced by the mass transfer coefficient /ZD, the area of the interface ( Aae ) and the partition coefficient. The most interesting part of this section is the mass transfer coefficient (hv($,d)), defined for an emulsion with a specific droplet size (d in um) and oil fraction (40, which Harrison and co-workers described with the equation M4>, d) = M0)exp -b(ln 10)

~

(7. 13)

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In equation (7.13), //o(0) is the mass transfer coefficient of the compound in water and is modified by the oil fraction, the droplet size and the exponent s. related to the contact time of the bulk elements of the emulsion and the surface and a constant b. Using these equations and assuming constant values for all but one parameter, it is possible to determine the extent to which a given parameter may influence volatile behaviour within a range of values (e.g. an oil fraction range from 0.0 to 1.0). The theoretical relationship between oil fraction and droplet size showed that, at a given oil fraction, the mass transfer coefficient should increase as the droplet size increases. Equally, they showed that, for an emulsion with a specific droplet size, the mass transfer coefficient should increase as oil fraction decreases. The model can also be used to determine the actual value of parameters such as the exponent s from experimental data for the rate of equilibration, where the characteristics of the system (<j>, d, etc.) are known. This is possible despite the fact that there are many terms in equations (7.12) and (7.13) because most of them have fixed values that are easily measured, leaving very few to be determined mathematically via the solution of equations. It is interesting to note the different methods used in the estimation of the overall mass transfer coefficients (equations 7.13 and 7.9). Both are dependent on a modification of values for liquid-phase mass transfer coefficients. Equation (7.9) (with Kae substituted for ATaw) does not have any components related to droplet size but does express the influence of oil fraction via K^. Such factors were not necessary in this estimation of overall mass transfer, since in the system studied the particle size was small and uniform such that it did not restrict or influence volatile partitioning. Extrapolating this approach for mass transfer estimation to systems with a higher oil fraction (where droplet coalescence is likely to occur), may result in error. This highlights one important aspect of models—it is important to remember how they were derived and, in the case of models based on data, the limits of the data set.

7.4 In vivo consumption There are fewer models that describe volatiles release during the eating process, mainly because of the number of variables that need to be considered and the limited amount of experimental data available. The more variables present in an equation, the harder it is to solve mathematically. Defining the values of constants in the equation is also a problem, a situation only made worse by limited data sets. Many of the dynamic headspace models that have been produced were generated in an attempt to describe the behaviour of volatiles under nonequilibrium conditions, such as might be found in vivo. The volatile concentrations in the breath during the consumption of standard foods (cheese, biscuits, etc.) can be substantially lower (about 100-fold) than those in the headspace (Deibler etal.. 2001). Even simple systems such as aqueous solutions

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of volatiles, which have minimal restrictions on mass transfer (as described by de Roos and Wolswinkel, 1994), only produce maximum breath concentrations equal to 10% of those expected on the basis of their Kaw. This might be due to liquid or gas phase dilution in mouth or as volatile-laden breath passes through the upper respiratory tract. However, there are distinct differences between volatiles, which may suggest a physicochemical basis (Linforth, unpublished data). Whatever the reason, the substantial departure of breath volatile concentrations from those of equilibrium may have a significant impact on the relative importance of the parameters used to model the behaviour of volatiles. 7.4.1 Emulsion dilution during consumption Not all models that describe volatiles release take into account absolute partition coefficients and mass transfer. Some simply describe what is likely to happen in vivo and the implications for the food sample thereafter. One such model is that of McNulty (1987), which considers the fate of emulsions during consumption. He argued that, during consumption, saliva dilutes the sample, and since the air-emulsion partition coefficient of an emulsion is dependent on the oil and water fractions (equation 7.3), the partition coefficient will change during the eating process, thereby affecting release of volatiles. For this to happen, the aroma compounds need to partition readily between the oil and water phases (otherwise the volatiles would effectively be trapped in the lipid), a phenomenon that has been observed experimentally (Figure 7.6). The greater the dilution, the more significant this factor will become in determining the breath volatile concentration. In contrast to emulsions, the fate of volatiles in an aqueous solution is purely dependent on the extent of salivary dilution, more dilution resulting in a lower aqueous phase concentration, and hence lower breath volatile concentrations. The relative differences between an emulsion and water upon dilution can be substantial for a compound with a strong tendency to partition into the lipid phase, as shown in Figure 7.7. Do such processes affect volatile release in vivo! Is there sufficient dilution by saliva to affect volatile delivery? McNulty (1987) presented sensory data that were consistent with a difference in emulsion behaviour compared to purely aqueous systems, a difference also observed by de Roos and Wolswinkel (1994). Direct measurement of breath volatile concentrations also agrees with this hypothesis (Doyen et al., 2001). It is important to remember, however, that emulsions can also affect mass transfer (equations 7.9 and 7.13) independently of dilution by saliva, and the relative effect of these two factors has yet to be established for volatile release from emulsions. 7.4.2 Effect of gas flow on volatile equilibration above emulsions One model that predicted changes in mass transfer in vivo but did not take into account dilution of the sample by saliva was proposed by Harrison and Hills

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100 1

75-

50-

25-

10

20

30

Dilution factor Figure 7.7 Relative changes in headspace concentration that would be expected for a volatile (Kav. = 2 x 10~2 and K30 = 2 x 10~5) dissolved in water (O) or an emulsion (•) (initial oil fraction 0.1). upon dilution.

(1997) in an extension to their initial model. The initial model (equation 7.10) described gas phase equilibration under static headspace conditions, and was then expanded to incorporate the effects of gas flow rate, a key factor in in-mouth dynamics. As with the model that studied equilibration rates under static conditions, one of the key factors in the equation was the mass transfer coefficient. Changes in the mass transfer coefficient as the oil fraction increased resulted in predictions of higher steady-state gas phase volatile concentrations than would be expected on the basis of their oil-water partition coefficients. On the basis of the oil-water partition coefficients, the equilibrium headspace concentrations for heptanone at oil fractions of 0.1 and 0.5, would be approximately 20% and 2.5% those observed for water. The predicted steady-state gas phase concentrations (using the model), however, were 60% and 20% relative to those of water. Clearly there is a theoretical potential for salivary dilution and/or mass transfer to affect release in vivo. The true significance of the two factors can only really be determined by comparison of prediction with (in vivo) reality under a range of experimental conditions. 7.4.3 Modelling volatile transfer through the upper airway Harrison (2000) has attempted to model not only the in-mouth partition of volatiles but also their transfer through the upper airways. The potential complexity of the model was reduced by selecting chewing gum as the food system, since it shows minimal changes in surface area during the eating process. Release of volatiles from the chewing gum itself was described by equation (7.14). where the mass of volatile transferred across the interface with time (dw/d/)

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was dependent on the surface area of the gum (A), mass transfer (h®), the saliva/gum partition (Ksg) and the concentrations of the volatile in the gum and saliva (cg and cs respectively). dm — = A/i D [* S gCg(t)-c s (0]

(7.14)

Higher values for K&B (0.2) would (according to the model) result in higher initial volatiles release followed by a decrease over several minutes, whereas lower values (0.05) would result in a more constant release and a lower overall saliva volatile concentration. After modelling of the movement of volatiles from the gum into the saliva (based on the assumption that all volatiles release takes place via saliva), the effect of salivary dilution and the air-saliva partition coefficient was estimated to give the in-mouth gas phase volatile concentration. Thereafter the transfer of volatiles from the throat through the upper airway was modelled by an equation containing nine parameters describing factors such as the dimensions of the upper airway, breath velocity, mass transfer and partition coefficients. This latter equation effectively modelled volatile movement through a tube where the volatile partitioned in and out of the liquid surface. It was predicted that a single pulse of hydrophilic compounds entering the gas flow of the tube would be retarded and smeared (as the compounds partitioned in and out of the mucous membranes), relative to hydrophobic compounds, which would show much less retention and peak broadening. Unfortunately, no experimental evidence was available to confirm the predictions of the models. Experimental data might also have helped to define the useful operating range of some parameters, and allowed others to be reduced to constants (e.g. those related to diffusion in air, breath velocity and the length of the throat). This emphasises the fact that theoretical models need experimental data at some stage to allow further development of the model and, ultimately, to test the validity of the model. 7.4.4 Nonequilibrium partition model for in vivo release de Roos and Wolswinkel (1994) also considered in vivo release of volatiles from chewing gum using their nonequilibrium partition approach, and produced a model describing in vivo release during consumption. The initial model (based on the partition of volatiles from the gum to the saliva) contained only three parameters, and did not correlate closely with experimental data, so further physicochemical parameters were introduced into the equation. The final equation generated (equation 7.15), included both the gum-water (AT gw ) and airwater partition coefficients, since release into both saliva and the gas phase appeared to be important factors (in contrast to the model proposed by Harrison, 2000). The saliva-gum volume ratio (V w /V g ) was one of the key parameters

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incorporating the effect of volatile partitioning into saliva, whereas the volume ratio between the gum and gas phase Va/VR affected the extent of partitioning into the gas phase. *1 = _

^_

(715)

As with their model for the dynamic equilibrium (section 7.3.2), in vitro values for Vw/Vg and VJVg can be determined from experimental results. In this instance, there was 'natural variation' due to differences between people, and estimates for these parameters were based on the average panellist. Once these values had been determined, the amount of volatiles retained in the gum phase was estimated, based on ATgw and Kaw for each compound. In addition, it was possible to consider the effect of short or long eating periods by using different values for n . Thereafter, it may be assumed that the changes in breath volatile concentration will be proportional to that of the chewing gum volatile concentration over time. The one limitation with this approach to empirical modelling is the range of foods to which it can be applied. The data set (residual volatiles in bolus) was produced by the extraction of volatiles from chewing gum (after it had been chewed for different periods of time) followed by quantitative analysis. In the case of chewing gum, where the bolus remains intact and unswallowed, this is a relatively easy task. For foods where the bolus disintegrates, dissolves or is swallowed, the collection of suitable data presents more of a problem. In this instance, alternative experimental approaches are necessary. 7.4.5 Modelling flavour release using time intensity data Data sets for empirical modelling can also be gathered from individuals eating foods and recording the change in perceived flavour with time (Time Intensity (TI) methodology). Here the data set takes into account not only the processes of mass transfer, diffusion, etc. in-mouth but the whole process of volatile transport to the olfactory epithelium and perception. Moore and co-workers (2000) prepared a series of protein gels that contained fat droplets and a volatile. Samples of the gels were eaten by their panel, TI curves were recorded, and the parameters extracted from the TI curves (maximum intensity, etc.) were compared with those estimated from the model. The model took into account factors such as particle size reduction due to chewing, swallowing of volatileladen saliva, retronasal air flow, the size of the saliva-air interface, and mass transfer between the saliva and air. In addition, measurements of gel texture and fat droplet size were made, to see whether these had a significant impact on perception that could be described by the model. The best correlation between experimental values and those obtained from the model were for the area under the curve up to the point of maximum intensity

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and the time to maximum intensity. Other values did not show such strong correlations, which may have been due to the fact that at 30 s the panellists were allowed to swallow, substantially altering the amount of bolus retained in mouth. No correlations were found between the fat droplet size, or the hardness of the gels and release of volatile. This may have been due to the limited differences in these parameters, which had a range of x 10 and x5 respectively. This lack of variation in the samples (and subsequently the TI data set) is one of the key problems encountered in this type of work, because, without sufficient variation in the data set, it is virtually impossible to develop and evaluate empirically based models. 7.4.6

QSPR of in vivo volatile release from gels

Flavour release data sets can be generated by direct measurement of the volatile composition of the breath during eating (Linforth et al,, 1996). This does not take into account the perception of the compounds, but it does allow the study of a wide range of different compounds with different properties. Release of the different compounds can readily be compared in absolute terms (e.g. mg/m3), whereas it is difficult to express the perception of very diverse compounds on one unifying sensory scale. Linforth et al. (2000) used this approach to study flavour release from gelatin/ sugar gels. They found that the maximum breath volatile concentration (/max) observed varied by a factor of 10000 depending on the compound studied (all compounds were present in the gels at the same concentration), providing good variation in the data set. Since the gel matrix and the panellists were constant, and the key differences were due to the compounds themselves, QSPR was the obvious choice for model generation. Physicochemical parameters were calculated using chemical modelling software to describe each compound numerically. These were then analysed statistically to determine which parameters best described the variation in the data set. Three parameters were selected and an equation was produced (equation 7.16) that could be used to estimate 7max. The R2 of the model was 0.88 and the /?2V was 0.82, indicating reasonable predictive power. log /max = - 1.3 + 0.9 logP + 0.7 log PL - 0.06 Energy - 0.15(logP) 2 + 0.13(logp L ) 2 + (1.4x 1(T3) x Energy2 - (7.3 x 10~6) x Energy3

(7.16)

The parameters of equation (7.16) describe the hydrophobicity-hydrophilicity of a compound (log P), vapour pressure (log /?L; Liang and Gallagher, 1998) and the compound's size and shape (Hartree energy). These values were all calculated using chemical modelling software. From the model, it is possible to

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predict the behaviour of any other compound by calculating the three parameters from the molecular structure and substituting them into equation 7.16. The model can be represented as a two-dimensional contour plot (Figure 7.8), where the lines join regions of the plot where the value of Zmax are the same; log Zmax for a hydrophilic compound with a high vapour pressure (e.g. ethanol) would be found in the top left of Figure 7.8. In contrast, the Zmax for a hydrophobic compounds with lower vapour pressure (e.g. decanol) would be found towards the opposite corner. The contour lines clearly run from top left towards the lower right-hand corner, indicating that the Imax values would be the same for these two diverse compounds; this is indeed the case, with ethanol and decanol having virtually identical release characteristics in this system. The contour plot also shows the range over which parameters exert the greatest influence. For example, log P has a major effect on a compounds behaviour up to 1.40. Thereafter, increasing log P has virtually no effect on Zmax. The QSPR models describing release of volatiles may be simple linear relationships, where one factor directly influences another. However, such relationships rarely continue indefinitely and in many instances quadratic (or cubic) functions may best represent the relationship between a modelling parameter and the response being modelled. Hence, the power terms in equation (7.16) and the curved contours in Figure 7.8.

Image Not Available

Figure 7.8 Contour plot of log Zmax (mg/m3) as a function of log P and log p~ for a fixed Hartree energy value. Reprinted with permission from Linforth et al. (2000). 02000 American Chemical Society.

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0.5 Time (min)

Figure 7.9 Observed (•) and predicted (•) release curves for hexanol present at lOOppm in a 6% gelatin gel.

In addition to modelling the differences in /max it was also possible to describe the variations in the temporal dimension using the same parameters. Consequently, the entire flavour release curve could be estimated for any given compound (Figure 7.9). 7.5

Conclusion

There are clearly many different approaches to modelling. However, it is evident that there is one common theme, in that there are major differences between compounds that need to be taken into account in any model. This can be achieved using partition coefficients, mass transfer coefficients or physicochemical parameters, one or more of which can be found in every equation describing the behaviour of volatiles. Models can describe a range of systems including the static partition, dynamic partition or in vivo behaviour of volatile compounds and their interactions with different matrices. They can be generated by a theoretical consideration of the system or empirically from a data set. If models are generated via a data set, the model should (ideally) be validated using an experimental test set of samples not used in the development of the original model: the quality of the model is then determined by the accuracy with which the model predicts the values for the test set. This guards against models based on random correlations between parameters and data, which do not truly describe the variation in the data set. Likewise, theoretical models should be validated, and equations should be generated (using real data) and solved to find the values of parameters that are

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constant in a given system (e.g. gas flow rate or volume). Thereafter the model should predict all available experimental data, keeping the constants constant and using values for key variables in the equation (e.g. partition coefficients). It is important to remember the range of the data (volatiles and matrices) used to develop and test a model, since extrapolation beyond these limits has little (if any) validity.

References Buttery, R.G.. Guadagni. D.G. and Ling. L.C. (1973) Flavour compounds: volatilities in vegetable oil and oil-water mixtures. Estimation of odour thresholds. J. Agric. Food Chem.. 21 198-201. Carey. M.E.. Asquith, T., Linforth, R.S.T. and Taylor, A.J. The effect of a cloud emulsion matrix on volatile partitioning. J. Agric. Food Chem.. submitted for publication. Deibler. K.D., Acree, T.E.. Lavin. E.H., Taylor. A.J. and Linforth. R.S.T. (2001) Flavor release measurements with retronasal aroma simulator, in Flavour 2000 (ed. M. Rothe). Eigenverlag M Rothe. Bergholz Rehbrucke. pp 41-48. de Roos, K.B. and Sarelse. J.A. (1996) Volatile acids and nitrogen compounds in prawn powder, in Flavour Science. Recent Developments (eds A.J. Taylor and D.S. Mottram). Royal Society of Chemistry, Cambridge, pp 13-18. deRoos, K.B. andWolswinkel.C. (1994) Non-equilibrium partition model for predicting flavour release in the mouth, in Trends in Flavour Research (eds H. Maarse and D.G. Van der Heij). Elsevier Science. Amsterdam, pp 15-32. Doyen. K.. Carey, M., Linforth. R.S.T. Marin. M. and Taylor. A.J. (2001) Volatile release from an emulsion: headspace and in-mouth studies. J. Agric. Food Chem.. 49 804-810. Harrison. M. (2000) Mathematical models of release and transport of flavors from foods in the mouth to the olfactory epithelium, in Flavor Release (eds D.D. Roberts and A.J. Taylor). American Chemical Society, Washington. DC. pp 179-191. Harrison, M. and Hills. B.P. (1997) Effects of air flow-rate on flavour release from liquid emulsions in the mouth. Int. J. Food Sci. Techno!.. 32 1-9. Harrison. M.. Hills, B.P.. Bakker, J. and Clothier. T. (1997) Mathematical models of flavor release from liquid emulsions. / Food Sci.. 62 653-664. Katritzky. A.R.. Mu. L. and Karelson, M. (1996) A QSPR study of the solubility of gases and vapors in water. / Chem. Inform. Comput. Sci.. 36 1162-1168. Katritzky. A.R., Wang. Y.L., Sild. S.. Tamm. T. and Karelson. M. (1998) QSPR studies on vapor pressure. aqueous solubility, and the prediction of water-air partition coefficients. / Chem. Inform. Compu. Sci.. 38 720-725. Liang, C. and Gallagher, D.A. (1997) Prediction of physical and chemical properties by quantitative structure-property relationships. Am. Labo.. (March), 34-40. Liang. C.K., Gallagher, D.A. (1998) QSPR prediction of vapor pressure from solely theoretically-derived descriptors. J. Chem. Inform. Comput. Sci.. 38 321-324. Linforth. R.S.T. Ingham, K.E. and Taylor, A.J. (1996) Time course profiling of volatile release from foods during the eating process, in Flavor Science: Recent Developments (eds A.J. Taylor and D.S. Mottram), Royal Society of Chemistry, Cambridge, pp 361-368. Linforth. R.S.T. Friel, E.N. and Taylor, A.J. (2000) Modeling aroma release from foods using physicochemical parameters, in Flavor Release (eds D.D. Roberts and A.J. Taylor). American Chemical Society, Washington. DC. pp 166-178. Marin, M., Back. I. and Taylor. A.J. (1999) Volatile release from aqueous solutions under dynamic headspace dilution conditions. J. Agri. Food Chem.. 47 4750-4755.

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Marin, M., Back, I. and Taylor, A.J. (2000) Flavor release as a unit operation: mass transfer approach based on a dynamic headspace dilution method, in Flavor Release (eds D.D. Roberts and AJ. Taylor), American Chemical Society, Washington, DC, pp 153-165. McNulty, P.B. (1987) Flavour release - elusive and dynamic, in Food Structure and Behavior (eds J.M. Blanshard and P. Lillford) Academic Press, London, pp 245-258. Moore, LPT., Dodds, T.M., Turnbull, R.P. and Crawford, R.A. (2000) Flavor release from composite dairy gels: a comparison between model predictions and time-intensity experimental studies, in Flavor Release (eds D.D. Roberts and AJ. Taylor), American Chemical Society, Washington, DC, pp 381-394. Reid, R.C., Prausnitz, J.M. and Poling, P.E. (1987) Properties of Gases and Liquids, McGraw-Hill. New York. Roberts, D.D. and Acree, T. (1996) Model development for flavour release from homogenous phases, in Flavour Science. Recent Developments (eds A.J. Taylor and D.S. Mottram), Royal Society of Chemistry, Cambridge, pp 399-404. Sorrentino, F., Voilley, A. and Richon, D. (1986) Activity coefficients of aroma compounds in model food systems. J. Am, Inst. Chem. Eng., 32 1988-1993. Voilley, A., Espinosa Diaz, M.A., Draux, C. and Landy, P. (2000) Flavour release from emulsions and complex media, in Flavor Release (eds D.D. Roberts and A.J. Taylor), American Chemical Society, Washington, DC, pp 142-152.

8

Instrumental methods of analysis Gary Reineccius

This chapter will provide an insight into how one approaches the instrumental analysis of flavour. Only the aroma component of flavour will be discussed; this excludes both taste and trigeminal effects that are also universally considered to be components of flavour. The aim is to provide an understanding of the unique challenges faced in this analysis and how and why particular analytical approaches are taken to solve a particular flavour problem. Issues of sample preparation, aroma isolation, compound selection (when an objective), quantification and identification will be discussed. Since the topic is broad, it cannot be covered in detail—Ho and Manley (1993), Marsili (1997) and Mussinan and Morello (1998) are recommended for greater detail overall. 8.1 Analytical challenges The aroma component of flavour is due to a complex mixture of volatile organic chemicals. A 'simple' flavour may have 100-300 volatile constituents (e.g. strawberry or grape). Foods that are more 'complex' in flavour, for example, those resulting from the Maillard reaction (e.g. coffee, meat or chocolate), may involve 800 or more volatile constituents (Nijssen et al., 1996). Of these volatiles, a limited number may be adequate to characterise the aroma of a food. For example, benzaldehyde is recognised by most individuals as cherry and citral is recognised as lemon. While in some cases one chemical may provide a recognisable odour character, most food aromas require 10-30 additional volatile compounds to provide a more rounded aroma that is characteristic of the food. Thus, while the analytical chemists may wish to focus their efforts on a given chemical or a limited number of chemicals, they must attempt to deal with hundreds of chemicals simultaneously, many of which make little or no contribution to aroma. The complexity of the analytical task is further complicated by the large number of volatile compounds that make up the total 'pool' of aroma chemicals. The task would be sufficiently formidable if the total pool of aroma constituents equalled that of the most complex flavour, for example meat, and all other flavours were a result of a different balance of these components. Unfortunately, the total pool of aroma constituents identified to date now exceeds 7000 (Nijssen et al.. 1996). Thus, in aroma analysis, the potential number of aroma compounds one could conceivably encounter is immense.

Image Not Available

Figure 8.1 Influence of aroma isolation method on the recovery of selected aroma compounds from an aqueous solution (equal concentrations by weight). 1, Ethanol; 2, propanol; 3, butanol; 4, octane; 5, decane; 6, ethyl propanoate; 7, ethyl butanoate; 8, ethyl pentanoate; 9, 2-heptanone; 10, acetophenone; 11, benzyl acetate; 12, methyl salicylate; 13, L-carvone; 14, p-ionone; 15, methyl anthranilate; 16, ethylmethylphenyl glycidate; 17, isoeugenol. (Reprinted with permission from Leahy and Reineccius, 1984; copyright 1984 American Chemical Society.)

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The wide chemical diversity of aroma chemicals found in foods further complicates these studies. Aroma compounds comprise a large number of different chemical structures and, therefore, of physical and chemical properties. It is difficult to match an analytical method to a physical property, such as volatility or molecular size, when there is such a broad range in these properties. The only attribute that all aroma chemicals have in common is volatility, but the wide range in volatility one encounters renders this commonality of limited value. For example, hydrogen sulfide has a boiling point of — 60°C and vanillin one of 284°C: a method based on volatility designed to isolate hydrogen sulfide is not likely to be appropriate for the isolation of vanillin. It is equally difficult to design an isolation or analytical technique on a chemical property such as reaction with a derivatising reagent (e.g. 2,4-dinitrophenylhydrazine for carbonyls) when aroma constituents comprise so many different chemical structures. There often is little in common between chemicals known to be odour active, other than they contribute to aroma. It is also problematic that aroma compounds may be sensorially significant when present in extremely low concentrations. The analytical chemist may have to work with femtogram or attogram (10~ 18 ) quantities of flavour constituents (Acree, 1993). This problem is accentuated by the fact that these traces of aroma chemicals are generally in a complex food system that contains thermally labile constituents (e.g. sugars, proteins, lipids and vitamins), good emulsifiers (e.g. lecithins and proteins), aqueous and fat-soluble components, and other volatile components (water). A seemingly impossible task (complete aroma isolation) becomes even more complicated as one considers it. One can readily appreciate that the isolation and analysis of aroma from a food product is indeed challenging (Teranishi, 1998). No single method yields an accurate picture of the aroma constituents in a food. Every method produces some picture of the aroma profile, but the profile is strongly determined by the biases introduced by the methodology itself. The aroma profile obtained from using five different isolation techniques on the same standard mixture of volatiles is illustrated in Figure 8.1. Ideally, the bars would all be the same height if the methods were 100% efficient in the extraction of all volatiles. It is quite clear that each method gives its own unique profile. Thus, in method selection, the analyst must be knowledgeable in the field and have a clear understanding of the study objectives. There is no universal approach to the instrumental analysis of aroma; each analysis must have a uniquely designed protocol. With this introduction, approaches for aroma isolation will be discussed. 8.2 Aroma isolation Numerous methods can be used to isolate aroma constituents from other food components (proteins, carbohydrates, water, fats, minerals, vitamins, etc.). The

INSTRUMENTAL METHODS OF ANALYSIS

2i3

primary principles used to isolate aroma constituents from the major food components are volatility and/or solubility. It is problematic that water is, with few exceptions, the most abundant volatile constituent in a food. This creates a problem in aroma isolation since isolation methods based on volatility also include water from the food. The analyst does not obtain just aroma constituents but rather a dilute 'solution' of aroma constituents in water. The aroma components must then be further isolated from water to permit concentration and further analysis. This additional step introduces further errors to the method (such as aroma losses and artefact contamination) as well as adding time. Most (but not all) aroma compounds are soluble in organic solvents, while the bulk of the major food constituents are soluble in water. Unfortunately, this rule is complicated by the fact that food lipids are also soluble in organic solvents. Thus, a solvent extract of a food yields not only the aroma constituents but also triglycerides, mono- and diglycerides, phospholipids, vitamins, chlorophyll, carotenoids, and so on. A useful aroma isolate cannot be prepared via solvent extraction if lipids are present in the food unless further steps are taken to separate the aroma from the lipids. Again, this additional handling adds the potential for more error (loss of volatiles and potential for artefact formation) and adds time to the analysis. The ability to utilise either volatility or solubility as a basis for aroma isolation is further complicated by the range in volatility and/or solubility displayed by aroma constituents. Earlier it was noted that hydrogen sulfide has a boiling point of — 60°C while vanillin boils at 284°C (with some degradation). Compounds such as hydrogen sulfide have little solubility in organic solvents, while vanillin is soluble in more polar solvents (e.g. ethanol). It is evident that no physical or chemical properties are uniquely and universally held by aroma constituents to permit their isolation from foods, nor are the 'somewhat unique' properties (e.g. boiling points) similar enough to permit an efficient total aroma isolation. The following section will focus on biases in methodology but present little detail on the methodology itself. There are numerous in-depth reviews describing approaches for flavour isolation that the reader may consult (e.g. Reineccius and Anandaraman, 1984; Parliment, 1986, 1997; Werkhoff et al., 1989-1990; Widner, 1990; Reineccius, 1989, 1993). Detailed methodology can be found only in original research publications. 8.2.1 Aroma isolation methods based on volatility One property that an aroma constituent must inherently possess is volatility. It must exhibit sufficient vapour pressure to be present in the gas phase at a concentration detectable by the olfactory system. Thus, it is understandable that numerous aroma isolation techniques are based on volatility, such as static headspace, dynamic headspace, molecular distillation, steam distillation, and

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FOOD FLAVOUR TECHNOLOGY

direct injection techniques (where food is placed in the apparatus itself and heated to volatilise aroma constituents). Some initial generalisations are made about these approaches, and methodspecific comments follow. All of the methods based on volatility will be strongly biased towards those aroma constituents that are most volatile in the food system to be studied. This qualification about 'the system to be studied' must be included, since volatility is dependent upon food composition. Efficiency of recovery by these methods does not follow in order of volatility (vapour pressure) of the pure compounds but, rather, of their vapour pressure over the food system. Generally, compounds with the greatest vapour pressure in the pure states are low molecular weight substances that often have some water solubility. Dissolution in water reduces vapour pressure according to Raoulf s law and, thus, these very volatile constituents may not be easily recovered from aqueous-based foods. The medium-volatility compounds, which still are very volatile but have lower water solubility, often have the highest vapour pressure in aqueous-based food systems. High molecular weight compounds have little vapour pressure in the pure form or in aqueous systems and, thus, are not readily recovered. One can apply similar reasoning to volatiles that are lipophilic when placed in lipid-containing food systems. Thus, methods based on volatility are very biased towards aroma constituents that have the greatest vapour pressure over a given food. 8.2.1.1 Static headspace methods Direct analysis of the equilibrium headspace above a food product would appear to be an ideal method for aroma studies. The method analyses exactly what the nose receives. Also, the method is very simple and gentle—one simply draws a few millilitres of vapour above a food into a gas-tight syringe and makes a direct injection into a gas chromatograph. Schaefer (1981) has illustrated the primary limitation of static headspace methodology—inadequate sensitivity (see Table 8.1). Since direct headspace Table 8.1 Minimum concentrations of a substance in a given volume of air required for gas chromatographic (GC) analysis or identification by mass spectrometry (MS) (Schaefer. 1981) Air volume2 1ml 10ml 100ml 1 litre 10 litres 100 litres 1000 litres (1m 3 )

GC(g/l)

10-5 _io~ 6 io~76 -io- 87 io- -io-

10-8-10-9 io-109-io-10

io- -io~"1 2 10-H-iO-

"Sample volume put into a GC or MS.

MS(g/l) IQ-^-lQ- 4 10-54-]0"56

io-A-io^ 7 io- -io~ io- 7 -io-98 !0 -«-ioio- 9 -io-'°

INSTRUMENTAL METHODS OF ANALYSIS

2! 5

injections into a gas chromatograph are generally limited to 10ml or less, one can see that only volatiles present at concentrations exceeding !CT7g/l (headspace) will be detected by gas chromatography, and only those at concentrations exceeding 10~~5 g/1 will be adequate for mass spectrometry. Since the concentration of volatiles above a food product generally ranges from about 1CT4 to 10~10 g/1 (or less) (Weurman, 1974), only the most abundant volatiles will be detected by direct headspace sampling. Trace component analysis will require some method of headspace concentration that permits sampling of large volumes of headspace (100-10001), thereby compensating for low headspace concentrations. Sensitivity of the method may be enhanced to some extent via headspace enrichment. This may be accomplished by preparing a distillate of a food product and analysing the distillate by headspace methods. Enrichment of the headspace may also be accomplished through the addition of soluble salts to the aqueous food product. The salts tend to drive the organic volatiles from solution into the vapour phase (Jennings and Filsoof, 1977). It is of interest that the enhancing effect is not similar for all volatiles (Roberts and Pollien, 2000). The use of sodium chloride to enrich headspace volatiles may quantitatively distort the headspace profile. A second disadvantage of headspace methods is that it is difficult to do quantitative studies using them. The analytical data one receives are on the amount of an aroma constituent in the headspace. The relationship between concentrations in the headspace versus those in the food can be very complex and must be determined experimentally. The issue of quantification is discussed later in this chapter. The advantages and shortcomings of static headspace sampling dictate its applications (Wampler, 1997). It is often used in quality control situations where only major components need to be measured. Although the components measured might not actually be responsible for the flavour attributes being monitored, if there is a good correlation between flavour quality and the component(s) measured, the goal has been accomplished. For example, Buttery and Teranishi (1963) used headspace analysis of 2-methylpropanal and 2- and 3-methylbutanal as indicators of nonenzymatic browning in potato granules. Sullivan et al. (1974) have used this technique to do additional work on the flavour quality of dehydrated potatoes. It is commonly used to indicate the oxidative quality of edible oils (by monitoring hexanal formation). 8.2.1.2 Methods based on purging and trapping (headspace concentration) Headspace trapping methods are commonly called dynamic headspace or purgeand-trap methods. In these methods, the sample is purged with an inert gas, such as nitrogen or helium, which strips aroma constituents from the sample (Figure 8.2). The volatiles in the purge gas must then be trapped (somehow

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Image Not Available

Figure 8.2 Schematic examples of apparatus for the isolation of volatiles via dynamic headspace techniques. The system in (a) uses a cryotrap (vacuum operation); that in (b) uses a Tenax trap (ambient pressure operation). (Reprinted with permission from Guntert et al.. 1998: copyright 1998 American Chemical Society.)

removed) from the gas stream. The aroma constituents may be trapped via a cryogenic, Tenax (or alternative polymer), charcoal, or other suitable trapping system. This approach favours the isolation of constituents with the highest vapour pressure, as has been discussed. Additional distortion of the aroma profile results from the aroma-trapping technique. A cryogenic trap is the least selective of the traps. It will remove and contain virtually any aroma constituent if properly designed and operated. The primary problem with a cryogenic trap is that it will also trap water—the most abundant volatile in nearly all foods. Thus, one obtains an aqueous distillate of the product, which must then be solvent extracted to recover the aroma fraction. Solvent extraction will again alter the true aroma profile. A Tenax trap is very widely used for aroma trapping. Despite its wide usage, it has a low surface area and, therefore, a low adsorption capacity. Additionally, Tenax has a low affinity for polar compounds (hence it does not retain much water) and high affinity for nonpolar compounds. An aroma compound such as hydrogen sulfide would not be retained at all on this material (Reineccius and Liardon, 1985). Buckholz et al. (1980) demonstrated the biases associated with Tenax traps during a study on peanut aroma. They found a 'breakthrough' of peanut aroma (through two traps in series) after only 15 min of purging at

INSTRUMENTAL METHODS OF ANALYSIS

21 7

40 ml/min. In an evaluation of the sensory properties of the material collected on the Tenax trap, they found that a representative peanut aroma had been collected by the trap after 4 h of purging. Shorter or longer purge times did not produce an aroma characteristic of peanuts. In fact, the majority of purging conditions did not yield an aroma isolate characteristic of the sample. The isolate was biased both by the volatiles preferentially going into the purge gas and by the Tenax trapping method. The work of Guntert et al. (1998) found that the Tenax trapping method (ambient pressure) did not yield as true an aroma profile as vacuum distillation (cryotrapping) (apparatus presented in Figure 8.2). This difference in performance may have been due in part to the operating conditions used for the Tenax system. As Buckholz et al. (1980) noted, operating conditions have a strong influence on the composition of the aroma isolate. Sucan et al. (1998) used a response surface methodology to optimise their purge and trap method for a study of dry dog food aroma. This approach, once optimised, yielded a very good quality aroma isolate. Activated carbon traps have a strong affinity and large capacity for most aroma constituents. As little as 1-lOmg of carbon will trap the volatiles from 10-100 litres of purge gas (Schaefer, 1981). The primary concern with charcoal traps is that they may not give up their aromatic components without artefact production. It has been suggested that this problem can be minimised through the use of a good quality coconut charcoal. Despite the concerns noted about this approach to aroma analysis, it is commonly used in the field today. Several manufacturers offer completely automated systems that simplify the task and add considerable precision to the data. Wampler (1997) has provided a review of this technique. 8.2.1.3 Distillation methods Distillation can be defined broadly to include high-vacuum molecular distillation (the vacuum headspace method apparatus shown in Figure 8.2 would be more appropriately termed high-vacuum distillation), steam distillation, or simple heating of the food and sweeping of the 'distilled' aroma constituents into a gas chromatograph. High-vacuum distillation may be applied to pure fats or oils, solvent extracts of fat-containing foods or aqueous-based foods (e.g. fruit). Since fats and oils are essentially anhydrous, additional extractions (or sample manipulations) would not be required to remove any co-distilled water. The use of high-vacuum distillation for the isolation of volatiles from solvent extracts has frequently been used to provide good quality extracts for aroma extraction dilution assays (to be discussed later). This distillation process is likely to require an additional solvent extraction step since diethyl ether is commonly used as the extracting solvent and this will extract some water as well. The high-vacuum distillation of fresh food products uses product moisture to codistill volatiles. Water is always the major part of the distillate and a secondary extraction is mandatory. Thus, an extraction is commonly a part of this type of

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FOOD FLAVOUR TECHNOLOGY

aroma isolation process. The primary sources of aroma profile distortion come from the distillation process and subsequent solvent extraction. Steam distillation may be accomplished in several ways. The product may simply be put in a rotary evaporator (if liquid, or initially slurried in water if solid) and a distillate collected. This distillate would be solvent extracted to yield an aroma isolate suitable for GC analysis. The most common steam distillation method employs simultaneous distillation/solvent extraction (Likens-Nickerson). This is one of the oldest and most popular methods for obtaining aroma isolates. Chaintreau (2001) has provided a very good review of this method and its evolution. Figure 8.3 shows an atmospheric pressure system (a) and a vacuum system (b). The primary difference is that the vacuum system has to have joints that are air-tight and all parts of the apparatus have to be under tight temperature control. In either approach, the aroma profile ultimately obtained is influenced by volatility of the aroma compounds (initial isolation), solubility during solvent extraction of the distillate and, finally, volatility again during the concentration of the solvent extract. The aroma isolate prepared by simultaneous distillation/extraction (atmospheric or reduced-pressure operation) contains nearly all

Image Not Available

Figure 8.3 Simultaneous steam distillation/solvent extraction apparatus: (a) atmospheric pressure: (b) vacuum operation (Chaintreau, 2001: John Wiley & Sons Ltd. reproduced with permission).

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INSTRUMENTAL METHODS OF ANALYSIS

the volatiles in a food, but their proportions may only poorly represent the true profile in a food (Figure 8.1). The popularity of this method derives from the fact that medium to high boilers are recovered well and a liquid isolate is obtained. This liquid isolate is quite concentrated, which facilitates mass spectrometric work or repeated injections for further studies. Distillation, as defined here, also includes direct thermal analysis techniques. These techniques involve the heating of a food sample in an in-line desorber (i.e. in the carrier gas flow of the gas chromatograph). Generally, aroma compounds are thermally desorbed from the food and then cryofocused on the GC column to enhance chromatographic resolution. This technique has been used for a number of years for the analysis of lipids and was later modified to include aqueous samples (Dupuy et al., 1971; Legendre et al., 1979). Aqueous samples were accommodated by including a water trap after the desorption cell. This general approach has been incorporated into the short path thermal desorption apparatus discussed by Hartman et al. (1993) and Grimm et al. (1997). In this apparatus, shown schematically in Figure 8.4, a sample of food is placed in the desorption tube and quickly heated.

Valve 1 Open

6

Helium carrier gas

2. i Sample tube

Valve 2 Closed To septum purge vent Split/splitless injector

To split vent

ToGC Figure 8.4 Short-path thermal desorption apparatus (Grimm et al.. 1997).

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FOOD FLAVOUR TECHNOLOGY

The volatiles are distilled into the gas flow, which carries them into the cooled injection system where they are cryofocused prior to injection into the analytical column. The issue of water in the sample often limits sample size even when it is a minor component of the food. Virtually all of these methods or instruments require cryofocusing prior to gas chromatography and small amounts of water will tend to freeze in the cryotrap, blocking the carrier gas flow. Thus, the sample size (and therefore, sensitivity) is often limited by the moisture content of the sample (the method is applied to samples < 5% moisture; Rothaupt. 1998). The primary bias inherent in this approach is again relative volatility of the aroma constituents. Additional concerns involve the technique used to remove water from the sample (if the sample is aqueous-based) and the potential for artefact production due to heating of the sample. There is a substantial body of information demonstrating aroma formation (in this case artefact formation) due to heating. Some reactions proceed rapidly at temperatures as low as 60~C: therefore, the aroma profile can be greatly altered via the addition of artefacts due to heating of the sample during isolation. 8.2.2 Aroma isolation methods using solvent extraction One of the simplest and most efficient approaches for aroma isolation is direct solvent extraction. The major limitation of this method is that it is most useful on foods that do not contain any lipids. If the food contains lipids, they will also be extracted along with the aroma constituents, and they must be separated from the solvent extract prior to further analysis. Aroma constituents can be separated from fat-containing solvent extracts using techniques such as molecular distillation, steam distillation, purge-and-trap or dialysis. A second consideration in solvent extraction is solvent purity. Solvents must be of the highest quality, which often necessitates in-house distillation prior to use. One must be mindful that various qualities of solvents can be purchased and GC grade is highly recommended (not HPLC or other quality). Furthermore, a reagent (solvent) blank must always be run to monitor solvent artefacts irrespective of the quality of the solvent. Solvent extraction can be as simple as putting the food sample (e.g. apple juice) into a separating funnel, adding a solvent (e.g. dichloromethane) and shaking. The dichloromethane is collected from the separating funnel, dried with an anhydrous salt and then concentrated for GC analysis. Alternatively, the process may be much more costly and complicated, involving, for example, a pressure chamber and supercritical CO2 (Jennings and Filsoof, 1977). Supercritical CO2 has the advantage that it is very low boiling (and so is efficiently separated from extracted volatiles), it leaves no 'residue' to interfere with any subsequent sensory analysis, it penetrates food matrices and its solvent

INSTRUMENTAL METHODS OF ANALYSIS

99'

properties can be altered through temperature and pressure changes or the use of chemical modifiers (e.g. methanol). Negative aspects of this solvent include its high cost due to pressure requirements, small sample sizes (most commercial extractors) and its highly nonpolar nature (without modifiers). The use of modifiers such as methanol negates some of the advantages noted earlier. Morello (1994) has provided a good example of its use and thoughtful discussion of the technique. The biases imposed on the aroma profile by solvent extraction relate to the relative solubility of various aroma constituents in the organic/aqueous phases. A graphic comparison of the recovery of aroma compounds from a model flavour system (in water) using pentane versus dichloromethane as solvent is presented in Figure 8.5. It is obvious that neither solvent gave 100% recovery of all aroma constituents and that dichloromethane extraction gave quite a different aroma isolate from that using pentane. Cobb and Bursey (1978) have made a similar comparison of solvent effect on recovery of a model aroma system from 12% (v/v) ethanol in water (Table 8.2). Recoveries of aroma constituents were low and variable, depending on the solvent chosen and aromatic component being extracted. While it is obvious that even a simple solvent extraction introduces substantial bias into an aroma profile, combining solvent extraction with another technique (e.g. to separate aroma components from extracts containing lipids) adds more bias. For example, applying a distillation technique to a solvent

Image Not Available

Figure 8.5 Batch solvent extraction of a model system showing the influence of solvent ((a) pentane; (b) dichloromethane) on recovery of volatiles. 1, Ethanol; 2, propanol; 3, butanol; 4, octane; 5, decane; 6, ethyl propanoate; 7, ethyl butanoate; 8, ethyl pentanoate; 9, 2-heptanone; 10, acetophenone; 11, benzyl acetate; 12, methyl salicylate; 13, L-carvone; 14, (3-ionone; 15, methyl anthranilate; 16, ethylmethylphenyl glycidate; 17, isoeugenol. (Reprinted with permission from Leahy and Reineccius, 1984; copyright 1984 American Chemical Society.)

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Table 8.2 Recovery of model compounds from an alcohol-water (12% v/v) system (Cobb and Bursey. 1978) Recovery in (%) a Compounds extracted

Freon II

Dichloromethane

Ether

Isopentane

Ethyl butanoate 2-Methyl- 1 -propanol 3-Methyl- 1 -butanol 1-Hexanol Benzaldehyde Acetophenone Benzyl formate 2-Phenethyl butanoate Methyl anthranilate

66 34 63 85 83 53 75 46 62

43 55 66 67 54 41 56 48 59

22 50 23 18 34 21 25 57

16 32 48 38 20 20 25 17 27

a

Batch separating funnel extraction; 757 ml of model system extracted 6 x 50ml solvent.

extract that contains lipids selects for the most volatile components (now from an oil phase). 8.2.3 Solid-phase microextraction Solid-phase microextraction (SPME) is a relatively new technique for the isolation of food aromas. Pawliszyn's group (1997) were the first to develop this method and applied it in environmental analysis. Since then, it has become a widely used technique for the analysis of volatiles in foods. It is of interest that Roberts et al. (2000) noted that 420 references were found related to SPME in the literature (as a whole) as of May 1999 while this author found over 1200 references to this technique as of April 2001. Harmon (1997) has provided a comprehensive review of this technique. For SPME, an inert fibre is coated with an adsorbent (of which there are several choices). The adsorbent-coated fibre is placed in the headspace of a sample and allowed to adsorb volatiles. The 'loaded' fibre is then thermally desorbed into a GC carrier flow and the released volatiles are analysed. A schematic of the device used to adsorb volatiles is presented in Figure 8.6 and the overall process is shown in Figure 8.7. The coated fibre is a modified syringe in which the needle is retractable and is coated with adsorbent. The feature of retractability affords protection to the fibre against physical damage and contamination. SPME is an equilibrium technique and, therefore, the volatiles profile one obtains is strongly dependent upon sample composition and careful control of all sampling parameters. While Harmon (1997) notes that the method can give excellent results, Coleman (1996) cautions that the fibres have a definite linear range and competition between volatiles for binding sites can introduce errors. Roberts et al. (2000), recognising the sensitivity of the method to sampling errors, proposed a stable-isotope method for the accurate quantification of volatiles by SPME.

Image Not Available

Figure 8.6 Schematic of a SPME device (reprinted with permission from Zhang et al., 1994; copyright 1994 American Chemical Society).

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Figure 8.7 Schematic showing the steps involved in the use of a SPME device; (a) extraction procedure; (b) desorption procedure. (Courtesy of Supelco Inc.)

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Similarly to all of the other methods described thus far, SPME affords a certain view of the volatile composition of the food. This view is determined by factors common to headspace techniques as well as the unique effects contributed by the adsorption process. To use the method effectively, one has to be very familiar with the factors that influence volatile recovery. These factors have been discussed in detail in the literature (references cited above). If it provides an isolate that has the component(s) one wishes to measure and it is adequately reproducible, the method is quite attractive. There are no solvents for contamination, it is simple (and has been automated), and it is sensitive and rapid. There are several other approaches for the isolation of food aromas; those that have been mentioned are the primary methods in use today. The reader is encouraged to go to more detailed reviews (e.g. Reineccius and Anandaraman, 1984), specialised books (e.g. Ho and Manley, 1993; Marsili, 1997) and original literature for more explicit information and a broader view of the topic. 8.2.4 General considerations in preparing aroma isolates While the isolation methods themselves are selective in their recovery of aroma constituents from foods, and therefore give an aroma profile unrepresentative of the food, the aroma profile may be further biased by other means. Some of these considerations are outlined below. 8.2.4.1 Sample preparation Many food products contain active enzyme systems, for example in unprocessed plant or animal tissue. These enzyme systems may become active if the food is chopped, ground or macerated to facilitate aroma isolation. If the enzyme systems are not inactivated (e.g. by heating, or addition of alcohol or sodium fluoride) prior to starting analysis, the aroma profile may change greatly. We may want this change to occur if this is how a food is normally consumed, or we may want to inhibit enzymatic changes. In either event, enzymatic action may introduce changes in the aroma profile and we must take this effect into consideration. 8.2.4.2 Contamination by artefacts The analyst is typically working in the ppm (mg/kg) or lower concentration ranges. There are numerous ways in which volatile constituents may contaminate the aroma isolate at such low levels. One must be extremely careful of water quality if the sample is mixed with any water (or steam). Organic solvents are seldom sufficiently pure to

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be used in aroma isolation without additional cleanup (typically by distillation). Any polymer-based materials (containers or tubing) are common sources of contamination. Antifoam additives may contribute as many components to an aroma isolate as does the food itself. Stopcock or vacuum greases are known sources of contamination. Bottle closures must be Teflon-coated rather than rubber to prevent the closure from both absorbing some aroma components and contributing others. Blanks must always be run to determine the magnitude of contamination contributed by the system. Every effort must be made to minimise contamination from all sources. 8,2.4.3 Thermally induced artefacts Foods are very good reaction systems. At least 3000 volatile compounds have been identified to date arising from the heating of foods. Many techniques for aroma isolation involve heating of the food sample. This is done in order to more effectively remove volatiles from the food matrix. Extreme care must be exercised in heating food samples to ensure that the aroma isolate collected truly represents that of the food and is not contaminated by thermally induced artefacts. We have a general rule that we do not heat raw (not thermally processed) foods above 60°C in aroma isolation. We may go above 60°C for thermally processed foods, but we always choose to use minimal heat exposure. 8.2.5 Aroma isolation summary Since every method preferentially selects those aroma constituents that meet certain physical or chemical criteria (e.g. solubility, volatility or affinity), one must 'make do' and compensate for having a very biased analytical view of the aroma constituents in a food product. That this view is biased does not mean that it is useless or even of lesser value than a truly accurate picture. We need to choose our methodology wisely so that we measure the aroma components we need to monitor to solve our problem, that is, so that they are contained in the aroma 'view' we consciously select. Furthermore, one must recognise that the most commonly used approaches in the literature may not be the best or even suitable for a given task. A particular task requires a unique method. The frequency of a method appearing in the literature is more often linked to the size of the research group than to any other factor. A particular research group may be large and doing similar work and thus, their particular methodology appears frequently. Also, individuals have certain biases—no two researchers will approach the same problem in the same manner. With that said, this author's view (including biases) of method selection is discussed in the following section of this chapter.

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Selection of aroma isolation method

One cannot choose a method for aroma isolation without first defining the objectives of the study. For example, one may wish 1. 2. 3. 4. 5.

to obtain a 'complete' aroma isolate to accurately identify and quantify every aroma constituent in a food; to identify only key components of an aroma profile, that is those components that are responsible for the characteristic sensory properties: to identify an off-note in a food product; to monitor aroma changes with time; or to predict sensory attribute(s).

Each of these tasks imposes different requirements on the methodology. 8.3.1

'Complete' aroma profile

Obtaining a complete aroma profile is one of the most difficult tasks to accomplish. It is a given that no individual isolation technique will yield an accurate analytical profile. Thus, one must use several isolation techniques in combination. A good combination would be a static headspace method to obtain a profile of the most volatile and most abundant volatiles. One can follow this with a purge-and-trap method to obtain data on the less volatile and less abundant constituents (no solvent to 'cover' early-eluting components of interest). The task would finish with a solvent extraction (if there are no lipids in the food) or simultaneous distillation/extraction method to obtain a profile of the least volatile aroma components. This combination of techniques should yield a reasonably complete view of the aroma profile (for example, see Qian, 2000). Each of the profiles will be biased (as discussed), so that quantitative data will have to be obtained using the food system plus some quantification approach. This might include the use of multiple internal standards (Siek and Lindsay, 1968), a method of standard addition (adding known quantities of each individual pure component and determining increases in GC peak areas versus amount added and relating this back to original peak area; Qian, 2000), or. ideally, use of isotopically labelled pure standard compounds (adding a known quantity of stable-isotope-labelled compound and monitoring aroma isolate via selected ion monitoring mass spectrometry to obtain a ratio between labelled and unlabelled compounds of interest; Milo and Blank, 1998). Traditionally, flavour chemists have been less than rigorous in this respect. It has long been a habit to simply use GC percentage area in tabulations of aroma compounds and their quantities found in foods. The data obtained and reported in this manner are typically grossly in error due to the many biases in aroma isolation and GC analysis. Occasionally a researcher will add an internal standard and report quantitative data in terms of the internal standard. This approach offers

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little or no improvement over GC peak area. Obtaining accurate quantitative data is a very formidable task that many researchers choose to short-cut. 8.3.2 Key components contributing to sensory properties Since the task of identifying these, in general, will be discussed later in the chapter, the immediate discussion will focus only on the methods one might use in preparing an aroma isolate for this purpose. While much of the early research done for this purpose used only one isolation method (most commonly high-vacuum distillation/sublimation), it was recognised that this method did not provide a satisfactory isolate of the very volatile arorna compounds. These volatiles were lost during sublimation, extraction, and/or concentration of the aroma isolate or with the solvent front during chromatography. Thus, the methods employed underestimated the importance of the more volatile aroma compounds. The work done today generally combines two or more isolation methods to provide a more complete view of the food aroma. Typically a rigorous technique such as high-vacuum distillation (or solvent-assisted flavor evaporation, Engel et al., 1999) is combined with a static headspace technique. Qian (2000), in fact, chose to use a solvent extraction method to determine free fatty acids, a static headspace method to see the more volatile aroma compounds, a purge-and-trap method for the intermediate volatility aroma compounds and a high-vacuum distillation/sublimation method for the least volatile compounds (in Parmesan cheese). 8.3.3 Off-notes in a food product The requirements imposed upon isolation methodology for identification of offnotes are much less stringent than those imposed by the first two tasks. Virtually any isolation method can be used that yields an aroma isolate containing the offnote. Selection of the isolation method can initially be guided by experience. For example, off-notes that have a less characteristic aroma but are more generically solvent-like (e.g. contamination by food packaging or printing inks) can initially be approached using a purge-and-trap or even perhaps a static headspace method. Off-notes that are very characteristic and heavier in sensory character (e.g. earthy, musty, cooked or burnt) may require more rigorous methods such as steam distillation or high-vacuum distillation. Irrespective of method, it is essential that the aroma isolate of a control (no offnote) and the off-flavoured product be produced and then sensorially evaluated to ensure that the chemical constituent(s) responsible for the off-notes have actually been isolated from the food. Method validation can be done in different ways. For example, if the isolate is a liquid, a blotter can be dipped in it, the solvent evaporated and the blotter smelled for the off-note. Alternatively, the

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isolate can be applied to a gas chromatograph and then the effluent smelled for the off-note. The analytical approach to finding the odorant(s) causing an off-flavour then involves analysing the isolates from the good and bad samples by gas chromatography while smelling the GC column effluent to locate the chemical constituent (GC peak) corresponding to the characteristic off-note. One must recognise that any food isolate may contain odorants that are 'unpleasant". However, the presence of unpleasant odorants in the GC effluent may simply be due to their concentration in the effluent. They may not be a source of off flavour in the food itself. Thus, it is essential that odorants (GC peaks) be selected by sniffing that are characteristic of the off flavour in the food. Ideally, the aroma isolation procedure should yield an isolate sufficiently concentrated for GC-MS identification of the tainting compound. If there is no GC peak in the chromatogram where the offending aroma is smelled (or too little compound is present for a good mass spectrum), then the aroma isolation procedure needs to be changed so that larger quantities of the tainting chemical are obtained, or identification may be attempted on gas chromatographic retention data (retention indices) and sensory description. 8.3.4 Monitoring aroma changes in foods There are many situations in which the flavour chemist wishes to monitor changes in food aroma over time. For example, one may wish to analytically monitor flavour losses from a food product during storage (e.g. from coffee), the formation of desirable flavours (e.g. in wine or cheese ageing) or the appearance of off flavours (e.g. through lipid oxidation). For these types of problems, one has to consider at least four factors: 1. 2. 3. 4.

Does the aroma isolation method provide data on the aroma compounds of interest? Is the method sufficiently robust to be stable over time? Is there adequate precision in the method to see the anticipated variation? Is the method rapid enough to be used in the study?

The importance of each factor depends on the task at hand. For example, a storage study with 10-20 samples to be analysed each day (or even each week) will preclude any method that is very time-consuming (distillation methods may be problematic). One would like to use an automated headspace method (static or dynamic). Precision is typically obtained through the use of automated methods and/or analytical standards. Automated headspace methods may have a coefficient of variation (CV) of only 2-3% (compound dependent), while a distillation method may have a CV ranging from 10% to 50%. Many of the analytical methods in flavour research suffer from poor reproducibility. The range of problems and priorities one may encounter in this type of study makes any further discussion difficult.

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8.3.5 Using aroma profiles to predict sensory response This task generally does not require a complete or an accurate aroma profile. The goal is to produce an aroma profile that is reproducible (precision aids prediction reliability) and contains some components related to the sensory attributes one desires to predict. The aroma constituents used in prediction are generally not causally but only statistically correlated. Most often this task is used in a production or quality control setting, so time, ease and reliability are additional factors influencing method choice. Static headspace and direct analysis isolation techniques are well suited for this task. Both approaches are routinely used in the quality control of fats and oils for the analysis of hexanal as an indicator of rancidity. In some cases, static headspace may be replaced by a purge-and-trap method to yield more information for obtaining valid statistical correlations. There is a great deal of literature available demonstrating correlations between GC aroma profiles obtained using purge-and-trap methodology and some sensory attribute (e.g. coffee attributes) or other information of interest (e.g. geographical origin of olive oils or wines). Purge-and-trap methods have been automated to greatly reduce operator time required and, to a limited extent, operator expertise. 8.3.6 Summary comments on isolation methods While aroma isolation from foods is generally based on either volatility or solubility of the aroma compounds, numerous methods have been developed to apply these principles to this task. Some methods depend solely upon volatility (e.g. headspace, direct analysis and molecular distillation) or solubility (solvent extraction). Other methods depend upon combinations of volatility and solubility for isolation (e.g. simultaneous steam distillation/solvent extraction). One must be conscious that none of these approaches yields an aroma isolate for further analysis that either qualitatively or quantitatively truly represents the aroma of the food. Each method provides a unique view of the aroma profile that may be more or less suited to addressing the problem faced by the flavour chemist. In method selection, the chemist must define the problem and consider which isolation method is best suited to his or her particular task. Most problems in the flavour area can be addressed with existing technology, but the chemist must choose wisely among the technologies to yield a solution. 8.4 Aroma isolate fractionation prior to analysis It may prove advantageous to pretreat the aroma isolate before GC analysis (Parliment, 1997). Even though high-resolution GC columns and specialised detectors are available to the analytical flavour chemist, the complex nature of the flavour isolates often makes complete resolution or adequate concentration impossible. Some prefractionation of flavour isolates prior to GC simplifies

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analysis. The fractionation of flavour isolates into classes of chemicals with similar chemical properties also permits the use of specialised gas chromatographic columns and detectors. For example, columns that are particularly adapted to the separation of basic compounds may be selected and nitrogenspecific detectors utilised. The following section discusses some of the more common methods applied to the fractionation of flavour concentrates. 8.4.1 Fractionation of concentrates prior to analysis 8.4.1.1 Acid/base separations This method utilises the differential solubility of ionised and non-ionised species in aqueous and organic solvents. lonisation of ionisable compounds is generally accomplished via pH control of the aqueous solvent. A typical acid/base separation scheme is presented in Figure 8.8. Whereas this scheme assumes one is starting with a flavour isolate in an organic solvent, the same technique is easily applied to acid/base fractionation of aqueous distillates. The distillate is initially adjusted in pH prior to solvent extraction and then a similar pH adjustment scheme is followed. Sub-fractionation of acidic compounds can be accomplished by extracting the organic phase with 5% sodium carbonate initially to extract the strong acids (e.g. carboxylic acids) and then 5% NaOH to extract weakly acidic compounds (e.g. phenols). In this case, there would be two aqueous phases on pH adjustment, one of which would yield an isolate of weak acids and the other of strong acids. Acid/base separations are relatively simple and rapid, and do not require sophisticated equipment or expensive reagents. This fractionation simplifies the following GC analysis by reducing the number of components to be separated, permits further concentration than might otherwise be possible, and allows a more specific column choice and tailoring of GC operating conditions. The acid/base/neutral fractions can be subjected to sensory analysis, which provides information on what type of flavour is responsible for the flavour notes of interest. However, this technique has two major drawbacks. The separation between classes is often incomplete, and poor recoveries are common. Efficient extraction requires large volumes of solvents and multiple extractions. Multiple extractions with large volumes of solvent can be tedious and result in dilute solutions of flavour compounds in the organic phase. This would mean greater losses during the concentration step. Artefact formation is possible under either alkaline or acidic conditions: esters are susceptible to hydrolysis under acid and alkaline conditions to yield the corresponding alcohols and acids: epoxides can yield diols in the presence of acid and water. 8.4.1.2 High-pressure liquid chromatography The fractionation of flavour concentrates using high-pressure liquid chromatography (HPLC) has been used to a limited extent. HPLC is an attractive method for

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Extraction with aqueous mineral acid pH4.0

Aqueous phase

Reextract with organic solvent Organic phase

Aqueous phase

Adjust to pH 9 with alkali Extract with organic solvent _J

I Aqueous phase

I Organic phase (Basic compounds)

Organic phase

Extraction with aqueous alkali pH 10 I , I I Aqueous phase Organic phase (Neutral compounds) Extract with organic solvent

Aqueous phase Organic phase Adjust to pH 4 with minimal acid Extract with organic solvent

Aqueous phase Organic phase (Acidic compounds) Figure 8.8 Fractionation scheme for the of volatiles into acid, neutral and basic fractions (Reineccius and Anandaraman, 1984).

fractionation of flavour concentrates since it utilises a different set of physical properties for component separation than does GC. The potential exists for flavour fractionation on molecular sieves, adsorption, and reversed-phase or normal phase chromatography. A logical sequence would be to use adsorption chromatography first since this technique has the greatest column capacity and is capable of handling the widest range of types of compounds (Teitelbaum, 1977). This would accomplish a fractionation based on adsorption affinity. These individual fractions could then be again fractionated on a normal or reversedphase HPLC column. At this point, substantial separation of the rather complex flavour concentrate has been accomplished and would definitely simplify the subsequent GC analysis.

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The general use of chemically bonded reversed-phase columns for HPLC makes this method attractive (Parliment, 1981). Reversed-phase columns are very inert and will not bleed into the eluting solvent. Therefore, they present minimal concern for contribution to artefact formation. Evaporation of the HPLC solvent may not be necessary for subsequent gas chromatography. Jennings (1979a,b) has demonstrated the applicability of splitless GC for the analysis of dilute solutions. Also, today, large-volume GC injection systems are available that further facilitate this approach. This means that solvent extraction and concentration of the collected fractions from the HPLC might not be necessary, eliminating concerns about solvent extraction efficiency, contamination and losses during concentration of the extracting solvent. There are a few disadvantages to the fractionation of flavour concentrates via HPLC. One concern is for contamination of the flavour concentrates due to HPLC solvents and repeated transfers. If the HPLC eluent must be extracted and concentrated for gas chromatography, then one has also to be concerned with extraction efficiency, solvent impurities and losses during solvent evaporation. The method requires expensive equipment, columns and high-purity solvents. 8.4.1.3 Silicic acid Silicic acid (H2SiO3) and the dehydrated derivative silica gel (SiOi) have been widely used in separating organic compounds via conventional column chromatography. Silica gel, depending on water content, will adsorb organic compounds with different affinities. Elution of these adsorbed compounds with an appropriate organic solvent or elution gradient results in a chromatographic separation. If suitable choices of silica gel activity (amount of water in the gel), solvents, and other chromatographic parameters (adsorbent/adsorbate ratio, column height, column diameter, elution rate, and so on) are used, efficient functional group separations can be accomplished. The separations are primarily due to differences in polarity of compounds in the mixture. Utilising this chromatographic technique, flavour concentrates can be fractionated into a number of less complex mixtures. The primary advantages of silica gel fractionations are the efficiency and simplicity of the method. The method requires only simple chromatographic columns and inexpensive silica gel and solvents. The separation achieved via silica gel chromatography can be quite efficient, depending on the chromatographic conditions chosen. Solvent choice, activity of silica gel, column dimensions and elution rates are the primary factors determining fractionation achieved. Silica gel fractionation also has some inherent disadvantages. A good understanding of these disadvantages is essential for the proper use of this technique. Silica gel may not release the adsorbed compounds quantitatively. Recoveries from a silica gel column can vary considerably (65-95%), depending on the nature of the compounds. Recovery of polar compounds is generally less than that of the less polar ones (e.g. hydrocarbons). Recoveries of the same compound

with the same eluting solvent can vary if the activity of the silica gel is not the same. This can lead to inaccurate quantitative data. The errors are cumulative if repeated silica gel fractionation is attempted prior to analysis. Compounds containing certain labile or reactive functional groups are known to undergo chemical transformation in contact with highly activated adsorbents. For example, active silica gel can promote isomerisation reactions. Epoxides formed from the oxidation of tri- and tetra-substituted double bonds are very susceptible to isomerisation by active silica even at room temperature. Doublebond migration is also known to occur in terpene hydrocarbons in the presence of silica gel. Artefact formation can be minimised or eliminated by carefully controlling the activity of the silica gel and keeping the contact time to a minimum. Faster elution rates can be used only at the expense of some fractionation efficiency. In the published literature, one may note many instances where the authors did not mention either the elution rate or the activity of the silica gel. Standard methods are available for the determination of activity and procedures are available to prepare silica gels of lower activities from fully active gel (Hernandez et al., 1961). Another means of reducing contact time is to use shorter columns when resolution permits. An unnecessarily large adsorbent/adsorbate ratio should not be used. If such unfavourable ratios are employed, isomerisation reactions will be facilitated due to increased contact with active centres or adsorption sites. 8.4.1.4 Preparative gas chromatography Preparative GC as a fractionation technique offers the greatest separating power of the methods discussed. To provide sufficient column capacity (load), either 3 mm or 6 mm (o.d.) packed columns are typically used for resolution of components. The liquid phase of the preparative column is generally chosen to be of opposite polarity to the column used for the analytical study. Column effluent is either split via an effluent splitter (10:1 exit/detector) or passed through a nondestructive detector. This effluent is usually trapped in a cold trap for rechromatography on an analytical column. The primary advantages to preparative GC fractionation are the extreme efficiency of separation and the fact that fractions collected are solvent-free. If a large number of chromatographic cuts are taken from a run, each fraction collected can contain only a few compounds for further analysis. If the preparative GC work is done on a column of opposite polarity to the analytical column, separation by the analytical column is simplified. Since the collected fractions are solvent-free, sensory analysis is easily done on individual fractions. This often aids in narrowing the flavour study to a relatively simple fraction or few fractions. The solvent-free aspect also means that trace components in a flavour isolate can be made the major components in the fraction collected. One chooses the chromatographic cuts such that trace components are collected

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separately from the major components. This greatly enhances the separation and identification of trace components of food flavour. Preparative GC may be criticised as a method that subjects the flavour isolate to high temperatures and active surfaces. A survey of early published literature in flavour fractionation reveals that invariably a packed metal column of over 3 m has been used in the preparative GC work. Large surface areas offered by the solid support and the inner metal surface of the column could prove extremely detrimental to labile compounds or could irreversibly adsorb other compounds. The residence time of high-boiling compounds may also promote artefact formation. Re-chromatography and sub-fractionation increase the concerns. Recent efforts have recognised these potential errors and have used deactivated solid supports, all-glass injection ports, glass columns and glass-lined transfer lines. Preparative GC generally complicates quantification such that it is used only for qualitative studies. 8.5

Flavour analysis by gas chromatography

Gas chromatography is the single most widely used technique in flavour studies. It is exceptionally uncommon to see any analytical flavour work that does not include gas chromatography. This is not unexpected, since compounds most commonly of interest are those that are volatile and therefore may contribute to aroma. Gas chromatography has tremendous separating power, sometimes in excess of 200000 theoretical plates per column. This attribute is essential for the separation of complex flavour isolates. Detectors available for GC work offer excellent sensitivity, providing picogram detection levels, and yet may be general (flame ionisation) or very specific (flame photometry, electron capture, and nitrogen/phosphorus). It is not unexpected, then, that flavour research advanced greatly in the mid-1960s when GC became readily available to the flavour chemist. A tabulation of flavour compounds identified in foods lists only 500 compounds found by 1963. Only 15 years later, this number had increased to over 3000 and, today, over 7000 have been identified. We will not go into a general discussion of GC. There are many excellent books on this topic (Matter, 1977; McNair and Miller, 1998; Jennings et al.. 1997; Scott, 1997). We will, however, go into some aspects of GC that are particularly relevant to GC studies in flavour research. 8.5.7 High-resolution gas chromatography (HRGC) The primary purpose of gas chromatography in flavour research is to separate aroma mixtures into individual components. Once they are separated, quantification and identification become primary concerns. Owing to the complex nature of most food flavours, resolving power is a critical need in GC. Therefore, capillary column GC (HRGC) is the standard today. The primary disadvantage

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of using capillary columns in flavour analysis is their limited capacity. Flavour studies may require the trapping of individual components for infrared, NMR, sensory evaluation, or other analyses. Column capacity is typically being enhanced through the use of thick-film coatings as opposed to wide-bore columns (smaller losses in efficiency). Whereas a typical fused-silica column is limited to less than 100 ng of each component, thick-film fused-silica columns will handle 500 ng of each component without overloading. While this is still not sufficient for some needs, the use of automated injection systems and capillary fraction collectors can result in the collection of substantial amounts of material. When HRGC does not offer adequate resolving power for a particular application, resolving power can be enhanced through the use of cold trapping and re-chromatography (off-line) or multidimensional GC (in-line). The former method employs a fraction collector that can take heart cuts out of a GC run; the operator will then manually re-inject each cut onto a second GC. The second GC run is typically equipped with a different GC column phase to enhance resolving power. The in-line version of this process automatically does the heart cutting and re-injection of the sample into a second GC column (and oven). Wright (1997) has presented a discussion and examples of the application of multidimensional chromatography in flavour analysis. 8.5.2 Gas chromatography-olfactometry (GC-O) Gas chromatography of flavour extracts provides the opportunity to use a rather unique detector, the human nose. The usual GC detector provides very little information for the flavour chemist—simply a line on a paper. A trained person can smell the GC effluent and tell the potency (sensory intensity) of a GC peak, character, and often even the chemical identity of a peak. Compounds with extremely low sensory thresholds (e.g. sulfur compounds) may be detected by smell but not detected by the GC. The nose may tell us that we have to concentrate the sample further or use a different isolation technique to prepare the sample for GC. A chromatogram obtained via smelling of the GC effluent, and therefore containing sensory information, is called an 'aromagram'. GC-O can be used in a different manner to provide information about key aroma contributors to foods. This has been a very active research endeavour for the last 20 years or more and is discussed below and by Blank (1997) and Leland et al. (2001). 8.5.2.1 GC-O to obtain aromagrams Simultaneous GC detection and odour profiling are accomplished using an effluent splitter, a nondestructive detector or chromatography without and with a detector (two GC runs). When an effluent splitter is used, the effluent is generally split such that a larger portion goes to the nose than to the GC detector (10:1). Whereas the nose is often more sensitive than the GC detector, dilution in air at the time of smelling and human response time necessitate a split in favour

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of the nose. The thermal conductivity (TC) detector is most commonly used for odour profiling when a nondestructive detector is desired. Whereas a TC detector may seem to be a better choice than effluent splitting, since the entire sample is available for GC detection and smelling, a TC detector is not as sensitive as other GC detectors. Flame ionisation detectors (FID) have a 104-106 times lower detection limit than a TC detector. Therefore, even with the 10:1 split, the use of an FID is preferred to a TC nondestructive approach. The final approach, that of making one GC run with effluent smelling (no GC detection) followed by a second GC run with GC detection (no smelling) is also done. This offers the maximum sample quantity to both the GC detector and the nose. The disadvantage is the time required for two GC runs and ambiguity of what odour goes with what peak when resolution is difficult. GC-O may be criticised as being a subjective method yielding inconsistent results. However, independent judgements of well-trained subjects can minimise this concern. The duration of a routine GC run is often in excess of 30 min. Odour fatigue can set in well before the end of the analysis, leading to incorrect odour descriptions. Thus, one must be conscious of the time a subject is asked to perform this task (it may be limited to 20 min). Odour characteristics of some flavour compounds tend to vary as a function of concentration. Skatole (3-methylindole) has a characteristic faecal odour at high levels but becomes pleasant, sweet, and warm at very low levels. Fortunately, there are not many aroma compounds exhibiting such a large concentration-dependent odour character. Furthermore, attempts to indicate the perceived intensity of a GC peak can be in error owing to masking in mixtures. Finally, in-line condensation of some compounds can result in persistent background odours. The splitter and the transfer lines should be well-conditioned and adequately heated to render them odour-free. Despite these potential pitfalls, GC-O is an invaluable tool to the flavour chemist and has found frequent application in this field (Leland et al., 2001). 8.5.2.2 GC-O to select key odorants in foods As mentioned earlier, the aroma, i.e. the volatile component, of a food is generally made up of a very complex mixture of volatiles. It is well accepted that not all of these volatiles contribute to sensory perception. Many compounds are present in a food at concentrations below the level needed to elicit a sensory response. Research has generally shown that 10-30 volatiles are adequate to reproduce the aroma of any food studied thus far. The analytical challenge is to determine which of perhaps 800+ aroma compounds (such as meat aroma) are needed to properly reproduce the sensory character of a food. Several different approaches to this challenge have appeared in the literature. The earliest work in this area is now more than 40 years old (Patton and Josephson, 1957). They proposed estimating the importance of a flavour compound by the ratio of the compound to its threshold concentration. This ratio is known as the odour activity value (OAV) (also as odour value, odour unit.

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flavour unit, or aroma value). This ratio indicates by how much the actual concentration of a compound exceeds its sensory threshold. They suggested that compounds present above their sensory threshold concentrations in a food are significant contributors to its aroma, whereas those occurring below threshold are not. Patton and Josephson (1957) proposed this method as a guide 'that may not hold in some instances'. The OAV concept was applied to mixtures by Guadagni et al. (1966), who suggested that if the perceived intensity of odorants in a mixture is additive, the relationship between OAV of single components in the mixture and the OAV of that mixture is OAV(W = OAVW

OAVn + OAV/

(8.1)

where /i , . . . , / „ represent compound 1 , . . . , n in mixture m. Thus, the relative contribution of a compound to a mixture could be described as the ratio of its OAV to the OAV of the mixture. Guadagni et al. (1966) noted that this implied nothing about the odour quality of the final mixture and nothing about the relationship between the stimulus concentration and sensation above threshold. Since the introduction of the OAV concept, GC-O and OAV approaches have been extensively used to screen for 'significant' odorants in food. Two major screening procedures for determining the key odorants in food are based on this concept. Grosch developed the Aroma Extract Dilution Analysis (AEDA; Ullrich and Grosch 1987) and a recent variation, the Aroma Extract Concentration Analysis (AECA; Kerscher and Grosch, 2000); Charm Analysis was developed by Acree et al. (1984) (Figure 8.9). These two methods evaluate

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Figure 8.9 Schematic of the GC-O system used in obtaining Charm data (Acree, 1993).

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FOOD FLAVOUR TECHNOLOGY

by GC-O a dilution (or concentration) series of the original aroma extract from a particular food and attempt to rank the key odorants in order of potency. The highest dilution at which a substance is smelled is defined as its dilution value. The dilution value is proportional to the OAV evaluated in air. Both AEDA and Charm methodologies originally proposed that the larger the dilution value, the greater the potential contribution of that compound to the overall aroma. With time, data interpretation has changed. Researchers now consider AEDA, OAV and Charm methodologies to be screening in nature. These methodologies are used to determine those aroma compounds most likely to make a contribution to the odour of a food, recognising that sensory work (e.g. recombination studies) needs to be done to determine which aroma compounds are truly contributory. Interpretation has changed owing to recognition that the methods violate certain sensory rules or psychophysical laws (Frijters, 1978; Piggot, 1990; Abbot et al.. 1993;Mistrye?a/., 1997). Two other GC-O methods have also found application for this purpose. One is called OSME and the other NIF (nasal impact frequency) or SNIP (surface of nasal impact frequency). OSME was developed by McDaniel et al. (1990) and has been applied to wine aroma studies. In her method, a panellist evaluates the aromas eluting from a GC column and responds by moving a variable resistor as aroma intensity changes (Figure 8.10). Thus, one is obtaining intensity and duration measurements of each GC peak. There are no dilutions made of the sample, which facilitates the use of a larger number of judges as opposed to

Filters J3as-liquid chromatograph isory detector

Figure 8.10 GC-O system used by McDaniel for obtaining OSME data (McDaniel et al.. 1990).

INSTRUMENTAL METHODS OF ANALYSIS

239

a single judge doing dilutions. This adds further validity to the method. The importance of an odorant to the overall aroma is judged on the basis of relative sensory intensities during sniffing. This is a fundamental difference between the dilution methods (AEDA, OAV and Charm) and OSME. The dilution methods assume that compounds present at the greatest multiple of their threshold are most important to aroma. This violates a basic law of sensory science in that there is a power function relationship between concentration and sensory intensity and that relationship is different from one aroma compound to another. Thus, one cannot unequivocally rank compound intensity based on OAV, Charm or AEDA value. This weakness is recognised and these values are now considered as screening techniques as opposed to providing 'hard' numbers: compounds with the highest values are candidates for further study to evaluate their true contribution. The NIF (or SNIP) method was developed by Pollien et al. (1997). In this method, 8-10 untrained individuals sniff the GC effluent (one at a time). They simply note when they smell an odour. The aroma isolate used is adjusted in strength such that about 30 odorants are perceivable to the sniffers. This adds an element of selection in that only the more intense aroma compounds will be evaluated. The number of sniffers detecting an odorant is tabulated and plotted. Those odorants (GC peaks) being detected by the greatest number of individuals are considered the most important. The method has its weaknesses. One problem is that, for two compounds, one may be barely over the sensory threshold of all sniffers while another may be a great distance above its sensory threshold for all sniffers and yet both of these compounds would be viewed as being equal by this methodology. As in searching for the ideal method of aroma isolation, there is no perfect method for selecting key odorants in foods. Each method has weaknesses. The result is that we find more sensory work being done to evaluate the analytical data. Very often, researchers are using recombination studies followed by sensory analysis to determine what is contributing to the aroma of a food and what is not. If we are to attempt any re-creation of a flavour, we must have a list of odorants to study and have some reasonable basis for their selection and ranking. The GC-O methods that have been developed have served this purpose quite well. 3.5.3 Specific gas chromatographic detectors The nature of aroma compounds being studied (elemental composition) and the type of analysis desired may govern the choice of GC detector. For example, an alkali flame ionisation detector (NPD) may be employed to facilitate detection of nitrogen-containing compounds, and a flame photometric detector (or atomic emission detector) to analyse sulfur-containing compounds. If a nondestructive detector is desired, a micro-cross-section thermal conductivity detector is one of

240

FOOD FLAVOUR TECHNOLOGY

the choices. Flame ionisation detectors (FID, a nonspecific detector) are the most commonly used detectors in flavour analysis because of their high sensitivity, long life and relatively low cost. However, specific or selective detectors are often used to good advantage in flavour analysis. Selective detectors have several advantages. For example, the relative sensitivity of a specific detector can be manyfold higher with respect to a certain element as compared with a FID. An alkali FID can have 35 000-75 000-fold greater response for nitrogen and phosphorus when compared with an equal amount (weight basis) of carbon. This illustrates how a detector can aid analysis of a trace pyrazine or any nitrogen-containing compound in aroma analysis. Selective detection can also aid in identification of compounds. At times, mass spectral data cannot be interpreted with adequate certainty to make identification in GC-MS analysis of flavours. Specific detectors can provide information that facilitates interpretation of such spectra and subsequent identification. Employing specific detectors in aroma analysis cannot be to the analyst's disadvantage. However, it should be mentioned that these detectors require precise calibration for quantitative analysis. One should also keep in mind that some specific detectors only enhance the response of selected elements. They may still give limited response to other elements. Therefore, the results could be misleading. 8.6 Flavour analysis by high-pressure liquid chromatography (HPLC) Application of HPLC in flavour analysis has been quite limited. Major reasons for this are lack of sensitivity and resolution. Although analysis of nonvolatile derivatives of flavour compounds and fractionation of flavour isolates have been achieved via this approach, a complete flavour analysis has not been done using HPLC. Gas chromatography has been used extensively in flavour analysis, and advancements in column technology and detectors have made extremely high resolution and sensitivity possible. This, to some extent, has overshadowed the progress in the development of techniques for flavour analysis using HPLC. 8.7

Identification of volatile flavours

In most flavour studies it is desirable and most often necessary to identify the flavour components of interest (note the qualification 'of interest'). The problem may be to determine the volatiles that are important to coffee flavour or those that produce a taint in green beans. A decision should be made prior to starting identifications as to what should be identified. Despite rather sensitive and sophisticated instrumentation to aid in identification, the task of identifying every volatile component in a flavour isolate should be restricted only to the

INSTRUMENTAL METHODS OF ANALYSIS

241

young and/or foolish. Decisions about what needs to be identified should be based on sensory evaluation—i.e. GC-O data. One can then often narrow the task to the identification of a limited number of components. Although there are a multitude of methods available to the chemist for the identification of unknowns, only a few are useful to the flavour chemist. Unfortunately, the choices are very limited because of the extremely small quantities of material available to work with. Some flavour chemicals make a contribution to sensory perception when present in the food at parts per trillion (or less). This translates into the flavour chemist having a few nanograms, at best, to work with following isolation and concentration procedures. That is a long way from the gram or milligram necessary for many classical identification techniques. While quantities of components are the primary limiting factors determining the identification method, one should also recognise that most identification methods require pure compounds. This means that they will generally have to be trapped pure (one hopes) from a GC run. HRGC provides limited sample capacity and, owing to the limitations discussed, the flavour chemist depends most heavily upon GC and GC-mass spectrometry (GC-MS) for identifications. Infrared and nuclear magnetic resonance may also be used but much less frequently. 8.7.1 Gas chromatography Gas chromatography is primarily used as a technique for the separation of mixtures. However, owing to its extreme sensitivity, substantial effort has been directed towards developing identification methods utilising GC. These methods are based primarily on retention time data. 8.7.1.1 Retention time/retention indices Retention time, in an absolute sense, is not a suitable parameter for compound identification. Minor variations in operator technique or operational parameters produce retention time shifts. Thus, relative retention times, based on the retention time of an unknown on co-injection of a homologous series of n-paraffins or ethyl esters, are used. The n-paraffin standard was developed by Kovats and has become the most widely used retention index system. Compilations of Kovats indices of aroma compounds are available from several sources including Acree (2001), Kondjoyan and Berdague (1996), and the LRI and Odour Database. The flavour industry often uses ethyl ester standards. Substantial work has been done on polar GC columns (Carbowax 20M) and the nonpolar n-paraffins do not provide as good retention references as the more polar ethyl esters on these columns. The use of Kovats indices alone for compound identification is considered weakly tentative even if Kovats indices agree with published standards on two different column phases. A polar and a nonpolar phase should be selected for evaluation. A necessary word of caution is that published Kovats indices will

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differ from those run in another laboratory. Differences arise due to operator technique, minor column differences, and operational parameters. Therefore, retention indices are accurate only when they are obtained on the same column and instrument. Published data are useful, however, in narrowing choices or giving ideas of possible identity. The analyst must have additional data supporting an identification beyond GC data. This may be aroma character, mass spectrometry or nuclear magnetic resonance data. 8.7.1.2 Selective GC detectors Information about elemental composition may be obtained by the use of specific GC detectors. The most commonly used GC detectors have been discussed earlier in this chapter, but several other detectors are also available. GC detectors available include flame ionisation, flame photometric, alkali flame ionisation, electrolytic conductivity, microcoulometric, microwave plasma (atomic emission), radioactivity, and electron capture detectors. Information on these can be found in the literature. The utility of selective detectors in aiding in the identification of an unknown is rather evident and will not be pursued in this chapter. 8.7.2 Infrared spectroscopy Infrared (IR) spectroscopy was at one time the second most commonly used method in flavour research (second only to GC). Compounds were separated by GC and trapped for IR to aid in identifications. Two factors have combined to move this technique now into a distant third position behind mass spectrometry. The relatively large sample requirements of IR (l-10ug) are not compatible with current HRGC. Also, the direct coupling of mass spectrometry and gas chromatography, and the development of low-cost low-resolution mass spectrometry instruments have made it an extremely desirable (and ubiquitous) tool for the flavour chemist. Infrared spectroscopy provides information on the functional groups present in an unknown. Basically, it measures the vibrational modes of a molecule, which are determined by structure and functional groups. The most common problem in applying IR to flavour research is obtaining enough pure sample to obtain a usable absorption spectrum. These requirements are, to some extent, mutually exclusive since HRGC is required for assuring purity but provides little capacity. The simplest approach has been to couple IR with GC. Innovations in IR detectors and gas cells, the use of interferometer mirrors as replacements for the monochromator, and Fourier transform infrared spectroscopy have permitted the direct coupling of GC and IR. Unfortunately, gas phase IR spectra of some compounds may differ greatly from their liquid phase spectra. This may complicate IR interpretation and compound identification since most of the IR

INSTRUMENTAL METHODS OF ANALYSIS

243

literature is based on liquid phase IR. This approach also suffers from running in real time, the result being poor (inadequate) sensitivity. The alternative approach has been to use systems in which the GC effluent is frozen directly on a rotating mirrored surface. The mirrored surface is then scanned by IR 'off-line' to give a liquid spectrum and greater sensitivity. These instruments tend to be expensive and require considerable skill in use and, thus, have found limited application in our field. 8,7.3 Mass spectrometry 8.7.3.1 Compound identification or quantification Mass spectrometry (MS) has become the second most commonly used instrumental technique in flavour research. Mass spectrometry is exceptionally well adapted to flavour research since it is readily connected to GC, has excellent sensitivity (10-100 pg), and provides more structural information than any other spectroscopic method. There is little question that MS has been so successful in flavour research that other identification techniques have not been developed or fully applied in this area. The widespread use of GC-MS has resulted in large comprehensive spectral libraries with efficient, cheap, computerised spectrummatching systems. This adds ease of interpretation to an already attractive method. Mass spectrometers may be classed as low-resolution (LR) or high-resolution (HR) instruments. The LR instruments provide mass measurements to the closest whole mass unit. Since many combinations of elements may give the same unit mass, LR may provide molecular mass but does not provide elemental composition. High-resolution instruments will provide sufficiently accurate mass measurements to permit determination of elemental composition. The majority of flavour work in the past has utilized LR instruments. This is primarily because LR instruments are cheaper to purchase and operate than high-resolution instruments. Mass spectrometry is generally used in the flavour area either to determine the identity of an unknown or to act as a mass-selective GC detector. As mentioned, MS as an identification tool is unequalled by other instruments. The systems have largely become turnkey systems that require little or no operator expertise. If the operator can do GC, he or she can do MS. Comprehensive MS libraries and efficient searching algorithms make identification simple. Herein lies a danger. Mass spectrometry will provide a best match (suggest the identity) for any unknown irrespective of the validity of the match. The neophyte often accepts the proposed identifications without question and obtains incorrect identifications. It is essential that any MS identifications be supported by other data, for example, GC retention data, IR, NMR or odour character. The use of a mass spectrometer as a GC detector can facilitate some quantification problems that would be difficult or impossible by other techniques. For this

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FOOD FLAVOUR TECHNOLOGY

purpose, the mass spectrometer is operated in the selected ion or multiple-ion detection mode. In this mode, it continuously measures only selected ions at very short time intervals throughout a GC run. This makes it possible to separately quantify two components that are co-eluting from the GC (this may be two different compounds or a compound and its stable isotope counterpart used in quantitative studies). The mass spectrometer is generally set up to measure two or three ions unique to each component being quantified. It is wisest to use two or three ions to minimise the probability of an individual ion being contributed by another compound. The computer will then reconstruct mass chromatograms for each of the masses quantified. The extreme specificity and sensitivity of this technique makes selected ion monitoring a very valuable technique. Selected ion monitoring can also be used to enhance GC detection sensitivity or do suspect screening runs. The MS detector is generally more sensitive than flame ionisation detection. Thus, a gain is made in instrument sensitivity. If one knows or suspects certain compounds (e.g. off-flavour taints) in a GC profile, selected-ion monitoring is an excellent means of screening the flavour extract for these compounds. Poor GC resolution or limited sample may make interpretation of complete MS scans difficult, whereas selected ion monitoring is quite simple in these situations. 8.7.3.2 Direct MS of food aromas to measure aroma release Mass spectrometry may be used for the direct measurement (no separation) of volatiles released from foods or in human breath during eating (Taylor. 1996). This has applications in understanding how food composition and texture influence flavour perception. The process has been evolutionary as one would expect. One of the earliest methods for measuring aroma release from foods by MS simply passed a carrier gas over a food in a vial that was shaken with glass beads while held at constant temperature. The gas eluting from the vial was fed directly into an electrospray ionisation mass spectrometer (Lee, 1986). This technique lacked sensitivity and measured total volatiles as opposed to individual aroma components. Oxygen and moisture in the system were limiting factors. Soeting and Heidema (1988) and Springett et al. (1999) chose to use a membrane MS inlet to remove the interfering components, but this adds elements of compound discrimination and reduces resolution, response time and sensitivity. The best approach to date was developed by Taylor et al. (2000b) and involves the use of an atmospheric pressure ionisation (API) inlet coupled to MS. Taylor et al. (2000b) interfaced this inlet to a quadrupole mass spectrometer, while Grab and Gfeller (2000) used an ion trap mass spectrometer. These inlet systems are extremely robust and tolerate the host of non-aroma components, water and oxygen prevalent in the human breath or above foods. The MS system and its performance can be best understood by the illustrations that follow. The inlet system designed by Taylor et al. (2000b) is presented

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INSTRUMENTAL METHODS OF ANALYSIS

in Figure 8.11. In this system, the breath is drawn into the ionisation source by a venturi effect created by high nitrogen gas flows (source gas). The volatiles in the breath are ionised by the corona discharge pin and drawn into the MS analyser. The mass spectrometer then monitors individual ions characteristic of the compounds of interest. The raw data look like that shown in Figure 8.12 for chewing a mint gum. The top line indicates chewing times, the acetone line is indigenous to the breath and indicates breathing times, the next two lines (carvone and menthone) reflect their release from the gum during chewing and the bottom line is time/intensity data from a panellist. These data are tabulated and smoothed to result in the view of aroma release presented in Figure 8.13. These types of data are exceptionally valuable in understanding the factors that determine aroma perception in foods.

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4 1

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I

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Menthone

Time intensity 3.4

3.6

Time (min) Figure 8.12 Raw data obtained on aroma release during chewing on a mint gum (Taylor et al., 2000a).

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120

0

1

2

3

4

5

6

Time (min) Figure 8.13 The release of aroma, sucrose and flavour perception in chewing gums (Taylor etal.. 2000a). -•- Sucrose release: -•- Menthone release: — Time-intensitv.

8.8

Electronic 'noses'

An electronic nose functions by analysing a sensor array response to a complete aroma, that is there is no separation of aroma components. The sensory array response to any given aroma is correlated (using pattern recognition software) to sensory panel data. Using neural network software and many 'acceptable' and 'unacceptable' samples, the system determines a sensor response pattern that is representative of a fresh milk versus a spoiled milk, for example. This technique is relatively young but has generated a substantial number of publications (Hodgins, 1997). The technique is particularly attractive for quality control applications where an acceptable/unacceptable decision is often needed. The sensors are key components of this system (Hodgins, 1997). Currently, there are several types of sensors including semiconductor gas sensors (metal oxides), surface acoustic wave devices, biosensors, conducting polymers and mass spectrometry-based sensors (Marsili, 1999). In the current instruments, it is common to combine sensor types to gain a wider range in responses. At first glance the technique appears to be ideal in that there is no need for separation of volatiles. This can result in very rapid analysis. Also, it seems to be based on a process similar to the human olfactory system in that both the electronic nose and human olfactory systems consist of a host of receptors (sensors) and yield a pattern of response to any given aroma. The brain, in the case of the human, and the computer, in the case of the electronic nose, make judgements based on a pattern recognition process as to the aroma and its quality. Thus, the speed is attractive and the theoretical foundation appears to be rational.

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The primary weakness of such instrumentation is that one has no clear idea of what the instrument is responding to in making a judgement. One chooses to evaluate some sensory parameter (e.g. staling or rancidity during storage) and then asks the instrument to develop a means of predicting that sensory parameter. In the end, the instrument uses some stimuli/response pattern to make a prediction, but we have no idea that what the instrument was measuring. For example, roast and ground coffee gives off CO2 during ageing. In a storage study where one is determining the sensory quality of coffee and obtaining electronic nose correlations, the instrument could be responding to CC>2 as opposed to any oxidised flavour. As long as CO2 outgassing is correlated to lipid oxidation, the relationship is good. If it is not correlated in all situations, then the relationship is likely to be invalid in other systems or studies. The point is that the human brain uses causative input/patterns to make judgements, while the electronic nose uses patterns that are not necessarily causative and may be only casually or haphazardly related. Another potential concern about this type of instrumentation is that sensors may respond to water vapour or CC>2 and these responses may dominate or unduly alter sensor patterns. The sensors also deteriorate with time (or can be 'poisoned'), therefore changing response, which makes shelf-life studies problematic or frequent calibration necessary. These weaknesses largely relegate the technique to quality control situations as opposed to research studies. However, one must recognise the inherent weaknesses of the technique so as not to misuse it.

8.9

Summary

Quantitative isolation and analysis of volatile flavour compounds is fraught with practical difficulties and an appreciation of the limitations this places on data interpretation is essential. This is well illustrated in Chapter 4, where the poor performance of flavours formulated from analytical data alone are obvious. Flavour analyses can be used successfully in comparing samples with the same matrix (e.g. a hydrocolloid solution), but extrapolating data beyond the limits of the analysis can lead to gross errors.

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Kerscher, R. and Grosch, W. (2000) Comparison of the aromas of cooked beef, pork and chicken, in Frontiers of Flavour Science (eds P. Schieberle and K.H. Engel), Deutsche Forschungsanstalt fiir Lebensmittel, Garching, pp 17-20. Kondjoyan, N. and Berdague, J.L. (1996) A Compilation of Relative Retention indices for the Analysis of Aromatic Compounds, Edition du Laboratoire Flaveur, Station de Recherche sur la Viande, Sainte Genes Champanelle, France. Leahy, M.M. and Reineccius, G. A. (1984) Comparison of methods for the analysis of volatile compounds from aqueous model systems, in Analysis of Volatile?;: New Methods and Their Application (ed. P. Schreier), DeGruyter, Berlin, pp 19-47. Lee, W.E. (1986) A suggested instrumental technique for studying dynamic flavor release from food products. J. Food ScL, 51 249-250. Legendre, M.G., Fisher, G.S., Fuller, W.H., Dupuy, H.P. and Rayner, E.T. (1979) Novel techniques for the analysis of volatiles in aqueous and nonaqueous systems. /. Am. Oil Chem. Soc., 56 552-555. Leland, J.J., Schieberle, P., Buettner, A. and Acree, T.E. (2001) Gas chromatography-olfactometry: The State of the Art, ACS Symposium Series 782, American Chemical Society, Washington DC. LRI and Odour Database, www.odour.org.uk Marsili, R. (ed.) (1997) Techniques for Analyzing Food Aroma, Marcel Dekker, New York. Marsili, R.T. (1999) SPME-MS-M VA as an electronic nose for the study of off-flavors in milk. J. Agric. Food Chem., 47 648-654. Matter, L. (ed.) (1977) Food and Environmental Analysis by Capillary Gas Chromatography: Hints for Practical Use, Huethig, Heidelberg. McDaniel, M.R., Miranda-Lopez, R., Watson, B.T., Michaels, N.J. and Libbey, L.M. (1990) Pinot noir aroma: a sensory/gas chromatographic approach. Flavors and Off-flavors (ed. G. Charalambous), Elsevier, Amsterdam, pp 23-36. McNair, H.M. and Miller, J.M. (1998) Basic Gas Chromatography: Techniques in Analytical Chemistry, Wiley, New York. Milo, C. andBlank, I. (1998) Quantiftcati on of impact odorants in food by isotope dilution assay: strengths and limitations, in Flavor Analysis (eds C.J. Mussinan and M.J. Morello), ACS Symposium Series 705, American Chemical Society, Washington DC, pp 250-259. Mistry, B.S., Reineccius, T.A. and Olson, L.K. (1997) Gas chromatography-olfactometry for the determination of key odorants in foods, in Techniques for Analyzing Food Aroma (ed. R. Marsili), Marcel Dekker, New York, pp 265-292. Morello, M.J. (1994) Isolation of aroma volatiles from an extruded oat ready-to-eat cereal, in Thermally Generated Flavors (eds T.H. Parliment, M.J. Morello and R.J. McGorrin), ACS Symposium Series 543, American Chemical Society, Washington DC, pp 95-101. Mussinan, C.J. and Morello, M.J. (eds) (1998) Flavor Analysis, ACS Symposium Series 705, American Chemical Society, Washington DC. Nijssen, L.M., Visscher, C.A., Maarse, H., Willemsens, L.C. and Boelens, M.H. (1996) Volatile Compounds Found in Food, 7th edn, TNO Nutrition and Food Research Institute, Zeist, The Netherlands. Parliment, T.H. (1981) Concentration and fractionation of aromas on reverse-phase adsorbents. J. Agric. Food Chem., 29841-845. Parliment, T.H. (1986) Sample preparation techniques for gas-liquid chromatographic analysis of biologically derived aromas, in Biogeneration of Aromas (eds T.H. Parliament and R. Croteau), American Chemical Society, Washington DC, pp 34-52. Parliment, T.H. (1997) Solvent extraction and distillation techniques, in Techniques for Analyzing Food Aroma(ed. R. Marsili), Marcel Dekker, New York, pp 1—26. Patton. S. and Josephson, D. (1957) A method for determining significance of volatile flavor compounds in foods. Food Res., 22 316-318. Pawlis/yn, J. (1997) Solid Phase Microextraction: Theory and Practice, VCH, New York.

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Piggot, J.R. (1990) Relating the sensory and chemical data to understand flavor. J. Sensory Studies. 4 261-272. Pollien, P., Ott, A., Montigon, F. Baumgartner, M.. Munoz-Box. R. and Chaintreau. A. (1997) Hyphenated headspace-gas chromatography-sniffing technique: screening of impact odorants and quantitative aromagram comparisons. J. Agric. Food Chem.. 45(7) 2630 -2637. Qian. M. (2000) Study of Parmesan cheese aroma. PhD dissertation. University of Minnesota, St Paul. MN. Reineccius, G.A. (1989) Isolation of Food Flavors. Flavors in Lipid Foods (eds D.B. Min andT. Smouse). American Oil Chemists Society, Champaign, IL. pp 26-34. Reineccius. G.A. (1993) Biases in analytical profiles introduced by isolation method, in Flavor Measurement (eds C.T. Ho and C. Manley), Marcel Dekker. New York, pp 61-76. Reineccius, G.A. and Anandaraman, S. (1984) Analysis of volatile flavors in foods, in Food Constituents and Food Residues: Their Chromatographic Determination (ed. J.E. Lawrence). Marcel Dekker. New York, pp 195-293. Reineccius, G.A. and Liardon. R. (1985) The use of charcoal traps and microwave desorption for the analysis of headspace volatiles above heated thiamine solutions, in Topics in Flavour Research (eds R.G. Berger, S. Nitz and P. Schreier), H. Eichorn, Marzling-Hangenham. pp 125-138. Roberts, D.D. and Pollien. P. (2000) Dependence of "salting out" phenomenon on the physical chemical properties of the compounds, in Frontiers of Flavour Science (eds P. Schieberle and K.H. Engel). Deutsche Forschungsanstalt fiir Lebensmittel. Garching, pp 311-315. Roberts, D.D.. Pollien. P. and Milo. C. (2000) Solid phase microextraction method development for headspace analysis of volatile flavor compounds. J. Agric. Food Chem.. 48 2430-2437. Rothaupt, M. (1998) Thermo Desorption as sample preparation technique for food and flavor analysis by gas chromatography. in Flavor Analysis (eds C.J. Mussinan and M.J. Morello). ACS Symposium Series 705, American Chemical Society, Washington DC. pp 116-122. Schaefer. J. (1981) Comparison of adsorbents in headspace sampling, in Flavour '81 (ed. P. Schreier). de Gruyter. Berlin, pp 301-313. Scott. R.P.W. (ed.) (1997) Introduction to Analytical Gas Chromatography. 2nd edn. Marcel Dekker. New York. Siek. T.J. and Lindsay. R.C. (1968) Volatile components of milk fat steam distillates identified by gas chromatography. / Dairy Sci.. 57(12) 1887-1896. Soeting, W.J. and Heidema, J. (1988) A mass spectrometric method for measuring flavor concentration/ time profiles in human breath. Chem. Senses, 13 607-617. Springett. M.B.. Rozier. V. and Bakker. J. (1999) Use of fibre interface direct mass spectrometry for the determination of volatile flavour release from model systems. J. Agric. Food Chem.,4H 1125-1131. Sucan. M.K., Fritz-Jung, C. and Ballam, J. (1998) Evaluation of purge-and-trap paprameters: optimization using a statistical design, in Flavor Analysis (eds C.J. Mussinan and M.J. Morello). ACS Symposium Series 705. American Chemical Society. Washington DC. pp 22-37. Sullivan. J.F.. Konstance. R.P.. Calhoun. M.J.. Talley. F.B. Cording. J. Jr. and Panasiuk. O. (1974) Flavor and storage stability of explosion-puffed potatoes. Nonenzymatic browning. J. Food Sci.. 39 58-60. Taylor. A.J. (1996) Volatile release from foods during eating. Crit. Rev. Food Sci. Nutr.. 36(8) 765-784. Taylor. A.J.. Linforth. R.S.T.. Back. I.. Marin. M. and Davidson. J.M. (2000a) Flavour analysis under dynamic conditions: measuring the true profile sensed by consumers, in Frontiers of Flavour Science (eds P. Schieberle and K.H. Heinze). Deutsche Forschungsanstalt fur Lebensmittel. Garching. pp 255-260. Taylor, A.J., Linforth. R.S.T.. Harvery. B.A. and Blake. A. (2000b) Atmospheric pressure chemical ionization mass spectrometry for /;; vivo analysis of volatile flavour release. Food Chem.. 71 327-338. Teitelbaum, C.L. (1977) A new strategy for the analysis of complex flavors. J. Agric. Food Chem.. 25 466-470.

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Teranishi, R. (1998) Challenges in flavor chemistry: an overview, in Flavor Analysis (eds C.J. Mussinan and M.J. Morello), ACS Symposium Series 705, American Chemical Society, Washington DC. Ullrich, F. and Grosch, W.Z. (1987) Identification of the most intense odor compounds formed during the autoxidation of linoleic acid. Z. Lebensm.-Unters. Forsch., 184 277—282. Wampler, T.P. (1997) Analysis of food volatiles using headspace-gas chromatographic techniques, in Techniques for Analyzing Food Aroma (ed. R. Marsili), Marcel Dekker, New York, pp 27-58. Werkhoff, P., Bretschneider. W., Herman, H. and Schreiber, K. (1989-1990) Flavor analysis (a series of 12 articles), Labor Praxis. Weunnan, C. (1974) Sampling in airborne odorant analysis, in Human Responses to Environmental Odors (eds A. J. Turk, W. Johnston and D. G. Moulton), Academic Press, New York, pp 263-328. Widner, H.M. (1990) Recent developments in instrumental analysis, in Flavour Science and Technology (eds Y. Bessiere and A.F. Thomas), Wiley, Chichester, pp 180-190. Wright, D.W. (1997) Application of multidimensional gas chromatography techniques to aroma analysis, in Techniques for Analyzing Food Aroma (ed. R. Marsili), Marcel Dekker, New York, pp 113—141. Zhang, Z., Yang, M.L. and Pawliszyn, J. (1994) Solid phase-microextraction: a solvent-free alternative for sample preparation. Anal. Chem., 66 844A-857A.

Sensory methods of flavour analysis Ann C. Noble

9.1

Introduction

Similarly to instrumental analysis of flavour, sensory tests must be conducted under standardised and controlled conditions. However, many factors influence subjects psychologically and physiologically, and so protocols for sensory studies require control of additional variables. For example, it is important to remove all external clues that can bias perception. Tests must be conducted in facilities that prevent distraction. Studies must be designed to account for sequence effects that influence responses. Further, sensory judges require training, unlike instruments, although both need to be calibrated and tested for reproducibility. The type of test and judge depends on the purpose of the test. Most analytical tests answer one of the following questions: 'Is there a difference?", 'What is the difference?' and 'How large is it?'. These are done in sequential fashion and can provide very detailed information about the sensory properties of flavours or flavoured products. In contrast to these analytical evaluations, subjective preference (hedonic) tests are used to determine consumer acceptance. The purpose of this chapter is to introduce the basic principles of sensory evaluation and the factors that must be considered and controlled in sensory analyses of flavour. Excellent references for standard sensory methods include Carpenter et al. (2000), Lawless and Heymann (1998). Stone and Sidel (1985) and Meilgaard et al. (1987a,b).

9.2 Types of tests 9.2.7 Discrimination tests 9.2.1.1 Difference tests Initially, informal or formal tests should be conducted to determine whether perceptible differences exist between products. When the products differ by differences that are too small to be described, the two most common difference tests are the duo-trio test and the triangle test (Lawless and Heymann, 1998). When the difference can be defined, pair tests are used to ask which sample is higher in a specified attribute.

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9.2.1.2 Threshold tests Threshold tests are performed to determine the sensitivity of subjects to a specific compound or to estimate the compound's contribution to flavour. The threshold value is the concentration of a compound at which a detectable difference in aroma or taste is found (detection threshold) or at which the characteristic odour or taste can be recognised (recognition threshold). Typically these values are determined by difference tests, with the sets presented in increasing order of concentration. A compilation of threshold values is available in an ASTM publication (Stahl, 1978) Determination of threshold values should be used in quality control tests of taints to select judges who are sensitive to specific compounds. Similarly, threshold determination is useful in studying factors that influence individual differences, such as age, gender or disease. In most flavour investigations, threshold values are of only limited use for several reasons. The value applies only to the tested product under specific testing conditions since the threshold level varies with temperature, sample composition and the method by which the values were determined. Guadagni et al. (1968) proposed the concept of odour units, whereby the concentration of a component is expressed as the concentration divided by the threshold value. However, as all flavourists and perfumers know, neither the intensity nor the nature of suprathreshold concentrations of an odorant can be predicted from the threshold value and the number of odour units present. Compounds increase in intensity at different rates and can demonstrate marked differences in quality at different concentrations. At best, only 'guestimates' of the contribution of individual compounds to flavour can be made from the threshold value and volatiles composition. 9.2.1.3 Gas chromatography—olfactometry Analogously to threshold value determinations, gas chromatographyolfactometry (GC-O) methods have been used to identify compounds that are 'impact' (characteristic) volatiles or at least 'odour-active' compounds. Interpretation of GC-O results is subject to problems similar to those discussed above in threshold testing. Although specific compounds may be 'odour-active', their contribution to the flavour in the mixture may not correspond to GC-O predictions. Despite this caveat, the utility of GC-O has been demonstrated by Guth (1997). Forty-four odour-active compounds identified in Gewiirztranliner wine were added to a model solution at concentrations equal to those in the wine. The solution was rated very similar in odour to the parent wine. When one of the most odour-active compounds, cw-rose oxide, was omitted, the solution was no longer perceived as similar to the wine, demonstrating the importance of this compound to the Gewiirztraminer aroma. Several GC-O methods, as discussed else where in this text (see Chapter 8), have been employed to detect compounds that contribute to aroma: Charm

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(Acree et al, 1984), AEDA (Ullrich and Grosch, 1987), OSME (McDaniel et al., 1990) and Olfactory Global Analysis (OGA) (Ott et al, 1997). The methods vary primarily in that Charm and AEDA are dilution techniques, whereas OSME and OGA rely on judge consensus in evaluation of one concentration. Sources of error that occur in GC-O applications include judges not being trained or becoming fatigued. Typically in GC-O studies, judges are required to sniff a run for a maximum of 20 minutes so as to minimise fatigue. Another source of error is the difficulty in perceiving peaks that elute very rapidly. Hanaoka et al. (2001) noted that subjects who had faster breathing rates showed a tendency for higher frequency of odour detection. In one experiment, when subjects were asked to breath faster, the primary effect was an increase in intensity of the odour. Usually the make-up air is humidified to minimise the discomfort of dry nasal passages experienced by judges. In contrast to observations made by others over the years and to observations made in our laboratory, Hanaoka et al. (2000) reported that omitting the moisture in the make-up gas did not affect subjects' comfort. For AEDA (or Charm), extracts are presented to a few individuals in serial dilution until no further odour is perceived. The dilution factor (FD) is reported for each individual as an indication of the 'strength' of the odour in AEDA. For OGA (Van Ruth et al., 1995) and OSME, odour-active volatiles detected by consensus are reported. Despite the different methodologies, Le Guen et al. (2000) found the three GC-O methods to yield similar results in evaluation of the impact compounds of mussels. 9.2.2

Intensity rating tests

To measure the size and nature of differences in flavours or flavoured products, the intensity of a specific attribute or attributes is rated by trained judges. There are several different scaling procedures and types of scales. Category scales or unstructured line (graphic) scales are used most frequently. Another method is magnitude estimation (ratio scaling) in which the intensity of a sensation is rated relative to the intensity of a reference. For example, if the reference is defined as an intensity of 10, and the sample is twice as intense, it is rated 20. Magnitude matching (or cross-modal matching) is a variation in which the intensity of tastes, smells or mouth feel is rated relative to a standard sound or light (Marks et al., 1988). More recently, the labelled magnitude scale (LMS). which is a combination of the ratio and category scales, was developed by Green et al. (1993). In contrast to category and graphic scales, which are anchored at the ends by the terms 'low' and 'high', the LMS is anchored by 'barely detectable' and 'strongest you can imagine' (see Figure 9.1). For most studies in which the sensory properties of flavoured systems are being measured, the unstructured line scales or category scales are ideal. For a normal range of intensities, the same results are obtained using ratio scaling as found with category or graphic scales, but the latter are simpler to

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Strongest imaginable

High

90

8 7 6 5 4 3 2

80 70 60 50 Very strong 40 30

Strong

20

Moderate Weak 0 Barely detectable

Very strong 9

10 1w

Low (b)

1 Very weak

(c)

Figure 9.1 Examples of scales used for rating intensity: (a) Labelled magnitude estimation (LMS); (b) category scale; (c) Unstructured (graphic) line scales.

use than magnitude estimation methods (Giovanni and Pangborn, 1983). For descriptive analysis in which many attributes are rated, estimating each on a scale anchored by 'strongest you can imagine' seems difficult and use of the LMS scale has not been attempted. However, to measure responses of individuals who perceive stimuli very differently because of inherent physiological differences, the LMS scale or cross-modal matching better reflect the strength of the sensation perceived by the individual (Prutkin et al, 2000). In many studies in which category scales were used to rate bitterness, no differences in bitterness intensity were found between subjects who were sensitive (Tasters) to the bitter compound propylthiouracil (PROP) and those who were insensitive (Non-Tasters) (Cubero-Castillo, 1999; Hall era/., 1975; Mela, 1989). In contrast, Tasters rated intensity of bitterness, saltiness and the burn of capsaicin more strongly than Non-Tasters using cross-modal matching (Bartoshuk et al., 1994) or LMS (Bartoshuk et al., 2000). 9.2.3

Time-intensity

Time-intensity (TI) procedures are used to characterise persistent sensations, such as astringency and bitterness, and to monitor perceived intensity as flavour is released during ingestion and mastication. As the concentration of a single compound is increased, maximum intensity and total duration increase, whereas only small differences in time to maximum intensity occur. When the structure or composition of a system is altered, the rates of release of tastants or odorants are affected, as well as the maximum intensities. Examining the rate of onset and rate of decay of intensity can be used to model perception of taste and

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mouth feel (Pfeiffer et al., 2000) and suggest explanations for mechanisms of perception (Linforth et al., 1999). Recently, sensory TI has been coupled with instrumental TI to characterise flavours more dynamically. Subjects rate perceived intensity while the concentrations of volatiles in the nasal passage (breath by breath analysis) (Linforth and Taylor, 1993; Chapter 7) or compounds in the oral cavity (Davidson et al., 1998, 1999) are simultaneously being measured. 9.2.3.1 Analysis of time-intensity data Rating intensity continuously is a difficult task that requires more training than do simple scaling tests. The way in which judges move their tongues, the rate at which they chew and their salivary flow rates influence their perception of intensity and persistence of the sensation. Even with extensive training, individual judges have characteristic curves with idiosyncratic, yet reproducible, patterns. Despite the difference in temporal patterns, judges can be trained to give reproducible responses that are consistent across samples. In many studies. TI parameters (such as time to maximum intensity (TMAX), maximum intensity (IMAX) and total duration (TOT)) are extracted from the raw time-intensity curves for each sample and each judge and then averaged. Alternatively, the mean intensity rating at each time is calculated and the results are expressed as an average TI curve. Because of the differences in the shapes of the individual judges' curves, the average values for the TI parameters IMAX, TMAX and TOT are slightly different from those illustrated in the average TI curves (Noble. 1995; Noble et al., 1991). To address this issue, several different methods have been proposed for normalising the ratings over time. Nevertheless, the results from analysis of normalised curves (Overbosch et al., 1986; MacFie and Liu. 1992) or from principal component analysis of curves (Dijksterhuis. 1993) do not differ much from the 'un-normalised' average curves or from comparison of average TI parameters such as IMAX and TOT. 9.2.3.2 Effect of mode of sampling Although continuous rating of intensity of 'flavour by mouth' attributes before and after spitting or swallowing the stimulus has many applications. TI methods are inappropriate for analysis of aromas that are not continuously evaluated. When odorants are rated when sniffed (or inhaled), the sensation of aroma decreases almost immediately after the subject stops sniffing the samples, as illustrated in Figure 9.2. When a solution of 150ppm menthol was sniffed for 10s, overall menthol intensity increased rapidly, and then equally quickly decreased when the subject stopped sniffing (curve 4 in Figure 9.2). In contrast, the perception of intensity of menthol was more intense and lasted far longer when the same solution was sipped and then spat at 10s (curve 2) since the residual menthol in the mouth was still detected (Opet, 1989).

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Time (s) Figure 9.2 Average intensity ratings over time of 150 ppm menthol evaluated under four conditions: sipping menthol + 2g/l caffeine (curve 1) or menthol alone (curve 2); sniffing menthol for 10 s while tasting either 2g/l caffeine (curve 3) or water (curve 4); n — 20 judges x 2 replications. Modified from Opet (1989).

9.2.3.3 Taste-smell interactions In many TI studies, cognitive interactions between taste, trigeminal sensations and smell are found (Noble, 1996). For example, increasing the sourness or sweetness of orange-flavoured solutions, without altering the flavour concentration, produced an increase in the intensity and duration of fruitiness (Bonnans and Noble, 1993). The overall intensity of menthol, which has a bitter taste, minty smell and cooling trigeminal sensation, is similarly affected by the bitterness of caffeine (Opet, 1989). When subjects tasted solutions of 150 ppm menthol to which 2g/l caffeine had been added (curve 1, Figure 9.2), menthol intensity was rated more intense and lasted longer than when menthol alone was sipped (curve 2). In the same study, cognitive enhancement also occurred when aroma and taste were perceived independently using a dual-delivery system (Hornung and Enns, 1984). When menthol was sniffed and caffeine was tasted simultaneously (curve 3), the perception of menthol intensity was rated higher and lasted longer than when menthol was sniffed and water was tasted (curve 4) (Opet, 1989). A similar cognitive interaction was seen in a study in which both TI and 'breath by breath' analyses were conducted (Davidson et al., 1999). Subjects chewed menthone-flavoured gum and rated the intensity of minty flavour while the concentration of menthone in the nasal passage was monitored. At the same time, the residual concentration of sucrose in the oral cavity was also monitored. As shown in Figure 9.3, the perceived intensity of the menthone flavour in the gum matched the change in sugar concentration rather than the

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Figure 9.3 Sucrose release (•), menthone release (I), and perceived overall mint flavour intensity ( G ) from chewing gum. Reprinted with permission from Davidson et al. (1999).

high concentration of menthone in the nose. Subjects probably became fatigued to the menthone, yet continued to rate 'mintiness' that was enhanced by or associated with the residual sweetness. 9.2.4 Descriptive analysis The technique of descriptive analysis (DA) provides a quantitative analytical characterisation of aroma, taste and mouth feel as described in detail elsewhere (Heymann et al., 1993; Lawless and Heymann, 1998; Stone et al., 1974: Hootman, 1992). The first requirement for a meaningful DA is development of a vocabulary that will describe the differences in flavour among the samples in specific concrete terms. For examples of these, see the lexicons for flavour of beer (Meilgaard et al., 1982), wine (Noble et al., 1987), whiskey (Shortread et al., 1979) and cheese (Berodier et al., 1997; Pagliarini et al., 1991). For each DA study, terms are derived that describe the differences in aromas and flavours of the experimental samples. Reference standards are provided to define each term and train judges to rate each attribute consistently, as detailed for cheese (Murray and Delahunty, 2000), beer (Meilgaard et al., 1982) and wine (Noble et al., 1987). After the judges are trained, the intensity of each term is rated in each product. A variation of DA, known as free-choice profiling, in which judges each use their own terms, can also be used, but the results are very difficult to interpret (Beal and Mottram, 1993; Williams and Arnold. 1985: Williams and Langron, 1984). Plotting the ratings from the descriptive analysis data reveals the flavour profiles of the samples as shown in the following two examples. Heating wines

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Citrus LSD=0.75 4Tea/tobacco Tropical fruit 3 zN^^ LSD=0.59 \ 7 X>t LSD=0.87

Honey LSD=0.81

Oak LSD=0.86^

xJ(

*Floral LSD=0.91

Rubber LSD-0.75 A

0

—a— 15

—O— 30

Figure 9.4 Aroma profiles of Chardonnay wines heated for 0, 15 or 30 days at 40°C. Centre of the figure = low intensity; perimeter = higher intensity for each term; LSD = least significant difference. Wines differ significantly (p < 0.05) if their intensity means differ by more than the LSD; n = 15 judges x 3 replications. Modified from de la Presa Owens and Noble (1997).

accelerates normal ageing reactions and produces changes in wine flavour. To characterise the effect of heating, Chardonnay wines that had been stored at 40°C for 0,15 and 30 days were profiled by DA (de la Presa Owens and Noble, 1997). As shown in Figure 9.4, the intensity of the floral and fruity notes (tropical fruit, green apple) was reduced by 20% after storage for 15 days, although only the decrease in green apple was statistically significant. After 30 days, the intensities of citrus, tropical fruity, green apple and floral aromas were significantly decreased by nearly 80%. At the same time, the intensity of tea, honey, rubber and oak aromas increased. Products heated in a microwave do not have the typical nutty, toasted or caramel aromas that are produced by Maillard browning reactions in conventional thermal cooking. Descriptive analysis was used to monitor the differences in aroma between thermally heated solutions of cysteine-HCl and glucose and those heated in a microwave (Song, 1990). In one trial, the pH of the microwaved samples was adjusted to 2, 7 and 9. In Figure 9.5, the mean ratings for each term in each sample are plotted to illustrate the aroma profiles. The roasted, nutty, brown sugar and popcorn notes were higher in the pH 9 microwave treatment than in all other samples, including the thermally heated one. 9.2.5

Consumer tests

Consumer testing requires identification of typical consumers of the products or flavours being tested. Personnel at a work site or 'experts' should not be used, unless they represent the typical consumer. Because of the tremendous variation in preferences, a large number of target consumers must be recruited.

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Nutty

Brown sugar

Bell pepper

Popcorn

Burnt

Cooked onion

Figure 9.5 Aroma profiles of aqueous solutions of 0.25 mol/1 cysteine-HCl and 0.25 mol/1 glucose that were heated conventionally for 40 h at 100°C with refluxing or microwaved for 5min. Centre of the figure = low intensity: perimeter = higher intensity for each term: (n = 15 judges x 3 replications): 0 Thermal pH 9: --A-- Microwave pH 9:—•—Microwave pH 7: -»• Microwave pH 2 (Song. 1990).

Most frequently, pair preference tests are used or samples are rated or ranked for preference. Preference is usually rated on hedonic category scales anchored by terms such as 'dislike extremely' to 'like extremely'. Typically the tests are conducted in home or at a central location. Focus groups are very useful for identifying factors that influence the consumer's preference (McNeill et al., 2000). Consumers cannot describe why they like or dislike samples, because they do not have the vocabulary to accurately describe flavour notes. For example, the terms 'dry', 'bad', 'bitter', 'astringent' and 'sour' are used interchangeably to describe sour or bitter or astringent products. Thus, to profile the highly preferred flavours or identify the flavours preferred by different market segments, preference mapping methods are used. Using these techniques, consumer preference ratings are statistically related to the descriptive analysis data (McEwan, 1996; Schlich. 1995). For more information on consumer testing, consult Resurreccion (1998).

9.2.6

Quality control tests

For quality control tests, the difference tests described above are too sensitive. Many alternative methods are used in quality control (QC) or quality assurance (QA) programmes, as reviewed in detail in Munoz et al. (1992). (See also Yantis (1992) and Carpenter et al. (2000).) The QC manual developed for evaluating corks for off-odours describes the procedures and guidelines for making accept or reject decisions (Butzke and Suprenant, 1998). The 'in/out' method is a decision-making tool for evaluating daily production. On-line judgments are made as to whether a product is within or outside

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the product specifications. The disadvantage of this approach is the lack of information provided and the difficulty in defining the specification limits. 'Difference from control' tests (degree of difference tests) rate the size of the difference of a production sample from a 'control' or 'standard' one. Category scales labelled with terms describing the degree of difference (None, Very Slight, Slight, Moderate, Large, Extreme) or unstructured line scales anchored by 'no difference' and 'extreme difference' can be used. Although this method is simple, its disadvantage is that the overall rating for the size of difference from the standard product provides no information about the nature of the differences. Some production sites use descriptive analyses for a more comprehensive approach to quality control. Trained judges rate the attributes that have been found to be most important in describing differences among products. Pre~set specifications are determined to define the acceptable range of attribute ratings for making quality control judgements. 9.3 Sensory testing administration 9.3.1 Facilities Analogously to instrumental measurements, judges need to be calibrated and experiments should be conducted under reproducible conditions. However, unlike the response of instruments, the perception of judges is influenced by psychological and physiological factors that can cause additional sources of 'experimental noise'. Consequently, sensory experiments are conducted under conditions designed to minimise or exclude bias and distraction. For example, the evaluation area is separated from the preparation area so that the judges are not given clues about the experiment and so that odours can be controlled. The environment in which sensory tests are conducted should be temperaturecontrolled, quiet and odour-free. The testing facility should have a clean source of odourless air that is maintained at positive air pressure to prevent outside odours from interfering. Ideally, booths with partitions between the judges eliminate distraction and prevent interaction between judges (see Eggert and Zook, 1986). Controlled lighting should be used either to mask differences in appearance or to standardise the light source and conditions under which colour and appearance are tested. If the samples vary in colour or appearance when only flavour is being evaluated, products should be served in black glasses or evaluated under masking red light to remove visual cues. Conditions for conducting sensory tests that are linked with instruments, such as GC-O, breath by breath analysis or MRI, should also be optimised to exclude distraction by the laboratory environment. The area must be protected from noise and odour. If this is not possible, other activities in the laboratory should be curtailed during sensory testing.

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Test administration

Samples should be presented in identical containers coded with random numbers to prevent bias from extraneous clues such as brand or treatment. Standard tulipshaped clear wine glasses are optimal for evaluation of aroma, while plastic cups or beakers can be used when only taste or mouth feel is being evaluated. Providing watch glasses or Petri dishes as sample lids during aroma evaluations increases aroma intensity and reduces odours in the tasting facility. Whether their participation involves simple or complex tasks, subjects should not be the experimenters (sensory administrators). In cases in which subjects are required to pour or distribute their own samples, error can be introduced by nonuniform sample sizes and by mixing up of samples. Further, judges may learn about the design of the experiment. Asking subjects to perform complex tasks on their own, such as initiating timing with a stopwatch, tasting a sample, and turning a device on and off at specified times, distracts from their ability to concentrate on rating intensity and introduces error. Preferably, subjects should not be required to monitor time while performing a series of tasks. Software for acquisition of sensory data can be programmed to prompt subjects when to sniff or sip, when to initiate rating and when to spit. Without this automation, a sensory analyst needs to work one-to-one with the subject to control and regulate all such details. 9.3.3 Experimental design The nature and number of the experimental samples determine the specific sensory test as well as the design of the experiment. When there are too many products to be evaluated in one session without fatiguing the judges, experimental designs should be used to randomly assign samples to different sessions. For experimental designs, see texts such as Cochran and Cox (1957). The number of samples that can be tested in one session without fatigue is dependent on both the nature of the samples and the demands of the sensory test. Fewer samples can be rated in a session when strong, pungent or persistent flavours are being evaluated than when less intense flavours or those with shorter duration are being tested. Three to four samples may be the most that can be evaluated in one session for a descriptive analysis in which 10-15 attributes are being rated. To determine whether fatigue has occurred in the training sessions, serve the same sample first and last. If results are not reproducible, try presenting fewer samples. To reduce fatigue, subjects should breathe fresh air or rinse with water between samples. The specific inter-stimulus protocol varies with each product. Warm water should be used for rinsing between oily or fatty samples, while rinses of gelatin alternated with water help reduce astringency. In almost all analytical sensory tests, the samples are not swallowed but spat to avoid introducing a new variable: judge satiation or intoxication in the case

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of alcoholic beverages. Small cardboard or plastic containers with lids are an inoffensive way to facilitate this. Both the context of presentation and the sequence in which samples are presented affect perception, as discussed in Lawless and Heymann (1998). Hence, samples should be served in randomised orders to eliminate sequence and carryover effects. The order of presentation of difference tests (pair, duo-trio or triangle sets) should be randomly assigned to avoid fatigue. To minimise guessing, the position of the samples within each set should be randomised. Designs for difference tests are presented in Stone and Sidel (1985) and Meilgaard et al. (1987a,b). For rating tests, the order in which samples are presented should be randomised to eliminate errors that arise from systematic contrasts. For example, an acid solution at pH 3.25 was rated lower in sourness when presented after a pH 3.0 sample than when presented after a solution with pH of 3.5 (Morris, 1982). Similarly, shifts in intensity were also observed in sweetness ratings of a fruit beverage. Midrange juices were less intense in the context of stronger items and more intense in the context of weaker items (Diamond and Lawless, 2001). Samples presented in the first position are usually rated higher, and often significantly higher, than subsequent samples irrespective of sample identity. This occurs with analytical rating as well as in preference tests. In an inter!aboratory study of preference, eight coffees were presented in a Latin square design. The preference score for the first position, regardless of coffee identity, was significantly higher than for the other positions (Network, 1996). To eliminate the effect of the first sample being rated differently, a warm-up can be presented first and its scores discarded (Stone and Sidel, 1985). In evaluation of some products, time-order or carryover effects occur (Amerine et aL, 1965). In the evaluation of astringent or bitter products, the second sample is almost always perceived as higher in intensity. Thus the design must randomise the order of presentation as well as balance the number of times each specimen is presented in a specific sequence. This can be done using William's Latin squares designs, which are balanced for first-order carryover effects (MacFie and Bratchell, 1989; Schlich, 1993). In some cases, the carryover effect is compound-specific. In a study of bitterness perception, different compounds were presented in sequence. No matter what compound was presented first, the perceived bitterness of all bitterants in the study increased when presented after another bitter stimulus. Yet caffeine increased bitterness of compounds that followed it by the largest amount, although bitterness of caffeine was least affected by other compounds. In contrast the increase in bitterness of quinine was higher than that of the other bitter compounds in the study (Cubero-Castillo and Noble, 2001). Several compounds that stimulate the trigeminal sense show both sensitisation (increased perception of intensity) and desensitisation, depending on the time between presentation of samples, as well as on the specific chemical (Green and Lawless, 1991; Karrer and Bartoshuk, 1991). Pungency and bum

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Image Not Available

Figure 9.6 Perceived burning intensity of capsaicin over time. Sensitisation increases the burning intensity as capsaicin is applied 25 times at I-min intervals. After a 15-min break, the intensity rating is greatly decreased, showing that desensitisation has occurred. Reprinted with permission from Green (1989).

of compounds such as capsaicin initially cause a Sensitisation in which each subsequent sample is perceived as more intense when samples are presented at 1- to 5-minute intervals. When the sample is presented again after a break of 15 to 20 minutes, desensitisation occurs. The intensity of burn is greatly reduced. as shown in Figure 9.6 (Green, 1989). Thus evaluation of 'hot' products must be made carefully for valid results. 9.4

Selection and training of judges

9.4.1 Human subject consent forms and regulations Government regulations require that researchers respect and protect the rights and welfare of individuals participating as subjects in studies. In conducting research involving human subjects, the individual's rights and well-being must be the primary concern. Judges may stop participating without penalty and cannot be coerced into participating against their will. Although the guidelines for approval of sensory protocols vary with country and company, all judges must sign a human consent form before participating in a study. In this form, subjects must be informed of risks and benefits of the study, such as the presence of sulfur dioxide in case the subject is allergic to it. In this form, it must be explained in general terms what the judges will be evaluating without revealing the purpose of the experiment or the exact nature of the variables to be tested.

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265

9.4.2 Judge selection Initially, judges should be selected on the basis of availability and motivation. Recruit more judges than are needed. The specific requirements for selecting judges vary with the type of test. If a QC test involves detection of a specific off-odour, such as the mouldy or musty notes of geosmin in water or of trichloroanisole in wine corks, select judges who are very sensitive to these compounds. To determine judge sensitivity, a series of pair tests can be presented to the judge in which a blank is compared with low concentrations of the specific chemical, or a formal threshold test can be done. For more general studies, judge performance should be examined. Retain subjects who perform reproducibly and are consistent with other judges, as discussed below. 9.4.3 Training At the beginning of any new testing, introduce each judge to the scorecard, the rating scale and the procedures. Initially present samples that are very different from each other. This is effective as a training tool, since the specific attributes of interest are more easily perceived, such as a 'floral' note in a set of Riesling wines. More importantly, it helps morale. For unspecified difference tests, once judges are familiar with the testing procedure, only one training session, if any, is required. For pair tests or scaling tests, training is needed to define the specific attribute being tested. For descriptive analysis, one to six weeks of training are required. The first sessions are discussions, in which reference standards are presented and their appropriateness for rating the flavour of the experimental products is reviewed. Subsequent sessions focus on refining the standards and training judges to use the terms consistently. Complex tests, such as time-intensity studies, require extensive training to train the judge to coordinate activities such as initiating the recording, sipping, spitting and possibly sipping subsequent samples, while focusing on rating the attribute. When TI is combined with breath by breath analysis, even more training is needed so that the judge can breathe with controlled cadence, while focusing on rating intensity. 9.4.4 Reference standards For pair tests, scaling, descriptive analysis or time-intensity tests, reference standards should be developed that define the attribute(s), such as those that have been used for cheese (Murray and Delahunty, 2000), beer (Meilgaard et al, 1982) and wine (Noble et al., 1987). Presenting 'high' and 'low' intensity standards for each attribute helps to train panellists in the use of the scale and align the concepts.

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9.4.5 Evaluating judge performance Keeping records on judge performance will permit selection of judges who are consistent, sensitive and reproducible. For difference tests, the cumulative number of correct judgements can be tracked (Amerine et a/., 1965). In descriptive analysis or time-intensity tests, practice sessions should be conducted that use the same protocol as that used in formal testing. Prior to each DA session, the judge should smell and/or taste each of the references, then rate each attribute. Inspect the data immediately to detect judges who are using a term inconsistently with the other subjects. This judge should be presented with samples or reference standards that illustrate 'low' and 'high' intensity of the term(s) that are not being used correctly. After familiarising himself or herself with these, the judge should rate more coded samples, and the results should be reviewed again. The data from the training sessions should be analysed before initiation of the formal data collection, as described below. This saves time and money. Rating 40 attributes and then finding that only 4 varied significantly across the samples is a waste of time. For any type of test, give rewards (cookies, money, gifts), and provide positive feedback. Motivated judges are more focused and have better performance. 9.5 9.5.7

Statistical analysis of data Difference tests

Tables constructed from binomial distributions provide the number of correct responses needed for statistically significant differences at specific levels of confidence. Alternatively, tables based on normal distributions indicate the probability that the number of correct responses could have occurred by chance alone. Tables for determining significance levels and exact probability levels are provided by Roessler et al. (1978), O'Mahony (1986), Lawless and Heymann (1998) and Brockhoff and Schlich (1998). 9.5.2 Analysis of variance Data from simple scaling tests, descriptive analysis or time-intensity studies are evaluated by analysis of variance (ANOVA) to determine whether the samples are significantly different. If the judges have scored the samples more than once, the ANOVA can determine whether judges are reproducible and consistent with each other (i.e. rate the attribute in the same manner). In ANOVA, the main effects, such as judges, samples and replications can be considered fixed (results apply only to this experiment) or mixed (the same results would apply if other cases or judges were tested). Unless the experiment is focused on individual judges (such as comparing responses of PROP Tasters vs NonTasters) instead of testing differences among samples, sensory data are analysed

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267

Table 9.1 Summary of analyses of variance of two aroma terms: F-ratios, degrees of freedom (df), significance levels and interpretation F -ratios Source of variation

df

Berry

Apricot

Wine (W)

27

3.58***

1.81**

20

14.63***

Rep(R) W x R I xR

I 27 20

W x J

540

1.23 1.04 1.05

2.56 0.91 1 .09 1.06

Interpretation Wines differ significantly in intensity of 'berry' and 'apricot' aromas Judges use different part of the scale (not a problem) Replications do not differ No difference between replications Judges did not vary differently between replications Some judges rated 'berry' differently from others Judges rated 'apricot' in the same way across wines

** and *** denote significance at p < 0.01 and p < 0.001 respectively.

by mixed model ANOVA. In mixed models, judges are treated as a random factor, while the samples and replications are fixed (Lawless and Heymann, 1998; Lundahl and McDaniel, 1988; O'Mahony, 1986). When significant differences are found, tests such as Duncan's multiple range test or Fischer's least significant difference (LSD) identify which products differed significantly from each other, as illustrated in Figure 9.4. In Table 9.1, the summaries of two ANOVAs are shown for a study in which 21 judges rated 28 wines in duplicate for intensity of berry and apricot aromas. In most sensory tests, judges use different parts of the scale and are a significant source of variation. For both terms, this occurred and can be ignored. However, if there is a significant Judge x Product interaction, it means that the judges are using the term inconsistently (Stone et al., 1974). In the mixed model ANOVA, this error is accounted for. Thus, it can be reported that there was a significant difference in intensity across wines for the berry attribute, despite the significant Judge x Wine interaction. Discussions of analysis of sensory data with emphasis on judge performance can be found elsewhere (Gay and Mead, 1992; Schlich, 1994). 9.5.3

Power

Power is the probability that the experimental results are correct: you have identified a real difference or you did not fail to find a real difference. Testing of power is seldom done, but it should be done since it validates the results or indicates that you have too low power to allow you to rely on your results. Most frequently, data from difference tests or analyses of variance are considered significantly different if a (the probability that the results could have occurred

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by chance alone) is less than 5% (this is also expressed as p < 0.05). Few studies test for p\ which is the chance that you did not find a real difference. Power is determined by three factors: the number of judges and replications, the magnitude of the differences among the samples (effect size), and the a level chosen. Power can be increased by using more judges and conducting more replications. Experiments conducted with a very low number of judges almost always have very low power. In most studies, the size of the differences among the samples cannot be changed. However, trained, sensitive judges can perceive very small differences and are more reproducible, thus increasing power over untrained judges. For more detail, see Cohen (1988) or Lipsey (1990). 9.5.4 Principal component analysis: an exploratory tool Principal component analysis (PCA) is an efficient way of looking at large amounts of data. For example, we may have many samples for which we have a large number of chemical variables (such as concentrations of 100 volatiles) or we may have intensity ratings for a number of attributes. A principal component analysis can reduce the information contained in the large number of variables to two (or three) principal components or factors. A first factor or principal component (PC) is extracted. The first PC is the combination of the original variables that best explains the variation among the samples. The second PC is then derived to account for the maximum variation in the remaining unexplained variability, and so on. The relationship of the original variables and the samples can then be seen by their projection on these few PCs. For example, a PCA of the sensory profile data plotted in Figure 9.5 was conducted. In Figure 9.7 the loadings for the sensory terms are shown as vectors and the factor scores of the samples as points for the first two principal components. The first PC (which accounts for 73.4% of the total variation) contrasts samples high in cooked onion and bell pepper aroma on the left against those on the right which are low in these two terms but high in popcorn, roasted, nutty and brown sugar. The second PC separates samples at pH 7 which were higher in burnt aroma from the rest. The small angles between cooked onion and bell pepper indicate that they are correlated with each other and negatively correlated with the brown sugar and nutty terms, which are also correlated with each other. Consistent with the flavour profiles in Figure 9.5, the pH 9 microwaved sample is high in brown sugar and low in bell pepper and cooked onion, whereas pH 7 and pH 2 microwaved samples are the converse. It must be emphasised that PCA is an exploratory method that facilitates data interpretation. Normally PCA is not used to test whether samples differ significantly. As shown in Figure 9.4. one should apply the LSD values to ratings for individual terms to determine which samples differ significantly in each attribute or do a canonical variates analysis (Heymann and Noble, 1989).

269

SENSORY METHODS OF FLAVOUR ANALYSIS 1.15-

Cooked onion

:oasted Nutty

1.15 Factor 1 (73.38%) Figure 9.7 Principal component analysis of descriptive analysis data for heated 0.25 mol/1 cysteine-HCl and 0.25 mol/1 glucose solutions. Projection of the attributes (vectors) and factor scores for sample means and for average values for three replications on principal components I and II. —— =0.4 Sample; <—= 0.2 Term; O Thermal; V pH 9; • pH 7; • pH 2; D Means (Song, 1990).

9.6

Relating sensory and instrumental flavour data

Several approaches have been utilised to seek causal and predictive relationships between sensory and instrumental data. Multiple regression analysis has been used to predict intensity of specific sensory attributes in terms of the 'aroma significant' volatile components. However, the volatiles are usually highly correlated; thus several solutions to each equation are possible and the method is of little use. Principal component analysis is a better tool for analysis of highly correlated data, but it should not be done on a data set in which both instrumental and sensory variables are included since the results are not meaningful. There are many multivariate methods for relating sensory and instrumental data in which the configurations of sensory and instrumental data matrices are compared. Calculation of the RV coefficient has been used as a measure of similarity between the matrices of sensory data and instrumental data (Schlich and Guichard, 1989; Schlich et al, 1987). Principal component analysis of instrumental variables (PCAIV) selects a small subset of volatiles that yield the configuration closest to that of the principal component analysis of the sensory data (de la Presa Owens et al., 1998). Procrustes analyses is a technique by which the spaces derived by principal component analyses are matched (Dijksterhuis and Gower, 1991). The relationship of chemical and sensory loadings in the

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consensus configuration indicates which volatiles are related to each sensory attribute, as shown in an analysis of 24 Bordeaux wines (Williams el al., 1984). The method of partial least squares (PLS) analysis of latent variables (Martens and Martens, 1986) is used to uncover relationships between sensory and instrumental data sets. The technique indicates how well variables in one data set predict or model the variation among variables in the second. PLS was used to relate the descriptive analysis ratings of heated cysteine-HCl/glucose solutions with their headspace volatiles. The PLS loadings for the sensory terms and the volatiles are shown for PLS factors 1 and 2 in Figure 9.8a. In Figure 9.8b, the factor scores for the means and three replications are plotted for each sample. This two-dimensional solution models 84% of the variation in the volatile data and 66% of the sensory variation. PLS factor 1 separates the pH 9 microwaved sample and the thermally heated one from the pH 2 and pH 6 microwave heated solutions, while the second factor separates the pH 9 microwaved from the thermal. The 2-ethylfuran (2'efr) is not weighted heavily, indicating that it does not model variation between the data sets, whereas the rest of the volatiles were loaded heavily and contribute to modelling variation in both data sets. The bell pepper and cooked onion aromas are most closely associated with unknown peak 5, while the burnt note is associated with the 3-methyl (3'mfr) and 2-acetylfuran (2'acf) and peaks 25, and 26.

5 4 3 co co co
1 0 -1 -3

u_

-4 -5 -6 -

(a)

3

-

2

-

1

0

1

2

Factor 1 (21%, 33%)

Figure 9.8 (a) Partial least squares regression of sensory descriptive analysis data and headspace volatiles data for heated 0.25 mol/1 cysteine-HCl and 0.25 mol/1 glucose solutions. Factor scores for sample means and for average values for three replications for factors I and II. O Thermal: T pH 9: • pH 7: • pH 2: D Means (Song. 1990). (b) Factor loadings for attributes and volatiles for factors I and II (percentage explained variation shown in bold for instrumental data and not bold for sensors data).

SENSORY METHODS OF FLAVOUR ANALYSIS

27 I

0.502'mthp 45' dmoxz BROWN SUGAR NUTTY POPCORN ROASTED

3'mfr BURNT

2'acfr p25+26 •-c4

co

p5

P emprz etlnslf

BELL PEPPER COOKED ONION

PYRAZINES -0.50 L -0.50

(b)

0.50

Factor 1 (21%,

33%)

Figure 9.8 (continued)

Analogously to the selection of odour-active compounds by GC-O, these multivariate methods reveal compounds that contribute to aroma. If a large number of samples are evaluated, than the patterns found by PLS, Procrustes or PCAIV can be robust. 9.7

Summary

Sensory analysis can provide objective, quantitative information about the sensory properties of flavours by the use of properly designed tests, trained judges and appropriate sensory protocols. With sophisticated sensory and instrumental techniques, the sensitivity, reproducibility and precision of both methods will increase. With more complete and accurate information, and the strengthened link betweeen flavour chemistry and sensory science, our understanding of the relationship between flavour stimuli and perceived flavour should help explain the mechanisms of flavour perception. References Acree, T.E., Barnard, J. and Cunningham, D.G. (1984) The analysis of odour-active volatiles in gas chromatographic effluents, in Analysis of Volatiles. Methods and Applications, (ed. P. Schreier), Walter de Gruyter, New York, pp 251-268. Amerine, M.A., Pangborn, R.M. and Roessler, E.B. (1965) Principles of Sensory Evaluation of Foods, Academic Press, New York. Bartoshuk, L.M., Duffy, V.B. and Miller, I.J. (1994) PTC/PROP tasting: anatomy, psychophysics, and sex effects. Physiology and Behavior, 56 1165-1171.

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Bartoshuk. L.M., Fast. K.. Duffy. V.B.. Prutkin. J.M.. Synder. D.J. and Green. B.G. (2000) Magnitude matching and a modified LMS produce valid sensory comparisons for PROP studies. Abstract from Society- for the Study of Ingestive Behavior. Dublin. 2000. Appetite. 35 277. Beal. A.D. and Mottram. D.S. (1993) An evaluation of the aroma characteristics of malted barley by free-choice profiling. J. Sci. Food Agric.. 61 17-22. Berodier. F., Stevenot. C. and Schlich. P. (1997) Description of the flavour of Comte cheese. LebensWissens. Technol.. 30 298-304. Bonnans. S. and Noble. A.C. (1993) Effect of sweetener type and of sweetener and acid level on temporal perception of sweetness, sourness and fruitiness. Chem. Senses. 18 273-283. Brockhoff, P.B. and Schlich. P. (1998) Handling replications in discrimination tests. Food Quality Preference. 9303-312. Butzke. C. and Suprenant. A. (1998) Cork Sensory QC Manual. University of California. Division of Agricricultural and Natural Resources. Oakland. CA. Carpenter. R.P.. Lyon. D.H. and Hasdell. T.A. (2000) Guidelines for Sensory Analysis in Food Product Development and Quality Control. Aspen Publishers. Gaithersburg. MD. Cochran. W.G. and Cox. G.M. (1957) Experimental Designs. Wiley, New York. Cohen. J. (1988) Statistical Power Analysis for the Behavioral Sciences. Lawrence Erlbaum Associates. Hillsdale, NJ. Cubero-Castillo. E.M. (1999) The effect of compound-specific sensitivity and carry-over effects on bitterness perception. PhD thesis. University of California. Davis. CA. Cubero-Castillo. E.M. and Noble. A.C. (2001) Effect of compound sequence on bitterness enhancement. Chem. Senses, 26419^24. Davidson. J.M.. Linforth, R.S.T. and Taylor. A.J. (1998) In-mouth measurement of pH and conductivity during eating. J. Agric. Food Chem.. 46 5210-5214. Davidson. J.M.. Linforth, R.S.T. Hollowood. T.A. and Taylor. A.J. (1999) Effect of sucrose on the perceived flavour intensity of chewing gum. J. Agric. Food Chem.. 47 4336-4340. de la Presa Owens. C. and Noble. A.C. (1997) Effect of storage at elevated temperatures on aroma of Chardonnay wines. Aw. J. Enol. Viticidt.. 48 310-316. de la Presa Owens. C.S.. P.. Wada. K. and Noble. A.C. (1998) Using sensory and instrumental data to interpret the effect of storage at elevated temperatures on aroma of Chardonnay wines. Ann. \Y Acad.Sci., 855 954-859. Diamond. J. and Lawless. H.T. (2001) Context effects and reference standards with magnitude estimation and the labeled magnitude scale. J. Sensory Studies. 16 1-10. Dijksterhuis. G.B. (1993) Principal component analysis of time-intensity bitterness curves. J. Sensory S/i«//Vs.8317-328. Dijksterhuis, G.B. and Gower. J.C. (1991) Interpretation of generalized procrustes analysis and allied methods. Food Quality Preference. 3 67-87. Eggert. J. and Zook. K. (ed.) (1986) Phvsical Requirement Guidelines for Sensory Evaluation Laboratories. ASTM Special Publication 913. American Society for Testing and Materials. Philadelphia. PA. Gay. C. and Mead. R. (1992) A statistical appraisal of the problem of sensory measurement. J. Sensory Studies. 1 205-228. Giovanni. M.E. and Pangborn. R.M. (1983) Measurement of taste intensity and degree of liking of beverages by graphic scales and magnitude estimation. J. Food Sci.. 48 1175-1182. Green. B.G. (1989) Capsacin sensitization and desensitization on the tongue produced by brief exposures to a low concentration. Neurosci. Lett.. 107 173-178. Green. B.G. and Lawless. H.T. (1991) The psychophysics of somatosensory chemoreception in the nose and mouth, in Smell and Taste in Health and Disease (edsT.V. Getchell. R.L. Doty. L.M. Bartoshuk. and J.B. Snow). Raven Press. NY. pp 235-254. Green. B.C.. Shaffer. S. and Gilmore. M. (1993) Derivation and evaluation of a semantic scale of oral sensation magnitude with apparent ratio properties. Chem. Senses. 18 683-702.

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Meilgaard, M, Civille, G.V. and Carr. B.T. (1987a) Season Evaluation Techniques. Vol I. CRC Press. Boca Raton. FL. Meilgaard, M.. Civille. G.V. and Carr. B.T. (1987b) Sensory Evaluation Techniques. Vol II. CRC Press. Boca Raton. FL. Mela. D.J. (1989) Bitter taste intensity: the effect of tastant and thiourea taster. Chem. Senses. 14(2| 131-135. Munoz, A.M., Civille. G.V. and Carr, B.T. (1992) Sensory Evaluation in Quality- Control. Van Nostrand Reinhold. NY. Murray. J.M. and Delahunty, C.M. (2000) Selection of standards to reference terms in a cheddar-type cheese flavour language. J. Sensory Studies. 15 179-199. Network. E.S. (1996) European Sensory and Consumer Study. A case study on coffee. Campden & Chorleywood Food Research Association. Chipping Camden. Noble. A.C. (1995) Application of time-intensity methodology for evaluation of taste and mouthfeel. Am. J. Enol. Viticult.. 46 128-133. Noble, A.C. (1996) Taste-aroma interactions. Trends Food Sci. TechnoL. 7 439—444. Noble, A.C.. Arnold, R.A.. Buechsenstein. J., Leach. E.J.. Schmidt. J.O. and Stern. P.M. (1987) Modification of a standardized system of wine aroma terminology. Am. J. Enol. Vnicult.. 38 143-146 Noble, A.C.. Matysiak, N.L. and Bonnans, S. (1991) Factors affecting the time-intensity parameters of sweetness. Food Technol., 45( 11) 121-126. Norris. M.B. (1982) Salivary and gustatory responses in aqueous solutions as a function of anionic species, titratable acidity and pH. MSc thesis. University of California. Davis. CA. O'Mahony. M. (1986) Sensory Evaluation of Food: Statistical Methods and Procedures. Marcel Dekker. New York. Opet. J.M. (1989) Effect of caffeine, ethanol, and sucrose on temporal perception of menthol. MSc thesis. University of California, Davis. CA. Ott. A.. Fay. L.B. and Chaintreau. A. (1997) Determination and origin of the aroma impact compounds of yogurt flavour. J. Agric. Food Chem.. 45 850-858. Overbosch. J.C.. Erden, V.d. and Keur. B.M. (1986) An improved method for measuring perceived intensity/time relationships in human taste and smell. Chem. Senses. 3 331-338. Pagliarini. E.. Lembo, P. and Bertuccioli. M. (1991) Recent advancements in sensory analysis of cheese. It. J. Food Sci.. 2 85-89. Pfeiffer. J.F.. Boulton. R.B. and Noble. A.C. (2000) Modeling the sweetness response using time-intensity data. Food Quality Preference, 11 129-138. Prutkin. J.. Duffy, V.B., Etter. L.. Gardner. E.. Lucchina, L.A.. Snyder. D.J.. Tie. K... Weiffenbach. J. and Bartoshuk. L.M. (2000) Genetic variation and inferences about perceived taste intensity in mice and men. Physio. Behavi.. 69 161-173. Resurreccion. A.V.A. (1998) Consumer Testing for Product Development. Aspen. Gaithersburg. MD. Roessler, E.B.. Pangborn. R.M.. Sidel. J.L. and Stone. H. (1978) Expanded tables for estimating significance in paired-preference, paired-difference, duo-trio and triangle tests. J. Food Sci.. 43 940-943. 947. Schlich. P. (1993) Uses of change-over designs and repeated measurements in sensory and consumer studies. Food Quality Preference. 4 223-235. Schlich. P. (1994) Graphical representation of assessors performances. J. Sensory Studies. 9 157-169. Schlich. P. (1995) Preference mapping: relating consumer preferences to sensory or instrumental measurements, in Biofavour'95 (eds P. Etievant and P. Schreier). INRA Editions. Paris. pp 135-150. Schlich. P. and Guichard. E. (1989) Selection and classification of volatile compounds of apricot using the RV coefficient. J. Agric. Food Chem.. 37 142-150. Schlich. P.. Issanchou. S.. Guichard. E.. Etievant, P. and Adda, J. (1987) RV coefficient: a new approach to select variables in PCA and get correlations between sensory and instrumental data, in Flavour

SENSORY METHODS OF FLAVOUR ANALYSIS

Science and Technology (eds M. Martens, G.A. Dalen and H. Russwurm), Wiley, New York, pp 469-473. Shortread, G.W., Richards, P., Swan, J.S. and Hurtles, S. (1979) The flavour terminology of Scotch whiskey. Brewers Guardian, 2-6. Song, W.L. (1990) Sensory and chemical evaluation of aroma formed via Maillard reaction upon microwave irradiation and conventional heating, MSc thesis, University of California, Davis, CA. Stahl, W.H. (ed.) (1978) Compilation of Odour and Taste Threshold Values Data, American Society for Testing and Materials, Philadelphia, PA. Stone, H., Sidel, J., Oliver, S., Woolsey, A. and Singleton, R.C. (1974) Sensory evaluation by quantitative descriptive analysis. Food Techno!., 28( 11) 24-34. Stone, H. and Sidel, J. (1985) Sensory Evaluation Practices, Academic Press, New York. Ullrich, F. and Grosch, W. (1987) Identification of most intense volatile flavour compounds formed during autoxidation of linoleic acid. Z. Lebensm.-Unters.-Forsch., 184 277—282. Van Ruth, S.M., Roozen, J.P. and Posthumus, M.A. (1995) Instrumental and sensory evaluation of the flavour of dried French beans (Phaseolus vulgaris) influenced by storage conditions. /. Sci. Food Agric., 69 393-401. Williams, A.A. and Arnold, G.M. (1985) A comparison of the aromas of 6 coffees characterised by conventional profiling, free-choice profiling and similarity scaling methods. J. Sci. Food Agric., 36 204-214. Williams, A.A. and Langron, S.P. (1984) The use of free-choice profiling for the evaluation of commercial Foils. J. Sci. Food Agric., 35 558-568. Williams, A.A., Rogers, C., Noble, A.C. (1984) Characterization of flavouring alcoholic beverages, in Flavour Research of Alcoholic Beverages. Instrumental and Sensory Analysis (eds L. Nykanen and P. Lehtonen), Foundation for Biotechnical and Industrial Research, Helsinki, pp 235-254. Yantis, J.E. (ed). (1992) The Role of Sensory Analysis in Quality Control, American Society for Testing and Materials, Philadelphia, PA.

10 Flavour legislation Jack Knights

10.1

Introduction

Flavourings represent a class of food additives that have had comparatively little legislation. This is probably due to the relatively large number of ingredients used in flavourings, the very small quantities involved and the difficulty of regulating added substances (which are also naturally present in foodstuffs) by analytical methods. It was estimated about 20 years ago that only 50 defined flavouring substances were used to the extent of over 50 kg/year in flavourings in the UK. This figure represents an average consumption of about 1 mg/person/ year, at which level many of the commonly available natural toxic materials, such as arsenicals or cyanides, are harmless. A corresponding figure for the USA appears to be about 200 substances. One of the characteristics of food additives in general and flavouring substances in particular is the low degree of risk they pose to the consumer in spite of the public perception of their undesirability. Around 1965, national regulators in several countries began to become interested in controlling food flavourings, although they had no evidence of untoward risks from their use. However, they felt that, without detailed knowledge regarding the composition of flavourings, they were laying themselves open to criticism by food activists. Initially it was believed that all compound flavourings would have to be registered and approved by the appropriate authority, but the flavour industry managed to convince the legislative authorities that this was unnecessary and impractical.

10.2

Methods of legislation

There are fundamentally two basic methods of legislation: 1.

Positive—only those materials that are listed are permitted to be used, to the exclusion of all others. This type of legislation has the disadvantage that research into possible new materials is frustrated by the requirement for publication before use, unless the time required for commercialisation is built into the regulation. However, regulatory authorities generally prefer positive listing because they believe that it offers the consumer maximum protection and is relatively easy to police.

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8000 r—

1965

1970

1975

1980

1985

1990

Figure 10.1 Total volatile compounds in food, 1965-1990, derived from Volatile Compounds in Food (TNO Biotechnology and Chemistry Institute, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist, The Netherlands) and from FEMA/GRAS listing 1965-1990. "Postulated" is estimated as 10% nonpublished flavour industry research. & • "Q Postulated; • t Published; ;*•—-•<< GRAS listed.

2.

Negative—all materials are allowed except those that are listed. In the case of food legislation this needs to be supplemented by a statement that none of the materials used is injurious to health. This firmly puts the requirement for safety of the materials onto the manufacturer, but is strongly objected to by most national legislative bodies on the basis that they do not have control over what is used. The system encourages the research and development of novel materials to provide more authentic flavourings, to the overall benefit of the consumer.

The above systems may be combined so that some groups of materials are controlled by positive lists while other groups are covered by negative lists. This is still the system in use in the European Union, although it is gradually being superseded by total positive lists. The relative effects on research of positive and negative legislation systems are illustrated in Figure 10.1. It is inconceivable that with so many new volatile compounds being identified in foods, so few were added to the FEMA/GRAS list. Many more were found of flavour utility in Europe. 10.3

Legislation in the United States

The system that has been adopted in the USA since 1965 is the positive list. The initial work was undertaken by the US Food and Drug Administration

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FOOD FLAVOUR TECHNOLOGY

(FDA) as part of Chapter 21 of the Code of Federal Regulations. Some 27 flavouring substances were evaluated and classified as 'Generally Recognised as Safe' (GRAS), presumably on the basis of long usage without untoward effect. At that time the Flavor and Extract Manufacturing Association (FEMA) proposed to assist the FDA by using independent experts to evaluate the other flavouring ingredients that were known to be in use at the time, classifying most of them as GRAS and allocating the numbers 2001-3124 to them (Hall and Oser, 1965, 1970). FEMA later set up a panel of independent experts to evaluate flavouring materials on behalf of the FDA (Hallagan and Hall. 1995). This group was later named FEXPAN and consists of independent international toxicologists who undertake the safety evaluation of novel flavouring ingredients. This procedure has continued to the present and there have been a further 16 reports allocating GRAS status to a further 840 flavouring ingredients (Burdock et al, 1990; Newberne et a/., 1998, 2000; Oser and Hall. 1972-1979; Oser et al., 1984, 1985; Smith and Ford. 1993; Smith et al.. 1996. 1997). It should be noted that GRAS listing applies to all ingredients for use in flavourings, including nonflavouring materials such as solvents, carriers, emulsifiers and antioxidants. The GRAS number makes no distinction between natural and artificial ingredients in the list; a given substance has the same entry irrespective of its status. However, foods may be used in the formulation of flavourings without being GRAS listed. An entry in the GRAS list is accompanied by average usual and average maximum levels in ppm that may be used in 34 categories of foodstuffs. These levels are provided for novel flavour materials by the submitter and agreed by FEXPAN. In the USA the only alternative designations are 'natural flavor' and 'artificial flavor' and the latter has to be displayed directly with the name of the food as well as in the ingredients list. It is therefore a highly undesirable designation for a food and is avoided wherever possible. The term 'natural flavor' or 'natural flavoring' is defined in Title 21 of the Code of Federal Regulations, Chapter 1 §101.22 (a) (3) as the essential oil, oleoresin, essence or extractive, protein hydrolysate, distillate, or any product of roasting, heating or enzymolysis, which contains the flavouring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products or fermentation products thereof, whose significant function in food is flavouring rather than nutritional. Natural flavours include the natural essence or extractives obtained from plants listed in §§182.10, 182.20, 182.40, 182.50 and Part 184 of this chapter and the substances listed in §172.510 of this chapter (Code of Federal Regulations. 1990. Title 21). It has become normal in the USA to designate various categories of natural flavourings as follows.

FLAVOUR LEGISLATION

•

•

•

279

FTNF (from the named fruit). As the name suggests, these consist solely of extracts or distillates derived from the named fruit. For instance, strawberry FTNF could consist of concentrated strawberry juice with added strawberry distillate. It may not contain material from any other natural source. Flavourings of this category tend to be very expensive in use (they are usually very weak) and not very stable. WONF (with other natural flavourings). These must contain more than 51 % derived from the named source but may contain other natural flavour ingredients. For instance, strawberry WONF could consist of 51 % concentrated strawberry juice fortified with other fruit juices or natural chemicals. These flavourings are still expensive in use because of the price of the named ingredient. Natural flavour. These must contain only natural ingredients, but the type or source is not defined. For instance, natural strawberry flavour may contain ingredients from any source so long as they are classified as natural.

The solvent or carrier has to be on the GRAS list (or a food) but does not affect the natural or artificial status of the flavouring. In recent years a great deal of research effort has been directed to the preparation of natural versions of many of the significant, defined chemicals that are important in flavours. This has included such techniques as direct esterification using enzymes as catalysts and heat-induced reactions often at elevated temperatures and high pressures, on the assumption that if the reactants are natural, then the finished product may be so designated. It should be noted that process flavours and smoke extracts are also regarded as natural under US law whereas in Europe both these categories are separately designated and specifically nonnatural. 10.4

International situation: JECFA

JECFA is the Joint Food and Agriculture Organisation of the United Nations (FOA)AVorld Health Organisation (WHO) Expert Committee on Food Additives. It was set up in 1956 to evaluate the safety of food additives, residues of veterinary drugs in food, and naturally occurring toxicants and contaminants in food. JECFA serves as a scientific advisory body to FAO, WHO, their Member States, and the Codex Alimentarius Commission, primarily through the Codex Committee on Food Additives and Contaminants (CCFAC), regarding the safety of food additives including flavouring substances. The Committee establishes acceptable intakes on the basis of toxicological data and related information on the substance being evaluated. It also develops general principles for assessing the safety of chemicals in food. The requirement to keep up-to-date with scientific developments in toxicology and related disciplines necessitates

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FOOD FLAVOUR TECHNOLOGY

a constant review of evaluation procedures (Munro et a/., 1999). JECFA has to date evaluated some 300 flavouring substances and, more importantly, indicated that many flavouring agents are used in such small quantity that a full evaluation may not be appropriate. JECFA evaluations and procedures are of international significance since they are not influenced by any particular group. JECFA evaluations are taken into account by both FEXPAN and the Scientific Committee for Food in their evaluation of the safety of flavourings for USA and Europe respectively. 10.5

Council of Europe

The Council of Europe ad hoc Working Party on Natural and Artificial Flavouring Substances was set up in 1965 as a subsidiary body to the Sub-Committee on the Health Control of Foodstuffs. Its aims were • •

to draw up a list of natural and artificial flavourings that could be used in foodstuffs without hazard to public health; to draw attention to those flavourings that presented a hazard to public health.

It is important in the context of the Council of Europe lists to understand the definitions they have used in classifying flavourings in their initial publication (Council of Europe, 1974). (i) (ii)

(iii)

A flavouring is a substance that has predominantly odour-producing properties and that possibly affects the taste. A natural flavouring is a substance obtained from vegetable and sometimes animal sources exclusively through the appropriate physical processes. Those biological processes that occur spontaneously, and roasting, are assimilated to physical processes. An artificial flavouring is a substance that has flavouring properties and that has been obtained by a chemical process. This term includes: (a) (b)

substances that exist in natural products; substances not present, or as yet undiscovered in natural products.

These definitions are unsatisfactory in a number of ways and were modified in the 1981 publication (Council of Europe, 1981) as follows. These definitions refer to materials and flavouring substances considered acceptable as flavourings and do not necessarily apply to flavourings found in commerce. (i) (ii)

Flavouring properties are those that are predominantly odour-producing and that possibly affect the taste. A flavouring substance is a chemically defined compound that has flavouring properties. It is obtained either by isolation from a natural source or by synthesis.

FLAVOUR LEGISLATION

(iv)

28 1

Natural sources of flavourings are products of plant or animal origin from which flavourings may be obtained exclusively through appropriate physical processes or by biological processes that occur spontaneously (e.g. fermentation). Natural flavourings may be defined as complex mixtures derived from natural sources that have flavouring properties.

These definitions were carried on more or less unchanged in the 1992 edition (Council of Europe, 1992). In the first two editions of the list the natural source materials were classified into the following groups. N1: N2:

N3:

N4:

Fruits and vegetables or parts thereof consumed as food. No restriction on the parts used under the usual conditions of consumption is proposed. Plants and parts thereof, including herbs, spices and seasonings commonly added to foodstuffs in small quantities, the use of which is considered acceptable with a possible limitation of an active principle in the final product. Plants and parts thereof which, in view of their long history of use without evidence of acute untoward effects, are temporarily acceptable for continued use, in the traditionally accepted manner, in certain beverages and other foodstuffs. The Committee of Experts, however, stresses that insufficient information is available for an adequate assessment of their potential long-term toxicity. Plants and parts thereof which are used for flavouring purposes at present but which cannot be classified owing to insufficient information.

A number of N2 and N3 were listed with active principles such as coumarin or safrole and these data have been the basis of Annex II in the EU Flavourings Directive. The 1992 edition does not have a section on natural source materials. In the 1974 and 1981 editions the artificial flavouring substances were classified into lists as follows: 6. 7. 8.

Flavouring substances that may be added to foodstuffs without hazard to public health. Flavouring substances that may be temporarily added to foodstuffs without hazard to public health. Flavouring substances not fully evaluated. This category was dropped from the 1981 edition in spite of containing a number of commonly used flavouring substances several of which were on the FEMA/GRAS list.

The 1994 edition took a totally different approach to the listing of artificial flavouring substances. Here they are classified into chemical groups and for each substance a summary consisting of name, category, structure, CoE, FEMA and CAS numbers, upper levels of use, natural occurrence and toxicity data is provided.

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FOOD FLAVOUR TECHNOLOGY

The Council of Europe Expert Committee has also produced guidelines on the production of processed flavourings, their ingredients and processing conditions (Council of Europe, 1995) as follows. 1.

Ingredients added prior to processing (a) A protein nitrogen source (b) A carbohydrate source (c) A fat or fatty acid source (d) Other ingredients. Herbs and spices and their extracts, water, thiamin, ascorbic, citric, lactic, fumaric, succinic and tartaric acids, guanylic and inosinic acids, inisitol, lecithin. pH regulators and siloxanes as antifoaming agents.

2.

Ingredients added after processing (a) (b)

3.

Flavourings Authorised food additives

Processing conditions (a) The temperature of the product should not exceed 180CC. (b) The duration of thermal processing should not exceed 15 minutes at 180°C with correspondingly longer times at lower temperatures. Heating up and cooling down time should be as short as practical (c) The pH during processing should not exceed the value of 8.0 (d) Flavourings and food additives should only be added after processing is complete. Flavour enhancers may be added before processing but only in minimum amounts necessary for flavour generation.

4.

Purity criteria (a) (b) (c) (d)

5.

Heavy metals as in EU Directive. Benzo [a] pyrene not more than 1 ug/kg, Benzo [a] anthracene not more than 2 ug/kg Amino-imidazo-azaarenes not detectable by the most sensitive routine method available.

Safety evaluation. As a minimum requirement the following toxicological studies should be performed: (a) A gene mutagenicity test (b) A test for chromosome damage in vivo or in vitro (c) A 90-day feeding study in animals

It should be noted that the Council of Europe lists have no legal standing. However, the work of the Council of Europe Committee of Experts has provided

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283

much of the basis for the current and future European Union legislation on flavourings. Many members of the committee are members also of the Flavourings Sub-Committee of the EU Scientific Committee for Food responsible for the evaluation of flavouring substances for a future EU positive list. 10.6

European Community

Several countries in Europe have had flavouring regulations in some form going back many years. In the then West Germany the initial regulation governing the use of artificial flavouring substances was enacted in 1959 (Essenzen VO, 1959). In this sense the term 'artificial' refers to substances that are not chemically identical to materials present in natural products. This regulation listed only five artificial flavouring substances that were permitted and a short list of processed foods in which they were allowed. This regulation was amended in 1970 (Essenzen VO, 1970) to allow several more artificial substances in the same restricted list of foods. In the UK in 1965, the Ministry of Agriculture, Food and Fisheries (MAFF) published a report on flavouring agents (MAFF, 1965) and a further report in 1976 (MAFF, 1976). Both these reports recommended that flavouring ingredients should be controlled by positive listing, but no action was taken to implement this. However, the latter report acknowledged that substances chemically identical to natural substances ('nature identical') are different from artificial flavouring substances. It also drew attention to the work of the Council of Europe in the flavouring field. In 1988 the first attempt was made to harmonise the flavour regulations of the Member States of the European Community. This took the form of a Council Directive on the approximation of laws of the Member States relating to flavourings and to source materials for their production (EC 1988a). The primary reason for the Directive was the protection of human health, but within these limits to take account of economic and technical needs. The intention of the Directive was to lay down a framework including (a) General purity criteria (b) Definitions (c) Labelling (d) Appropriate provisions for the inventories created by Decision 88/3897 EEC(EC1988b) (e) Specific purity and microbiological criteria (f) Limitation of certain components of vegetable or animal raw materials (g) Drawing up of lists of additives, solvents and diluents for flavourings In the following section only those Articles that deal with nonprocedural matters are included.

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Article 1 (Definitions) ' 1. 2.

This Directive shall apply to "flavourings" used or intended for use in or on foodstuffs to impart odour and/or taste, and to source materials used for the production of flavourings. For the purposes of this Directive: (a)

"Flavouring" means flavouring substances, flavouring preparations, smoke flavourings, process flavourings or mixtures thereof/

Unlike most earlier definitions and normal commercial practice, this definition does not include the solvent or carrier but only the active part of the finished flavouring. '(b)

"Flavouring substance" means a defined chemical substance with flavouring properties which is obtained: (i)

By appropriate physical processes (including distillation and solvent extraction) or enzymatic or microbiological processes from material of vegetable origin either in the raw state or after processing for human consumption by traditional foodpreparation processes (including drying, torrefaction and fermentation), (ii) By chemical synthesis or isolated by chemical processes and which is chemically identical to a substance naturally present in material of vegetable or animal origin as described in (i). (iii) By chemical synthesis but which is not chemically identical to a substance naturally present in material of vegetable or animal origin as described in (i).' These three definitions refer to flavouring substances that, in practice, are called natural, nature identical and artificial respectively. The initial part of the definition refers to a defined chemical substance but is understood to include a defined mixture of defined substances, e.g. citral, which is a mixture of cis- and transisomers. The natural substance definition includes non-foods, e.g. cedarwood. and thus the nature identical definition includes non-food-identical substances. There is no definition of 'a traditional food preparation process' or 'appropriate physical process', which leaves it open to individual interpretation. '(c)

"Flavouring preparation" means a product, other than the substances defined in (i) above, whether concentrated or not. with flavouring properties, which is obtained by appropriate physical processes (including distillation and solvent extraction) or by enzymatic or microbiological processes from material of vegetable or animal origin, either in the raw state or after processing for human consumption by traditional food-preparation processes (including drying, torrefaction and fermentation)/

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285

This definition covers such products as essential oils, concentrated essential oils, essential oil terpenes and isolates, oleoresins, resinoids, absolutes, extracts and tinctures of natural source materials (including the extraction solvent if this is a flavour carrier), distillates of natural source materials, and fruit juices either concentrated or not used for their flavouring properties. It suffers from the same imprecision as in the foregoing paragraph. "(d)

"Process flavouring" means a product which is obtained according to good manufacturing practices by heating to a temperature not exceeding 180°C for a period not exceeding 15 minutes a mixture of ingredients, not necessarily themselves having flavouring properties, of which at least one contains nitrogen (amino) and another is a reducing sugar.'

This definition is defective in that it does not apply to the majority of commercial process flavours, which are heated for 1-4 hours, albeit at lower temperatures. By negotiation with the Commission it has been unofficially agreed that longer times at lower temperatures are appropriate and a doubling of the time for each 10°C decrease in temperature is acceptable. The definition does not specify what other flavouring ingredients may be added or whether sources that provide reducing sugars are covered. '(e)

"Smoke flavouring" means a smoke extract used in traditional foodstuffs smoking processes.'

This is an impossible definition since traditional smoking processes do not use smoke extract. It is assumed that it refers to smoke generated by a method similar to that used in traditional smoking processes that is then condensed or absorbed in a carrier to form a smoke extract. This is the meaning that is used by the European flavour industry. '3.

Flavourings may also contain foodstuffs as well as additives necessary for the storage, use, dissolution or dilution, or processing aids where these are covered by other Community provisions.'

These materials are not within the ambit of paragraph 2(a) above and thus do not form part of the flavouring but are additives to it. Article 2 (Scope) 'The Directive shall not apply to: • • •

Edible substances and products intended to be consumed as such, with or without reconstitution. Substances which have exclusively a sweet, sour or salt taste. Material of vegetable or animal origin, having inherently flavouring properties, where they are not used as flavouring sources.'

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FOOD FLAVOUR TECHNOLOGY

The first indent excludes such products as fruit juices and the third herbs and spices. The position of flavour enhancers such as monosodium glutamate and other amino acids is not clear. They are not really in accordance with the second indent since they certainly have some taste apart from being salty. The latest inventory of flavouring substances includes some amino acids and they are also included in the proposed raw materials for the preparation of process flavourings. Article 4 (Restricted substances) 'Member States shall take all measures necessary to ensure that: (a)

(b)

•

Flavourings do not contain any element or substance in a toxicologically dangerous quantity • Subject to any exceptions provided for in the specific criteria of purity referred to in Article 6(2) third indent, they do not contain more than 3mg/kg of arsenic, lOmg/kg of lead. 1 mg/kg of cadmium and 1 mg/kg of mercury. The use of flavourings does not result in the presence in foodstuffs as consumed of undesirable substances listed in Annex I [see Table 10.1 ] in quantities greater than those specified therein/

These limits are extremely low when compared with the levels of the same compounds in the surface layers of smoked or roasted meats (around 1 ppm). '(c)

The use of flavourings and of other food ingredients with flavouring properties does not result in the presence of substances listed in Annex II [see Table 10.2] in quantities greater than those specified therein.'

Annexe II represents the position as at March 2001. It is likely that capsaicin. estragole and methyleugenol will be added to the list. Article 5 (Inventories) This is concerned with making appropriate decisions concerning the inventories created by Council Decision 88/389/EEC (EC 1988b) as follows. Table 10.1 ANNEX I: Maximum limits for certain undesirable substances present in foodstuffs as consumed as a result of the use of flavourings Substance

Foodstuffs

Beverages

Benzo-[a]-pyrene Benzo-[«]-anthracenea

0.03|jg/kg 0.06ug/kg

0.03ug/kg 0.06|jg/kg

a

Not currently limited (3/2001) but will be added.

FLAVOUR LEGISLATION

>87

Table 10.2 ANNEX II: Maximum limits for certain substances obtained from flavourings and other food ingredients with flavouring properties present in foodstuffs as consumed in which flavourings have been used Substance Agaric acid

Foodstuffs (mg/kg) 20

Beverages (mg/kg) 20

Exceptions and/or special restrictions 100 mg/kg in alcoholic beverages and foodstuffs containing mushrooms

Aloin

0.1

0.1

50 mg/kg in alcoholic beverages

fi-Asarone

0.1

0.1

1 mg/kg in alcoholic beverages and seasonings used in snack foods

Berberine

0.1

0.1

10 mg/kg in alcoholic beverages

Coumarin

2

2

10 mg/kg in certain types of caramel confectionery 50 mg/kg in chewing gum 10 mg/kg in alcoholic beverages

Hydrocyanic acid

1

1

50 mg/kg in nougat, marzipan or its substitutes or similar products 1 mg/% alcohol by volume/kg in alcoholic beverages 5 mg/kg in canned stone fruit

Hypericine

0.1

0.1

10 mg/kg in alcoholic beverages 1 mg/kg in confectionery

Pulegone

25

100

250 mg/kg in mint or peppermint flavoured beverages 350 mg/kg in mint confectionery

Quassine

5

5

10 mg/kg in confectionery in pastille form 50 mg/kg in alcoholic beverages

Safrole and isoSafrole

1

1

2 mg/kg in alcoholic beverages with less than 25% alcohol by volume 5 mg/kg in alcoholic beverages with more than 25% alcohol by volume 15 nig/kg in foodstuffs containing mace and nutmeg

Santonin

0.1

0.1

1 mg/kg in alcoholic beverages with more than 25% alcohol by volume

Thujone (a and (3)

0.5

0.5

5 mg/kg in alcoholic beverages with less than 25% alcohol by volume 10 mg/kg in alcoholic beverages with more than 25%; alcohol by volume 25 mg/kg in foodstuffs containing preparations based on sage 35 mg/kg in bitters

None of the substances listed above may be added as such to flavourings or to foodstuffs. They may be present in a foodstuff either naturally or following the addition of flavourings prepared from natural raw materials.

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FOOD FLAVOUR TECHNOLOGY

The Commission shall, within 24 months of the adoption of this Decision and after consultation of the Member States, establish an inventory of: (a) flavouring sources composed of foodstuffs and of herbs and spices normally considered as foods (b) flavouring sources composed of vegetable or animal raw materials not normally considered as food natural flavouring substances synthetic flavouring substances chemically identical to substances in foodstuffs, herbs and spices (e) synthetic flavouring substances chemically identical to substances in vegetable or animal raw materials not normally considered as foodstuffs, herbs and spices artificial flavouring substances (f) source materials used in the production of smoke and process flavourings and the reaction conditions under which they are prepared Groups (c), (d) and (e) have been combined into a single inventory under Commission Regulation 2232/96 (EC, 1996). This regulation required Member States to notify the Commission of those flavouring substances allowed in their territory. In practice the local flavour industry, under the guidance of the European Flavour and Fragrance Association (EFFA), supplied a list of the substances that were being used in their territory to the local regulatory organisation for that body to examine and, if appropriate, pass on to the Commission by 23 November 1996. The Commission was required to consolidate the submissions into a single inventory representing the flavouring substances that were currently being used in the EU (EC, 1999). It was permitted to disclose substances under code to maintain confidentiality of their identity (EC. 1998), but this route has been taken only for about 12 substances. The Standing Committee for Foodstuffs was required to examine and, if necessary, amend the inventory by 23 September 1999. The final inventory contains approximately 2500 substances and contains almost 500 substances that are not on the US GRAS lists. The final permitted list will be dependent on safety evaluation of each individual substance by the Scientific Committee for Food. This has been delegated to its flavouring sub-committee, which is examining the substances by chemical grouping under the Scientific Co-operation Procedure (SCOOP) (EC, 1994). The data required for evaluation are being provided by EFFA on the form illustrated in Figure 10.2. The substances contained in the inventory are meant to be permitted in each Member State of the EU until a final permitted list is established in about 2005. However, to date, both Germany and Italy have maintained their restricted lists of artificial flavouring substances that were in force before 1988. Some preliminary progress has been made on group (f). The European Commission has not yet published any guidelines on process flavourings but it is likely that they will follow the Council of Europe recommendations. The

289

FLAVOUR LEGISLATION

flavour industry stance, as agreed by IOFI (1989), is somewhat similar to those guidelines except for item ' 1 (d) Other ingredients' where 'Herbs and spices and their extracts' is expanded to include 'flavouring substances identified therein'. There can be no argument that this significantly increases the risk, since the

Flavouring Substance EFFA No

Butyi but-2-enoate CAS No

CoE No

0301

FEMA No

EINECS NO

JECFANo -

2-966X

o Me'

^

591-63-9

FL No

09.324

^ OBu-n

Chemical Name on Register

Butyl but-2-enoate

IUPAC Name

2-butenoic acid, (E)-, butyl ester

Synonyms

Crotonic acid, butyl ester, (E)-

Physical Form

liquid

Boiling Point ( °C, 76 Torr)

80(42 T)

Melting Point ( °C)

Ret. Index Lower Value (20 °C)

1 425

Ref. Index Upper Value Density Upper Value

Density Lower Value (25 °C) Minimum Assay Value Solubility

Food Category

1 Beverages, excl. dairy products

Sensory Description Food Source

Interpretive Study

95 insoluble in water

Chemical Group

1 431

Identity Test

BI 721 ses

Solubility in Ethanol

1 mi in 1 ml 95%EtOH

Origin (Codex, CAC)

nat /nat ident.

Normal Dosage in ppm

s

Maximum Dosage in ppm

25

Volume of use by EFFA kg/a

14

colourless liquid with fruity banana odour

Pawpaw (Asimina Triloba Dunal.)

in ppm

0.024

2000-3

Recent Studies Figure 10.2 Example of initial data submission required by SCOOP for flavouring substances, and volume of use by EFFA.

290

FOOD FLAVOUR TECHNOLOGY

Butyl but-2-enoate Volume of use by EFFA: kg/year 14 Application Normal dosa

Food Category

(ppm)

Dairy products (excluding those listed elsewhere) Fats and oils and fat emulsions

7

9e

Maximum dosage (ppm) 35

5

25

10

50

Processed fruits and vegetables

7

35

Sugar and chocolate confectionery

10

50

Edible ices including sherbets and water ices

5

25

10

50

Meat and meat products

2

10

Fish and fish products

2

10

5

25

10

50

Cereals and cereal products Bakery wares

Soups, sauces and seasonings Foodstuffs intended for particular nutritional purposes Beverages excluding dairy products Ready-to-eat savouries Composite foods (e.g. casseroles, etc.)

5

25

20

100

5

25

Figure 10.2 (continued).

presence of these materials is already permitted owing to their occurrence in herbs and spices. There has been an initial draft document concerning smoke flavourings (EC, 2001). This sets out the types of wood that may be used to produce the smoke used in the preparation of the smoke extract, the conditions under which the smoke may be generated, the general specification for the product and the toxicological data necessary for its approval. The way the document is written suggests that specific smoke flavourings from specified suppliers are the only ones that will be approved. This is currently the position in Sweden, where there is a positive list of named smoke flavourings to the exclusion of all others. This is not in accord with the Community anticompetition legislation.

FLAVOUR LEGISLATION

29 1

Article 6 (Additives for flavourings) This Article requires that the following lists of authorised substances be agreed: • • •

Additives necessary for the storage and use of flavourings Products used for dissolving and diluting flavourings Processing aids where these are not covered by other Community provisions

The first two indents above have proved extremely difficult in obtaining agreement with the Member States. Discussions have been taking place with the Commission since 1990 to try to thrash out a system that will satisfy all parties involved. These have consisted of permitting automatically all the additives that are permitted for use in food or those that are quantum satis and the others by positive list. Neither of these alternatives is regarded as satisfactory, mainly on the grounds that they would allow additives that are not permitted in a particular foodstuff to be added without declaration through the flavour composition. The discussion is ongoing and agreement is of considerable importance since individual Member State rules are being used to frustrate free trade in flavourings. Article 6 also deals with methods of sampling and analysis and microbiological criteria, none of which has been implemented to date. Article 9 (Labelling) 1.

Flavourings not intended for sale to the final consumer may not be marketed unless their packagings or containers bear the following information, which should be easily visible, clearly legible and indelible: (a) (b)

The name or business name and address of the manufacturer or packer, or of a seller established in the Community. Either the word 'flavouring' or a more specific name or description of the flavouring.

This enables descriptions such as 'Lemon Flavouring' or 'Natural Flavouring' if appropriate. (c) (d)

Either the statement 'for foodstuffs' or a more specific reference to the foodstuff for which the flavouring is intended. A list in descending order of weight of the categories of flavouring ingredients present as follows: • • •

Naturalflavouringsubstances Nature identicalflavouringsubstances Artificialflavouringsubstances

292

FOOD FLAVOUR TECHNOLOGY

• • • (e)

Flavouring preparations Process flavourings Smoke flavourings

In the case of mixtures of flavourings with other substances referred to in Article 6, a list in descending order of weight in the mixture of: • •

The categories of flavourings classified as in (d) above The names of each of the other substances or materials or their E numbers

It is not clear whether this means a single list or two lists. In general the flavour industry uses the latter. (f) (g) (h) 2.

An indication of the maximum quantity of each component contained in Annexes I and II or sufficient information to enable the food producer to comply with the limits for the finished food. An indication identifying the consignment. The nominal quantity in units of mass or volume.

The word 'natural', or any other word having substantially the same meaning, may only be used for flavourings in which the flavouring component consists of exclusively flavouring preparations and/or natural flavouring substances.

Paragraph 2 means that flavourings containing process flavourings or smoke flavourings cannot be labelled as natural, in contrast to the US position. The above requirement is complied with in all EU Member States except Italy, where flavourings containing nature-identical flavouring substances are also designated as natural. If the sales description of the flavouring contains a reference to a foodstuff or flavouring source, the word 'natural', or any word having substantially the same meaning may not be used unless the flavouring components have been isolated solely or almost solely from the flavouring source concerned. This means that in 'natural lemon flavouring' the flavouring components have to be natural and derived solely or almost solely from lemon. The term 'almost solely' is not defined but, by agreement with the 1988 Commissioner, a level of greater than 90% from the named source would be acceptable to both the Commission and industry. This leaves the question of how to designate natural flavourings that are not derived solely or also solely from the named source. This is of course a matter for the individual Member States. In the UK wording such as 'natural lemon flavour flavouring' or 'natural flavouring lemon type'

FLAVOUR LEGISLATION

293

have been used, but the matter has never been tested in the courts to decide whether these are acceptable. 3.

By derogation from paragraph 1, the information required by paragraph 1 (d), (e) and (f) may appear merely on the trade documents relating to the consignment supplied prior to the delivery, provided the phrase 'intended for the manufacture of foodstuffs and not for retail' appears in a conspicuous part of the packaging or container of the products in question.

It is not clear whether this phrase should replace that in paragraph l(c) or be in addition to it. It is normal for the former to be used. 4.

Member States shall refrain from laying down requirements more detailed than those contained in this Article concerning the manner in which the particulars provided for are to be shown. These particulars shall be given in terms easily understood by the purchaser. This shall not prevent them being given in various languages.

This provision has been ignored by at least one Member State by invoking the Reserved Dairy Descriptions Directive to prevent the use of milk, cream, butter or cheese in the description of flavourings. After complaints by other Member States this was abandoned, as was the insistence that solely their own language be used on label of flavourings for their country. 10.7

The future

It is widely recognised both among regulators and the international flavour industry that the lack of harmonisation of flavouring legislation represents a significant barrier to trade. The acceptance in one country of a flavouring material banned in another cannot possibly be on the basis of safety; it is usually on the basis of commercial interest. The International Organisation of the Flavour Industry (IOFI) has been pressing legislators to accept flavouring materials on the basis of safety evaluations performed for other groups, e.g. the acceptance by European Scientific Committee for Food of evaluations done by FEXPAN. The international nature of the flavour industry is providing data to FEXPAN to enable the evaluation of the 500 or so additional flavouring substances that are on the European inventory so that they may be granted GRAS status for use in the USA. References Burdock, G.A., Wagner, B.M., Smith, R.L., Munro, I.C. and Newbeme, P.M. (1990) Recent progress in the consideration of flavoring ingredients under the Food Additive Amendment. FEMA GRAS Substances 15. Food Techno/., 44(2) 82. Code of Federal Regulations Title 21(1990) - Food and Drugs. Chapter 1. § 101.22 (a) (3).

294

FOOD FLAVOUR TECHNOLOGY

Council of Europe (1974) Natural Flavouring Substances. Their Sources, and Added Artificial Flavouring Substances, Council of Europe. Maisonneuve. Council of Europe (1981) Flavouring Substances and Natural Sources of Flavourings. 3rd edn. Council of Europe, Maisonneuve. Council of Europe (1992) Flavouring Substances and Natural Sources of Flavourings, 4th edn. Council of Europe, Maisonneuve. Council of Europe (1995) Guidelines on the Production of Thermal Process Flavourings. Council of Europe Publishing. ISBN 92-871-2811-1. EC (1988a) Council Directive of 22 June 1988 on the approximation of the laws of the Member States relating of flavourings and to source materials for their production (88/388/EEC). Official Journal of the European Communities. No. L 184/6K15/7/88). See also UK implementation: The Flavouring in Foods Regulations S.I. 1992 No. 1971. The Flavouring of Food (Amendment) Regulations S.I. 1994 No. 1486. EC (1988b) Council Decision of 22 June 1988 on the establishment, by the Commission, of an inventors of the source materials and substances used in the preparation of flavourings (88/389/EEC). Official Journal of the European Communities. No. L 184/67 (15/7/88). EC (1994) European Commission Decision of 20 September 1994 establishing the inventory and distribution of tasks to be undertaken within the framework of co-operation by Member States in the scientific examination of questions relating to food (1994/652/EC). Official Journal of the European Communities. No. L 253/29 (29/9/94). EC (1996) European Parliament and Council Regulation (EC) of 28 October 1996 laying down a Community procedure for Flavouring Substances used or intended for use in Foodstuffs (2232/96). EC (1998) European Commission Recommendation 98/282/EC. EC (1999) European Commission Decision of 23 February 1999 adopting a register of flavouring substances used in or on foodstuffs (1999/21 I/EC). Official Journal of the European Communities. No. L 084/1 (27/3/99) amended by Decision 2000/489/EC. Official Journal of the European Communities. No. L 197/53 (3/8/00). EC (2001) European Commission Working Document WGF/007/01. Draft European Parliament and Council Regulation tor smoke flavourings used or intended to be used in or on foodstuffs (22 February 2001). Essenzen VO (1959) Verordnung uber Essen/en und Grundstoffe (Essen/en VO) vom 19 Dezember 1959 (BGB1.IS.747). Essenzen VO (1970) Verordnung uber Essenzen und Grundstoffe (Essenzen VO). Der Neufassung vom 9 Oktober 1970 (BGBL I S. 1389). Hall. R.L. and Oser. B.L. (1965. 1970) Recent progress in the consideration of flavoring ingredients under the Food Additive Amendment. FEMA GRAS Substances 3. Food Technol.. 19(2). Part 2. 151: FEMA GRAS Substances 4. Food Technol.. 24(5) 25. Hallagan. J.B. and Hall. R.L. (1995) FEMA GRAS—A GRAS Assessment Program tor Flavor Ingredients. Regulatory Toxicology and Pharmacology. 21 422-430. IOFI. International Organisation of the Flavour Industry (1989) Code of Practice Section III IOFI guidelines for the production and labelling of process flavourings (October 1989). MAFF (1965) Food Standards Committee Report on Flavouring Agents. London. HMSO. MAFF (1976) Food Additives and Contaminants Committee Report on the Review of Flavourings in Food. FAC/REP/22. London. HMSO. Munro. I.C.. Kennepohl. E. and Kroes, R. (1999) A procedure for the safety evaluation of flavouring substances. Food and Chemical Toxicology. 37 207-232. Newberne. P.. Smith. R.L.. Doull. J.. Goodman. J.I.. Munro. I.C.. Portoghese. P.S.. Wagner. B.M.. Weil. C.S.. Woods. L.A.. Adams. T.B. and Hallagan. J.B. (1998) GRAS flavoring substances 18. Food Technol.. 52(9) 65.

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295

Newberne, P., Smith, R.L., Doull, J., Feron, V.J., Goodman, J.I., Munro, I.C., Portoghese, P.S., Waddell, W.J., Wagner, B.M., Weil, C.S., Adams, T.B. and Hallagan, J.B. (2000) GRAS flavoring substances 19. Food TechnoL, 54(6) 66. Oser, B.L. and Hall, R.L. (1972-1979) Recent progress in the consideration of flavoring ingredients under the Food Additive Amendment. FEMA GRAS Substances 5. Food TechnoL, 26(5) 35 (1972): FEMA GRAS Substances 6. Food TechnoL, 27(1) 64 (1973); FEMA GRAS Substances 7. Food Techno!., 27(11) 56 (1973); FEMA GRAS Substances 8. Food Techno!., 28(9) 76 (1974); FEMA GRAS Substances 9. Food TechnoL, 29(9) 70 (1975); FEMA GRAS Substances 10. Food TechnoL. 31(1) 65 (1977); FEMA GRAS Substances 11. Food TechnoL, 32(2) 60 (1978); FEMA GRAS Substances 12. Food TechnoL, 33(7) 65 (1979). Oser, B.L., Ford R.A. and Bernard B.K. (1984, 1985) Recent progress in the consideration of flavoring ingredients under the Food Additive Amendment. FEMA GRAS Substances 13. Food TechnoL. 38( 10) 65 (1984); FEMA GRAS Substances 14. Food TechnoL, 39( 11) 108 (1985). Smith, R.L. and Ford, R.A. (1993) Recent progress in the consideration of flavoring ingredients under the Food Additive Amendment. FEMA GRAS Substances 16. Food TechnoL, 47(6) 104. Smith, R.L., Newberne, P., Adams, T.B., Ford, R.A., Hallagan, J.B. and the FEMA Expert Panel (1996) GRAS flavoring substances 17. Food TechnoL, 50(10) 72. Smith, R.L., Newberne, P., Adams, T.B., Ford, R.A., Hallagan, J.B. and the FEMA Expert Panel (1997) Correction to GRAS flavoring substances 17. Food TechnoL, 51(2) 32.

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Index

absorption, of flavours 170 abstract flavours 23 activated carbon, for aroma trapping 217 activity coefficient 169,187 acyloins, bioformation of 72 acyltransferases, genes for 86 adsorption, of flavours 170 AEDA 254 aldehydes, bioformation from alcohols 66 alliinase 138 genes 87 allium species, and flavour bioformation 81 allylic hydroxylation of terpenes 63 analysis byGC 234 of flavours 210-251 ANOVA, in sensory analysis 266 antiplasticisation, in polymers 156 API-MS 245 apples 142 apricot 146 aroma changes in foods, analysis of 228 extract dilution analysis (AEDA) 237 profiles 259 aroma extraction general considerations 224 principles of 213 selection of method 226 aroma extracts and characteristic aroma 227 and sensory response 229 aromagrams 235 artefact formation, and silica gel 233 artefacts, in aroma extraction 224-225 artifical flavor, definition 278 ASOG 188 Atmospheric Pressure lonisation-MS 244 barrier principles, of hydrophilic polymers 162 beer 130 undesirable flavours 131 benzaldehyde. bioformation of 70

binding of flavours 11, 170 biocatalysis, advantages of 61-62 bioreactor design, for flavour production 89 biotechnology of flavour generation 60-104 blackcurrant 144 blotters 1,227 advantages of 5 bouillon flavours 125 Brassica fl avour 140-141 bread crust aroma 134 breakthrough, on Tenax 217 butter aroma, composition of 121 C9 compounds, flavour impression of 141, 142 callus tissue, and bioformation of flavour 78 carbohydrate cleavage, in Maillard 33, 34 carbohydrate, interaction with flavour 172 category scales 260 cereal flavour, formation 41 cereal products 134 challenges, in analysis of flavours 210 character impact compounds 28, 108,253 Concord and Lambrusco 133 from Maillard 37,41 Sauvignon 133 yeast extracts 40 charm analysis 237, 253 cheese flavour 122 cheese ripening, effect of microbes 124 chemical binding 170 chemical nature of flavours, effect on behaviour in flavourings 105 cherry flavour, composition of 112 chocolate 126 flavour, via Maillard 51 citrus 146 processing 147 cocoa mass, odorants in 126 coffee 129 flavour composition 130 flavour formation 40 complete arorna composition 226 complexity, of flavours 210

298 concentration of flavours in food 19 conducting polymer sensors 246 consumer preference ratings 260 consumer tests 259 consumption indices 109 controlled release 163 Council of Europe and flavour definitions 280 and legislation 280 cream and butter flavour 121 cross modal matching 254-255 cryotrapping, of flavours 216 customer requirements 25 cyclodextrins 174 dairy flavours 120 definitions . for regulating flavours 284 degree of risk, posed by flavours 276 deoxysones 30 descriptive analysis 258 detection threshold 253 detectors for GC 239 deterpenation of lemon oil 10 dicarbonyl compounds, in Maillard 33 difference tests 252 diffusion from polymers 160 in flavour release 176 dilution factor 254 direct MS. of flavour compounds 244 discrimination tests 252 dispersed flavours 15 distillation for aroma extraction 217 for flavour extraction 10 diversity, of flavour chemicals 212 duo-trio test 252 eating behaviour, effect on flavour release 180 economics of bioflavour production 91 eddy diffusion 177 electronic noses 246 electronic noses, limitations 247 emulsion based flavours 15 encapsulation 153 methods of 153 principles of 154 environment, for sensory testing 261 enzyme technology 73-77 epoxidation, by cytochrome P450 65 ethanol. solvent for flavours 13

INDEX ethics, in sensory analysis 264 EU Flavourings Directive 281 experimental design, for sensory analysis 262 extraction of aromas 212 of flavours, from plants 114 method, and effect on flavour profile 211 facilities, for sensory testing 261 fat content effect on flavour formulation 9 effect on flavour quality 9 fatigue, in sensory' testing 262 fatty acids, in cheese 122-123 FD factor 254 FEMA (Flavor and Extract Manufacturing Association) 278 Pick'slaw 160. 175 flame ionisation detector 236. 239 flavour

balance 7 blocks 45. 117 complexity 7 delivery 168 systems 153 kinetic aspects 175 formulation 1-26 generation, thermal 27-59 precursors. Amadori and Heyns compounds 28 glycosides 74. 117 in tea 118 meat 30,31.32.40 wine 134 quality, assessment of 110 release 168 effect of gas flow 202 methods for measuring 182 systems 153 skeleton raspberry 19 strawberry 4. 8 thresholds, of mint enantiomers 113 Flory interaction 158 focus groups 260 Food and Drug Administration 278 food matrix, effect on flavour delivery 168 formulation of flavours 1 -26 fractionation acid-base 230 disadvantages 232 HPLC 230

299

INDEX prior to aroma analysis 229 scheme 23! silicic acid 232 free choice profiling 258 FTNF (From The Named Fruit) 279 furaneol, biotbrmation of 67 garlic flavour 138 GC-IR analysis of flavour 242 GC-MS, of flavours 243 GC-oJfactometry 235, 253 for screening 236 identification of Maillard aromas 37 in off odour analysis 228 limitations of 239, 254 gels and flavour release 179 genetic engineering and biotbrmation of flavour 82, 86 Gibbs energy, in encapsulation 154 glycosides see flavour precursors, glycosides golden rice 84 grapefruit 144 GRAS (Generally Recognised As Safe) 17,18,61,278 process flavours 45 green aromas 108 biotbrmation of 81 in raspberry 20 types of 5, 141 Guggenheim approximation 157 gum acacia 16 HACCP for flavours 120 harmonisation, of legislation 283 headspace analysis 2 dilution, modelling of 196 heat, effect on flavour I 1 Henry's Law 158, 169 high resolution GC 234 Hildebrand solubility parameter, in encapsulation 155 history of flavours 1, 60, 111 hops 131 HPLC, for flavour analysis 240 human subject consent, for sensory analysis 264 hydration, by biotransformation 66 hydrolysed vegetable protein (HVP) 50, 125 hydroperoxide lyase, genes for 87

identification of flavour compounds 240 immobilisation, and bioflavour production 90 inclusion compounds 173 infrared spectroscopy, in flavour analysis 242 intensity rating 254 interactions, between flavour and ingredients 26 interactions, between plants and microbial infections 78 interfacial mass transport 176 internal standards, in aroma extraction 226 international tastes 21 TOFI and regulation 289 guidelines for process flavours 44 GMP for bioprocessed flavours 61 isolation, of aromas 212 isomerism, effect on flavour 107, 113 isothiocyanates, in Brassica flavour 140 JECFA (Joint Expert Committee on Food Additives) 279 judges for sensory analysis 264-265 monitoring performance 266 Kovats indices 241 labelled magnitude scale 254 labelling, of flavours 291 lactones biotbrmation of 68 in cheese 122 in fruit 1 18 legislation 276 in USA 277 methods 276 national 283 the future 293 Likens- Nickerson distillation 218 limits of substances, in flavourings 287 limonene, biotransformation 63 lipid and flavour binding 171 effect on flavour partition 189 hydrolase enzymes, role in ester synthesis 75 oxidation pathway, genetic engineering of 85 LoeP 191,205

300 magnitude matching 254 Maillard reaction 27 pathways 28-29 maltol and taste analogues 6 bioformation of 67 mass spectra, identification 243 mass spectral libraries 243 mass spectrometry. of flavour compounds 243 mass transfer coefficient 180 mass transfer, in boundary layers 197 mass transport, in polymer systems 161 matching of flavours 24 meat aroma origin of 30-31 species-specific differences 40. 49 meat flavour, through process flavours 49 microorganisms, for bioflavour production 62 mode of sampling, in sensory analysis 256 modelling and emulsions 198-200 and flavour release 185 approach to 185. 194 dynamic systems 192 flavour release in vivo 200, 201 flavour transport in vivo 202 time intensity data 204 models for flavour release, types 186 molecular size, and diffusion 162 monoterpene, bioconversion of 62 multidimensional GC 235 myrosinase 140 natural flavor consumer attitude 61 definition 278,279 sources 105-152 nature-identical flavourings 112 negative list, of flavours 277 NIF analysis 238 nitrogen phosphorus detector 239 nonequilibrium partition 178. 194,203 norisoprenoids. bioconversion of 66 odour active compounds 253 odour activity value (OAV) 236 odour threshold 212 of Maillard aromas 38-39. 42-43 of terpenoid enantiomers 107 odour unit 236. 253 odour value 236

INDEX off notes, and isolation 227 oil fraction, effect on flavour release 191 oil soluble flavours 14 oleoresin 114 olfactory global analysis 254 onion flavour 139 optimisation, of reaction flavours 28 orange flavour 146 orange oils, composition of 148 order of presentation, in sensory testing 263 OSME analysis 238.254 oxygen heterocycles. bioformation of 67 pair preference tests 260 partial least squares analysis 270 partition coefficient 168.197 air-emulsion 190 air-water 186. 190 in air and water 172 partition models 186 pattern recognition 246 PCA 268 PCAIV 269 peach 146 pears 143 penetration theory 178 perception thresholds, C6 aldehydes 110 pH. effect on Maillard 33 phenylacetaldehyde formation, via Strecker reaction 35-36 2-phenylethanol. bioformation of 70 physical chemistry, and flavour release 168 a-pinene biotransformations 64 plant cells, and flavour bioformation 80 plant organ culture, for flavour bioformation 79 plant tissues, for bioformation of flavour 77 plasticiser, in encapsulation matrices 156 polyphenol oxidase. in cocoa 127 polysaccharides. and flavour binding 173 positive list, of flavours 276 power testing, in sensory analysis 268 preparative gas chromatography 233 primary character 3. 18 principal component analysis (PCA) 268 process conditions, for bioformation of flavour 88 process flavours 27 effect of amino acids 45-46 effect of ph 46 effect of time/temperature 47-48 effect of water activity 47

INDEX legislation on 283 preparation of 44-51 regulations for 285 savoury 48 sweet 51 Procrustes analysis 269 production of flavour formulations 17 productivity issues in bioreactors 89 propylene glycol, solvent for flavours 13 proteins, interaction with flavour 174 pungent flavours 137 purge and trap, for extraction of flavours 215 QSPR 187 model, for partition coefficient 189 parameters in 188 procedure 188 quality control tests, by sensory analyis 260 quantification of aroma, problems of 226 racemates, resolution of 76 RAS 193 raspberry 145 aldehyde 145 flavour 18-21 ketone 71, 145 receptors aroma and taste 106 mechanism for 106 recognition threshold 253 recombinant DNA and flavour formation 84 recovery, of aromas during extraction 222 regional tastes 21 regulation 18 reproducibility, of aroma analyses 228 restricted substances, in flavourings 286 retention indices 241 standards for 241 retention time 241 retronasal aroma simulator, modelling release from 193 safety of flavours 120 sales of flavours. 1994 figures 119 saliva, effect on flavour release 178 salting out 173 sample order, in sensory testing 263 sample preparation for aroma extraction 224 for sensory testing 262 scale up, in bioprocessing of flavours 88 scatchard plot 174

301 SCOOP (Scientific Co-operation Procedure) 288 SCOOP, data submission 289 secondary character 5, 19 selected ion monitoring (SIM) 243 semiconductor gas sensors 246 sensitivity, of direct headspace analysis 214 sensors, for electronic noses 246 sensory analysis, training of judges 264-265 sensory and instrumental data, analysis of 269 sensory methods 252 protocols for 252 sensory testing, administration of 261 sesquiterpenes, bioconversion of 66 short chain fatty acids, bioforrnation of 72 simplification of analyses 2 simultaneous distillation extraction 218 smoke flavouring, regulations for 285 SNIP analysis 238 solid phase micro extraction (SPME), for flavour extraction 222 solvents extraction, efficiency of solvent type 221 extraction, of aromas 220 for essential oil extraction 149 for flavours 12-13,25 GRAS status 279 sorption isotherms, theoretical 159 sorption of volatiles 157 spice flavours 136 SPME for flavour extraction 222 procedure 223 spray dried flavours 15-16 stable isotopes, as internal standards 226 stagnant film theory 177 standards, for sensory analysis 258. 265 starch, and flavour binding 173 static headspace method 214 quantification of 215 statistical analysis of difference tests 266 of sensory data 266 steam distillation 114 storage

302

INDEX

analysing aroma changes 228 changes in flavour 12 strawberries 145 strawberry aldehyde 8,145 Strecker degradation. 35 pathway 28 sugar cleavage, in Maillard 33 supercritical carbon dioxide, for aroma extraction 220 flavours surface acoustic wave sensors 246 synthetic flavour chemicals 112 taste as component of flavour perception 149 effects, in flavour formulation 6 thresholds, of terpenoid enantiomers 107 taste-smell interactions 257 in chewing gum 257 taurine. role in meat flavour 49 TCA 30 tea 128 odorants in 129 temperature, effect on perceived flavour 12 tenax trapping 216 thermal conductivity detector 236 desorption, of food 219 thermodynamics, of flavour delivery 168 thiazolidine carboxylic acid 30 threshold tests 253 threshold, aroma and taste 106 time intensity data 255. 256 trace flavour components 25 triacetin, solvent for flavours 13 triangle test 252 trigeminal senses, problems with sensory testing 263 UNIFAC 187 usage of flavours 290 vacuum headspace method 216 validation of models 186 validation, of flavour release models 207 Van der Waals forces, and flavour binding 171 vanilla bioformation of 69 flavour variation of 136 production 135 vapour pressure 170 correction 2. 18

variety-specific flavours, in apples 143 volatility and chemical properties 168 extraction using 213 water-soluble flavours crystal clear for beverages 14 12 wine 132 off flavours 134 WONF (With Other Natural Flavorings) 279 yeast extracts, as process flavour ingredients 50