Optimising Sweet Taste in Foods

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WOODHEAD PUBLISHING IN FOOD SCIENCE, TECHNOLOGY AND NUTRITION

The first part of the book reviews factors affecting sweet taste perception. It includes chapters on how taste cells respond to sweet taste compounds, genetic differences in sweet taste perception, the influence of tasteodour and taste-ingredient interactions and ways of measuring consumer perceptions of sweet taste. Part II discusses the main types of sweet-tasting compounds: sucrose, polyols, low-calorie and reduced-calorie sweeteners. The final part of the book looks at ways of improving the use of sweettasting compounds, including the range of strategies for developing new natural sweeteners, improving sweetener taste, optimising synergies in sweetener blends and improving the use of bulk sweeteners. With its distinguished editor and international team of contributors, Optimising sweet taste in foods will be a standard reference for the food industry in improving low-fat and other foods.

Optimising sweet taste in foods

A sweet taste is often a critical component in a consumer’s sensory evaluation of a food product. This book summarises key research on what determines consumer perceptions of sweet taste, the range of sweet-tasting compounds and the ways in which their use in foods can be optimised.

WOODHEAD PUBLISHING IN FOOD SCIENCE, TECHNOLOGY AND NUTRITION

Professor William J. Spillane works within the Chemistry Department of the National University of Ireland, Galway. He has published widely on non-nutritive sweeteners such as sulfamates as well as other aspects of sweet taste quality.

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Woodhead Publishing

ISBN-13:978-948-1-84569-0 ISBN-10:1-84569-008-7

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CRC Press LLC 6000 Broken Sound Parkway, NW Suite 300 Boca Raton FL 33487 USA CRC order number WP7603 CRC Press ISBN-10: 0-8493-7603-3

9 780849 376030 ISBN-13:978-948-1-84569-0 ISBN-10:1-84569-008-7

9 789481 845690

Spillane

Woodhead Publishing Ltd Abington Hall Abington ISBN-10: 0-8493-7603-3 Cambridge CB1 6AH England www.woodheadpublishing.com

Optimising sweet taste in foods Edited by W. J. Spillane

Optimising sweet taste in foods

Related titles: Flavour in food (ISBN-13: 978-1-85573-960-4; ISBN-10: 1-85573-960-7) The first part of the book reviews ways of measuring flavour. Part II looks at the ways flavour is retained and released in food. It considers the way flavour is retained in particular food matrices, how flavour is released during the process of eating, and the range of influences governing how flavour is perceived by the consumer. Flavour in food guides the reader through a complex subject and provides the essential foundation in both understanding and controlling food flavour. Improving the fat content of foods (ISBN-13: 978-1-85573-965-9; ISBN-10: 1-85573-965-8) Dietary fats have long been recognised as having a major impact on health: negative in the case of consumers' excessive intake of saturated fatty acids, positive in the case of increasing consumers' intake of long-chain n-3 polyunsaturated fatty acids (PUFAs). However, progress in ensuring that consumers achieve a nutritionally-optimal fat intake has been slow. This important collection reviews the range of steps needed to improve the fat content of foods whilst maintaining sensory quality. Texture in food, Volume 1: Semi-solid foods (ISBN-13: 978-1-85573-673-3; ISBN-10: 1-85573-673-X) Understanding and controlling the texture of semi-solid foods such as yoghurt and ice cream is a complex process. With a distinguished international team of contributors, this important collection summarises some of the most significant research in this area. The first part of the book looks at the behaviour of gels and emulsions, how they can be measured and their textural properties improved. The second part of the collection discusses the control of texture in particular foods such as yoghurt, ice cream, spreads and sauces. Texture in food, Volume 2: Solid foods (ISBN-13: 978-1-85573-724-2; ISBN-10: 1-85573-724-8) With its distinguished editor and international team of contributors, this authoritative book summarises the wealth of recent research on what influences texture in solid foods and how it can be controlled to maximise product quality. The first part of the book reviews research on understanding how consumers experience texture, and how they perceive key textural qualities. Part II considers the instrumental techniques used for analysing texture. The final part examines how the texture of particular foods may be better understood and improved. Details of these books and a complete list of Woodhead titles can be obtained by: · visiting our website at www.woodheadpublishing.com · contacting Customer Services (email: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext. 30; address: Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB1 6AH, England)

Optimising sweet taste in foods Edited by William J. Spillane

Published by Woodhead Publishing Limited Abington Hall, Abington Cambridge CB1 6AH England www.woodheadpublishing.com Published in North America by CRC Press LLC 6000 Broken Sound Parkway, NW Suite 300 Boca Raton, FL 33487 USA First published 2006, Woodhead Publishing Limited and CRC Press LLC ß 2006, Woodhead Publishing Limited The authors have asserted their moral rights. 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 authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book.

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Contents

Contributor contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. J. Spillane, National University of Ireland ± Galway, Ireland

xv

Part I 1

Factors affecting sweet taste perception

Stimulation of taste cells by sweet taste compounds . . . . . . . . . . . . M. Naim, The Hebrew University of Jerusalem, Israel, L. Huang, Monell Chemical Senses Center, USA, A. I. Spielman, College of Dentistry, New York University, USA and M. E. Shaul and A. Aliluiko, The Hebrew University of Jerusalem, Israel 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Peripheral organization of taste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 The sweet taste receptor(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Downstream signaling components . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Putative cellular mechanism(s) for the signal termination of sweet taste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

3 6 7 14 17 23 23 24

vi

Contents

2 Genetic differences in sweet taste perception . . . . . . . . . . . . . . . . . . . V. B. Duffy, J. E. Hayes and M. E. Dinehart, University of Connecticut, USA 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 PTC/PROP tasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Sweetness: relation to PROP, fungiform papillae density, T2R38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Relating genetic taste markers with dietary sweet behaviors . 2.5 Sweet liking: relation to PROP, fungiform papillae density, T2R38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Variation in other bitter markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 SAC Gene: human homolog and hedonic response to sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Children's liking of sweet tastes and its biological basis . . . . . . . . M. Y. Pepino and J. A. Mennella, Monell Chemical Senses Center, USA 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Ontogeny of sweet taste preferences: from fetal life to adolescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Role of experiential factors on sweet taste preferences . . . . . . 3.4 Physiological properties of sweet tastes . . . . . . . . . . . . . . . . . . . . . . 3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Taste±odour interactions in sweet taste perception . . . . . . . . . . . . . D. Valentin, C. Chrea and D. H. Nguyen, CSEG, France 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Overview of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Factors affecting taste±odour interactions . . . . . . . . . . . . . . . . . . . . 4.4 Mechanisms of taste±odour interactions . . . . . . . . . . . . . . . . . . . . . . 4.5 Implications for food product development . . . . . . . . . . . . . . . . . . 4.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Sources of further information and advice. . . . . . . . . . . . . . . . . . . . 4.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Taste±ingredient interactions modulating sweetness . . . . . . . . . . . . M. Lindley, Lindley Consulting, UK 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Interactions between sweeteners and bitter compounds . . . . . . 5.3 Interactions between sweeteners and acids . . . . . . . . . . . . . . . . . . . 5.4 Interactions of sweet and salty compounds . . . . . . . . . . . . . . . . . . .

30 30 36 37 41 42 45 46 49 49 54 54 55 58 59 60 61 61 66 66 67 69 75 79 80 81 81 85 85 86 88 92

Contents 5.5 5.6 5.7

Interactions of sweet compounds with other sweet compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 Measuring consumers' perceptions of sweet taste . . . . . . . . . . . . . . S. Issanchou and S. Nicklaus, INRA, France 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods to determine consumers' perceptions of sweet taste . 6.2 6.3 Overview of consumers' perception of sweetness . . . . . . . . . . . . 6.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Part II

vii 92 94 94 97 97 97 112 121 122 122

Types of sweet tasting compounds

7 Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. M. Cooper, British Sugar plc, UK 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Sugar manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Sugar products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Properties of sugar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Sugar functionality in food products . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Polyols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. E. Embuscado, McCormick & Company, Inc, USA 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Types of polyols, chemical structures and their manufacture 8.3 Physicochemical and functional properties . . . . . . . . . . . . . . . . . . . 8.4 Physiological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Advantages and disadvantages of using polyols . . . . . . . . . . . . . . 8.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7 Mixed sweetener potential of polyols . . . . . . . . . . . . . . . . . . . . . . . . 8.8 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Low-calorie sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. E. Kemp, Kemps Research Solutions Ltd, UK 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135 135 137 140 142 145 149 150 151 151 153 153 154 156 160 162 163 166 168 170 171 171 175 175

viii

Contents 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9

Low-calorie sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health-related developments in low-calorie sweeteners . . . . . . Market-related developments in low-calorie sweeteners . . . . . . Implications for food product design . . . . . . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of further information and advice . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

176 216 222 230 235 239 240 240

10 Reduced-calorie sweeteners and caloric alternatives . . . . . . . . . . . . G-W. von Rymon Lipinski, Consultant, Germany 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Reduced-calorie sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Alternative caloric sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Established caloric alternatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 10.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

252

Part III 11

252 254 259 266 275 276 276

Improving sweet tasting compounds and optimising their use in foods

Analysing and predicting properties of sweet-tasting compounds D. E. Walters, Rosalind Franklin University of Medicine and Science, USA 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 QSAR models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Pharmacophore models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Receptor models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 11.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12 Discovering new natural sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. D. Kinghorn, The Ohio State University, USA and N.-C. Kim, Lovelace Respiratory Research Institute, USA 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Commercially used natural sweeteners . . . . . . . . . . . . . . . . . . . . . . . 12.3 Approaches to natural sweetener discovery . . . . . . . . . . . . . . . . . . 12.4 Improvement of sweet taste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283 283 284 285 287 288 288 288 292 292 296 298 300 301 302 303

Contents 13 Molecular design and the development of new sweeteners . . . . . J. Polanski, University of Silesia, Poland 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 The historical development of sweetness consumption . . . . . . . 13.3 Commercial alternative sweetener discoveries . . . . . . . . . . . . . . . 13.4 Molecular design ± novel approaches novel chances . . . . . . . . . 13.5 Screening and visualising new sweeteners candidates . . . . . . . . 13.6 Molecular design in commercial sweetener development . . . . 13.7 From discovery to commercial products . . . . . . . . . . . . . . . . . . . . . 13.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.9 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 13.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Developing new sweeteners from natural compounds . . . . . . . . . . A. Bassoli, DISMA, University of Milan, Italy 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Importance of developing new sweeteners from natural compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Methods of designing new sweeteners from natural compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.4 Synthesising new sweeteners from natural compounds . . . . . . . 14.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Improving the taste of sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. E. Walters, Rosalind Franklin University of Medicine and Science, USA 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Blending sweeteners to provide synergy . . . . . . . . . . . . . . . . . . . . . 15.3 Blending sweeteners to improve taste profile . . . . . . . . . . . . . . . . 15.4 Blending sweeteners to improve temporal profile . . . . . . . . . . . . 15.5 Using additives to improve taste quality of sweeteners . . . . . . 15.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 15.8 Reference list . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Analysing and predicting synergy in sweetener blends . . . . . . . . . P. Laffort, Centre des Sciences du GouÃt, France 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 Sweet taste measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Response of a mixture vs. responses to its components: the perceptive models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.4 Response of a mixture vs. concentrations of its components: the ÿ (Gamma) family models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5 Other interaction models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix 307 307 308 309 310 313 317 318 320 321 322 327 327 327 329 336 340 341 344 344 344 345 346 346 347 347 347 349 349 350 356 361 366

x

Contents 16.6 16.7 16.8 16.9

17

18

Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix: precise technical details . . . . . . . . . . . . . . . . . . . . . . . . . .

369 371 371 373

Bulk sweet tasting compounds in food product development . . . M. Lindley, Lindley Consulting, UK 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Characteristics of bulk sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Sensory properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4 Physical functionalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 Bulk sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 17.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375

Hydrocolloid±sweetener interactions in food products . . . . . . . . . . D. Cook, The University of Nottingham, UK 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 Hydrocolloid±sweetener interactions . . . . . . . . . . . . . . . . . . . . . . . . . 18.3 Mechanisms of hydrocolloid±sweetener interactions . . . . . . . . . 18.4 Implications for food product development . . . . . . . . . . . . . . . . . . 18.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 18.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19

375 376 377 380 383 385 386 386 388 388 390 394 396 399 400 401

Future directions: using biotechnology to discover new sweeteners, bitter blockers and sweetness potentiators . . . . . . . . . R. McGregor Linguagen Corp., USA 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 Understanding taste at the molecular level enables discovery of novel sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 The use of bitter blockers to improve sweet taste . . . . . . . . . . . . 19.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 19.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

406 410 410 411 412

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

415

404 404

Contributor contact details

(* = main contact)

Editor Professor William J. Spillane Chemistry Department National University of Ireland Galway Ireland Tel: 353 91 492475 Fax: 353 91 525700 Email: [email protected]

PO Box 12, Rehovot 76-100 Israel Tel: 972-8-9489276 Fax: 972-8-9476189 E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] Dr Liquan Huang Monell Chemical Senses Center 3500 Market Str. Philadelphia, PA 19104 USA E-mail: [email protected]

Chapter 1 Professor Michael Naim,* Mrs Merav E. Shaul and Mr Alexander Aliluiko Institute of Biochemistry, Food Science and Nutrition Faculty of Agricultural, Food and Environmental Quality Sciences The Hebrew University of Jerusalem

Professor Andrew I. Spielman New York University College of Dentistry 345E 24th Str. New York, NY 10012 USA E-mail: [email protected]

xii

Contributors

Chapter 2

Chapters 5 and 17

Valerie B. Duffy,* J. E. Hayes and M. E. Dinehart School of Allied Health, University of Connecticut 358 Mansfield Road, Box U-101 Storrs, CT 06269-2101 USA

Dr Michael G. Lindley Lindley Consulting 17 Highway Crowthorne Berkshire RG45 6HE UK

E-mail: [email protected]

Tel: 01344 771015 Email: [email protected]

Chapter 3

Chapter 6

Professor Julie Mennella* Member and Director of Education Outreach Monell Chemical Senses Center 350 Market Street Philadelphia, PA 19104-3308 USA

Sylvie Issanchou and Sophie Nicklaus* INRA FLAVIC (UMR INRA-ENESAD) 17 rue Sully BP 86510 21 065 Dijon Cedex France

Tel: +01215 898 9230 E-mail: [email protected] Yanina Pepino Instituto de InvestigacioÂn MeÂdica Mercedes y MartõÂn Ferreyra CoÂrdoba Argentina

Chapter 4 Dominique Valentin,* C. Chrea and D. H. Nguyen CSEG Campus Universitaire 15 rue Hugues Picardet 21000 Dijon France Tel/Fax: 03 80 68 16 57 E-mail: [email protected]

Email: [email protected]; [email protected]

Chapter 7 Dr Julian M. Cooper Head of Food Science British Sugar plc Oundle Road Peterborough PE2 9QU UK Tel: +44 (0)1733 422478 E-mail: [email protected]

Contributors

Chapter 8 Dr Milda E. Embuscado McCormick & Company, Inc. 204 Wight Avenue Hunt Valley, MD 21031-1501 USA Tel: 410-527-6009 Fax: 410-527-6527 E-mail: [email protected]

Chapter 9 Dr Sarah E. Kemp Kemps Research Solutions Ltd 10 Overend Close Bradwell Village Milton Keynes MK13 9EJ UK Tel: +44 (0)1908 227575 E-mail: [email protected]

Chapter 10 Professor Gert von Rymon Lipinski Schlesienstraûe 62 65824 Schwalbach Germany Tel: +49 6196 9023651 Fax: +49 6196 9023652 E-mail: [email protected]

Chapters 11 and 15 D. Eric Walters Department of Biochemistry and Molecular Biology The Chicago Medical School Rosalind Franklin University of Medicine and Science

xiii

3333 Green Bay Road North Chicago, IL 60064 USA Tel: 847-578-8613 Fax: 847-578-3240 E-mail: [email protected]

Chapter 12 Professor A. Douglas Kinghorn Jack L. Beal Professor and Chair College of Pharmacy The Ohio State University 500 West 12th Avenue Columbus, OH 43210-1291 USA E-mail: [email protected]

Chapter 13 Professor Jaroslaw Polanski University of Silesia Institute of Chemistry ul. Szkolna 9 PL-40-006 Katowice Poland E-mail: [email protected]

Chapter 14 Dr Angela Bassoli DISMA Dipartimento di Scienze Molecolari Agroalimentari UniversitaÁ di Milano Via Celoria 2 I-20133 Milano Italy Tel: +39 02 5031 6815 Fax: +39 02 5031 6801 E-mail: [email protected]

xiv

Contributors

Chapter 16

Chapter 19

Dr Paul Laffort CESG-CNRS 15 Rue Hugues Picardet 21000 Dijon France

Richard McGregor Director, Technology Assessment Linguagen Corp. 2005 Eastpark Boulevard Cranbury, NJ 08512-3515 USA

E-mail: [email protected]

Chapter 18 Dr David Cook Division of Food Sciences University of Nottingham Sutton Bonington Campus Loughborough Leics LE12 5RD UK Tel: 0115 9516245 Fax: 0115 9516162 E-mail: [email protected]

Tel: 609 860 1500 Fax: 609 860 5900 E-mail: [email protected]

Introduction W. J. Spillane, National University of Ireland ± Galway, Ireland

The `sweetness' industry is a multimillion pound growth industry worldwide especially in the developed world. In 2004 over 2,000 reduced sugar and sugar free foods were introduced. In academe and in industrial laboratories there are numerous groups active in taste studies of many types and this is reflected in the volume of diverse scientific publications, patents etc. that have appeared. There have been quite a number of books and conferences on sweetness and taste and some of these meetings take place at regular intervals. It is a few years since a book has appeared that addresses the topic of sweetness and in this present volume, which we hope will be a timely contribution to the field, we have brought together an array of experts in the sweetness area. Each is an authority in his/her own area and in inviting scientists to contribute we have tried to cover areas that have not been touched on in recent times. We hope that this will make this volume of particular interest to a variety of researchers in the taste domain and to a wider circle of scientists who may wish to have an update in various sweetness fields. The book is in three sections: Part I deals with factors affecting sweet taste perception, Part II examines some types of sweet tasting compounds and Part III looks at improving sweet tasting compounds and optimising their use in foods. In Part I Chapter 1 has an important and timely discussion on the stimulation of taste cells by sweet tasting compounds including a useful resume on the stimulation of the TIR1, TIR2 and TIR3 sweet taste receptors. The intriguing subject of genetic differences in sweet taste perception is addressed in Chapter 2. Chapter 3 highlights the liking of sweet taste by children from infancy to adolescence. The surprising role of taste±odour interactions in sweet taste perception is assessed in Chapter 4. Taste±ingredient interactions that are responsible for modulating sweetness are considered in Chapter 5. Finally in this

xvi

Introduction

section of the book the sensory evaluation and factors affecting it of consumers' perceptions of sweet taste are focused on in Chapter 6. Some classes of sweet tasting compounds are described in Part II. Starting in Chapter 7 with the key sweetener sucrose emphasis had been put on its manufacture, processing, products, properties and myriad uses. The three general types of polyols (hydrogenated mono- and di-saccharides and mixtures of hydrogenated saccharides/polysaccharides) are dealt with in Chapter 8. The advantages and disadvantages of using polyols are weighed up and the applications and potential of these important sweeteners are looked at. One of the longer chapters in the book (Chapter 9) covers developments in the substantial area of low calorie sweeteners looking particularly at topics such as regulatory position and safety, health trends, their role in weight control, diabetes, dental caries, market economics and other important aspects of their wide usage. This part of the book concludes with Chapter 10 on reduced calorie sweeteners and caloric alternatives. This chapter includes a lengthy review of D-tagatose, the L-sugars and discussion of alternative caloric sweeteners, such as isomaltulose, trehalose, hydrolysed lactose and galactose, leucrose and starch hydrolysates among other types. In Part III the focus has been on the improvement of sweet tasting compounds and optimising their use. In Chapter 11 attention is given to analysing and predicting sweetness in which structure-taste relationships both qualitative and quantitative are reviewed and pharmacophore and receptor models are discussed. The discovery of new natural sweeteners is the thrust of Chapter 12. In it the authors give information on commercially used natural sweeteners, they detail the approaches to the discovery of such compounds and they show how improvements in sweet taste can be achieved using natural sweeteners. In Chapter 13 the molecular design and development of new sweeteners is discussed. The author looks at commercial alternative sweetener discoveries, molecular design, screening and visualising new sweetener candidates and the movement to commercial products. Chapter 14 outlines the development of new sweeteners from natural compounds. Methods of designing new sweeteners from natural compounds using QSARs and modelling and the synthesis of new sweeteners from natural compounds are addressed. Improving the taste of sweeteners through blending to provide better synergy, better taste profiles and better quality is examined in Chapter 15. In the next chapter synergy is the key topic and the author analyses and predicts synergy in various sweetener blends by examining a number of models available for synergistic analyses. Chapter 17 provides an interesting overview of bulk sweet tasting compounds in food product development. In this chapter many aspects of bulk sweeteners are delved into including flavour, sweetness, texture, appearance and physical properties. Hydrocolloid sweetener interactions are examined in Chapter 18 including discussions of the mechanisms of sweetener-hydrocolloid interactions and criteria for hydrocolloid selection in food product development. The use of biotechnology to discover new sweeteners, bitter blockers and sweetness potentiators is discussed as a future trend in Chapter 19.

Part I Factors affecting sweet taste perception

1 Stimulation of taste cells by sweet taste compounds M. Naim, The Hebrew University of Jerusalem, Israel, Liquan Huang, Monell Chemical Senses Center, USA, A. I. Spielman, College of Dentistry, New York University, USA and M. E. Shaul and A. Aliluiko, The Hebrew University of Jerusalem, Israel

1.1

Introduction

Perception of sweet sensation is known to be associated in humans and many mammals with a unique pleasure. Studies examining facial expressions of neonates following taste stimulation indicate appealing responses to sweet taste and aversion to bitter taste (Steiner, 1973), and newborns prefer sugar solutions to water and their preference increases with sugar concentration (Desor et al., 1973). Furthermore, their preference for different sugars corresponds to adult perception (Maller and Desor, 1974). These studies suggest that the response to sweet taste is innate. The high threshold for the appealing taste of sugar and actual preference (Cagan and Maller, 1974) lead mammals to select high-caloric foods. In contrast, as a counter defense and screening mechanism, a strong rejection of bitter stimuli is due to the fact that traditionally most bitter compounds were toxic and therefore are perceived at much lower concentrations than sweet compounds. These innate behavioral mechanisms have apparently had a significant effect on survival during phylogenetic development. Moreover, it has long been recognized that the preference for sugars may change with physiological state. Human subjects prefer a 5% sucrose solution to 30% sucrose solution, but can be induced to prefer the more concentrated solution once blood glucose level is decreased to 50 mg% following insulin injection (Mayer-Gross and Walker, 1946). Similar results are found in rats (Jacobs, 1958). A strong preference for sweet high-fat foods and fat is associated with opioids (MarksKaufman and Kanarek, 1981; Marks-Kaufman and Lipeles, 1982; Drewnowski

4

Optimising sweet taste in foods

et al., 1992; Drewnowski, 1991) which appear to be released under mild stress. Administration of naloxone, an opioid antagonist, reduces the hedonic preference for sugar (Drewnowski et al., 1995). Other studies have shown that oral stimulation by sweet taste substances may, via the cephalic phase of endocrine secretion, release insulin, which in turn may participate in stimulating eating (Brand et al., 1982). The intake of food items containing refined sugars and high caloric density constituents has increased significantly in the last century (Grand, 1974), and has been linked to metabolic disorders such as diabetes and obesity, termed `diabesity' (Shafrir, 1997), considered to be the main threats to human health in the 21st century (Zimmet et al., 2001). In response to such health hazards, during the last four decades many chemical studies were initiated to explore low-calorie alternative sweeteners with high sweet potency (DuBois et al., 1981; DuBois et al., 1993; Nofre and Tinti, 1987; Nofre et al., 1988; Tinti and Nofre, 1996). The resulting synthetic and some natural non-sugar sweeteners include a large collection of diverse compounds such as sulfamates, flavonoids, oximes, amino acids, peptides, proteins, guanidines and terpenoids. These discoveries were based on structure±function relationship studies which have essentially proposed a single sweet taste receptor for all sweeteners (Shallenberger and Acree, 1967; Kier, 1972; van der Heijden et al., 1985a, 1985b). Tinti and Nofre have prepared extremely potent sweeteners and proposed the multipoint attachment (MPA) model of sweetness (Tinti and Nofre, 1996; Nofre and Tinti, 1996). This model was in line with the notion of a single sweet taste receptor but with multiple binding sites between the high potency sweeteners and the receptor molecule. The nutritional justification of alternative sweeteners to sugars has led to increased consumption of low-calorie soft-drinks, creating a multi-billion dollar economic target for the food industry. However, the sweet taste of sugars, especially that of sucrose, is regarded as pure with optimal sensation in humans, whereas many non-sugar sweeteners possess inferior sweet quality. Psychophysical sensory studies in humans using the multi-dimensional similarity (MDS) analysis (Schiffman et al., 1979) clearly showed that the sweetness of a variety of non-sugar sweeteners may be located at a different site in the MDS sweet similarity map from that of sugar sweeteners. Cross-adaptation studies of sweeteners (Schiffman et al., 1981; Lawless and Stevens, 1983) have suggested for a long time that either different sweet taste receptors or different mechanisms are involved for sweet sensation. A further difference between non-sugar sweeteners and sugars is a phenomenon known as synergism. Sweet taste synergism occurs mainly between pairs of non-sugar sweeteners but not between pairs of sugars (Ayya and Lawless, 1992; DuBois, 2004; Schiffman et al., 1995). Moreover, in most cases, stimulation by non-sugar sweeteners does not lead to the maximal sweet taste intensity level achieved with sugars during psychophysical experiments, even though these sweeteners are more potent when intensity is evaluated on a weight basis (Moskowitz, 1977; DuBois et al., 1991). Sugars and sugar alcohols show linear concentration±response relationships while high-potency sweeteners yield hyperbolic dependence. According to

Stimulation of taste cells by sweet taste compounds

5

Moskowitz (1977), it may be related to the fact that as one increases the concentration of the non-sugar sweeteners, the additional taste qualities that such sweeteners possess become predominant and do not permit a further increase in sweetness intensity as occurs for sugars. In fact, it was suggested that the concentration-intensity slope be used as an additional parameter for evaluation of the sweet taste quality of a sweetener (Moskowitz, 1977). It is apparent that there are multiple reasons that may be related for the above dissimilarity in sweet taste between sugars and non-sugar sweeteners. It is well known that many (though not all) non-sugar sweeteners possess additional taste sensations such as bitter, metallic and licorice. Although the molecular basis for such sensations is not clear, the sweeteners saccharin and acesulfam K were recently found to stimulate both bitter and sweet taste receptors (Kuhn et al., 2004) (see more discussion on sweet taste receptors below). A further distinguishing factor that makes clearly non-sugar sweeteners inferior to sugars is their temporal properties. Time±intensity relationship studies indicated that, compared to sucrose, it takes a longer time for the sensation of a non-sugar sweetener to reach a maximal sweet taste intensity (slow taste onset), and more time (sometimes minutes) for the sweetness to be extinguished (lingering aftertaste) (Birch et al., 1980; Larson-Powers and Pangborn, 1978; Naim et al., 1986; DuBois et al., 1977). This lingering phenomenon is also known to occur for a variety of bitter stimuli. Interestingly, sweeteners which produce the sweet aftertaste may also induce `sweet water aftertaste', a phenomenon where water tastes sweet for several minutes after tasting a sweetener (van der Wel, 1972; Bartoshuk, 1987; Naim et al., 1986). This is also true for stimulation by some sweetener receptor antagonists (D'Angelo and DuBois, 1999). The delay in sweet taste termination following the tasting of nonsugar sweeteners is not unique to humans. It was also shown to occur in old world monkeys during behavioral studies (e.g., baboons, (Rogatka et al., 1985)). The mechanism of lingering taste is unknown. One question is whether such a delay is due to peripheral or central nervous system events. For instance, in olfaction, the memory of odors can persist, e.g., in the neuronal tissue of insects, even as cells go through metamorphosis (Ray, 1999). In the brain, taste sensation which is associated with malaise may form long-term conditioned taste aversion through memory which depends on identified biochemical mechanisms (Berman and Dudai, 2001). However, electrophysiological recordings of taste nerves at the periphery (Hellekant, 1994) have indicated `lingering aftertaste' in response to stimulation by non-sugar sweeteners. Furthermore, time-course measurements have shown that the transient onset and termination of IP3 release in taste cells following stimulation by some bitter tastants known to possess lingering aftertaste is delayed beyond 500 ms (Spielman et al., 2002). These results suggest that the delay in taste-signal termination induced by some non-sugar sweeteners and bitter tastants is at the periphery, at the taste-cell level. To date, the molecular basis for the `slow taste onset' and the `lingering aftertaste' phenomena is not known, even though it has significant implications regarding the acceptance of a variety of food products.

6

Optimising sweet taste in foods

In the current chapter we will discuss the complex signaling which leads to sweet taste sensation events occurring at the level of the taste bud cells. Furthermore, we will analyze current experimental data assumed to provide better understanding of biochemical events related to sweet taste quality.

1.2

Peripheral organization of taste

The peripheral gustatory system is exposed to a variety of physical, chemical and biological insults. Extreme hot, cold, irritating, acidic and non-sterile stimuli may have damaging effects on peripheral taste receptors. Therefore, unlike all other sensory systems that have peripheral neuronal receptors, the peripheral gustatory system has evolved to have receptor cells that are rapidly-renewing, specialized epithelial cells. This adaptation ensures rapid regeneration of taste receptor cells that become damaged. The peripheral organ in the gustatory system is comprised, in decreasing hierarchical order of: taste papilla, taste bud and taste cell. The largest is the taste papilla (Spielman and Brand, 1997). Visible to the naked eye, taste papillae in humans come in at least four different shapes and are located on the tongue, soft and hard palate, pharynx, epiglottis and larynx. The largest of the four types, the circumvallate (CV) papillae, are located on the dorsal surface of the tongue between the anterior 2/3 and posterior 1/3 along a V shaped line. The number of CV papillae varies from species to species. There are between three and 13 papillae in humans. In other animals, their number varies from just one in rodents to 25 in cows. On the postero-lateral border of the tongue are the foliate papillae, each of which is a pocket-shaped invagination lined with taste buds. The invaginations protect the taste buds from direct physical damage. The third type of papillae, the mushroom shaped fungiform papillae, cover the anterior dorsal surface of the tongue. Compared with other gustatory papillae, they are the largest in number with 50±200 in humans. Finally, the extralingual papillae are located on the soft and hard palate, the pharynx, epiglottis and larynx, with the most prominent being the taste stripes (or Geschmackstreifen from the original German term), located on both sides of the palatal midline at the transition of the soft and hard palate. Interestingly, the most abundant and recognizable structures, the filiform papillae are nongustatory. When overgrown and stained by beverages and food dye these threadlike papillae tend to provide a characteristic colour to the tongue. Similarly, excessive shedding of the papillae provides a `white coat' on the tongue. On the other hand, loss of the filiform papillae creates a dry and smooth appearance which is associated with a variety of oral and systemic conditions such as dry mouth, anaemia and Scarlet fever. The filiform papillae act like `VelcroÕ' to help secure food to the tongue so that the bolus can be moved around the oral cavity (Spielman, 1998). Within each taste papilla there are varying numbers of taste buds visible under light and electron microscopes. The number of taste buds varies from one

Stimulation of taste cells by sweet taste compounds

7

papilla to the next. In humans, fungiform papillae contain 1±10, CV papillae contain 100±200, while foliate papillae may have from a few hundred to a few thousand buds. The taste bud is the functional unit of the peripheral taste organ. It is onionshaped and contains 50±100 continuously-maturing taste receptor and supporting cells. Most of the taste cells in a bud are shielded from the oral cavity. Only the apical tip of a few taste cells contains taste receptor proteins that is exposed to the oral cavity through a 3±5 micron wide opening, referred to as the taste pore. Unlike any other sensory system, taste cells have a rapid turnover rate of 10.5 days, which is about twice as fast as the surrounding epithelial cells. The progenitor cells, also known as basal cells, are located in an epithelial layer at the base of the taste bud that corresponds to the germinal layer of the epithelium. As taste receptor cells continuously grow and mature, they migrate from the basal area of the bud toward the taste pore over a period of about 10 days, its turnover time. Exposure of an individual receptor cell to taste stimulants is limited usually to a single meal. Within a few hours of a chemosensory experience, the exposed taste receptor cells are shed into the oral cavity and the dead cells are washed away by saliva. The fungiform- and taste-stripe-containing taste buds are bathed in saliva from the bilateral major salivary glands (parotid, submandibular, sublingual). In contrast, taste buds in the circumvallate and foliate taste papillae are washed primarily by the saliva of the von Ebner's salivary glands located in the body of the tongue with openings into the trenches that surround these papillae. Within the taste bud, several taste receptor cells, certainly not all, will synapse with first order neurons belonging to either the facial (chorda tympani), glossopharyngeal or vagus nerves. In a recent study synapses were observed in a few taste receptor cells (Yang et al., 2000). In another study, the specific receptor cells, called Type II, associated with bitter, sweet and umami taste transduction was devoid of conventional synapse (Clapp et al., 2004). These cells either communicate first with adjacent taste cells in the bud before synapsing, or they have subsurface cysternae of smooth endoplasmic reticulum instead of traditional synapses (Clapp et al., 2004). The sensory pathway for taste involves a chain of neurons in series. First order neurons connect the periphery to the nucleus of the solitary tract in the central nervous system where they synapse second order neurons, cross the midline and connect with the thalamus on the opposite side, while the third order neurons connect the thalamus to the cortex. The sensory pathway for taste involves neural connections with the salivary nuclei to activate a reflex pathway and the perception of taste depends on the signals reaching the sensory cortex.

1.3

The sweet taste receptor(s)

1.3.1 Sweet taste is mediated by G protein coupled receptors Earlier biochemical studies indicated that sweet taste is detected by proteinaceous membrane receptors that are located on the tip of taste receptor

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Optimising sweet taste in foods

cells. Proteolytic (pronase) treatment of the surface of human tongue could temporarily remove sweet perception, indicating that the sweet receptors are the membrane-bound surface proteins (Hiji, 1975). Radiolabeled sugars and sweeteners such as sucrose, fructose, glucose, synthetic sweeteners (cyclamate and saccharin) and sweet-tasting proteins monellin and thaumatin could bind to the purified homogenate fractions from human and bovine papillae. This binding activity was taste papillae-dependent, saturable and reversible, could be competitively inhibited by unlabeled sugars and sweeteners, indicating that there were specific receptors on taste bud cells that could reversibly bind to sweet compounds (Cagan, 1971; Lum and Henkin, 1976; Cagan and Morris, 1979). Additionally, photoaffinity labeling demonstrated that the intensely sweettasting protein thaumatin I could bind to a protein of approximately Mr 50,000 from monkey circumvallate papillae (Shimazaki et al., 1986). This finding was further corroborated in other studies. Colloidal gold-labeled thaumatin was shown to bind to the microvilli in the taste pore of monkey taste buds, as expected, where the sweet receptors are located (Farbman et al., 1987; Menco and Hellekant, 1993). In an attempt to purify the sweet receptor proteins, affinity columns of covalently attached thaumatin and monellin were used to isolate thaumatin/monellin-bound proteins from cow, pig and rat gustatory membranes. These bindings could be displaced by the sweet peptide aspartame or the sweet taste modifier gymnemic acid, indicating that there is a common receptor for some sweet proteins and the peptide aspartame or gymnemic acid (Persaud et al., 1988). However, the isolation of sweet receptor proteins has been hampered largely due to three problems: the scarcity of taste buds, the lack of a tight binding between taste membranes and their presumed receptors (Bruch et al., 1988), and lack of cultured taste cell lines. Further biochemical studies indicated that sweet receptors are G-protein coupled receptors (GPCRs). Activation of these receptors in the membranes derived from taste bud-containing lingual epithelium by sucrose and other sugars significantly enhanced the activity of adenylyl cyclase (AC), one of the effector enzymes in G-protein-mediated signaling cascades. This response was concentration-dependent, and was mediated by guanine-nucleotide-binding proteins (G-proteins) found in the taste cell membranes (Striem et al., 1989; Bruch and Kalinoski, 1987). 1.3.2 Discovery of the sweet taste receptors Over the last three decades, animal behavioral assays demonstrated that inbred mouse strains such as C57BL/6 and DBA/2 displayed a marked difference in detection thresholds for synthetic sweet compounds such as saccharin and other sweeteners (Fuller, 1974). This trait exhibited a simple mendelian inheritance (Lush, 1989; Phillips et al., 1994; Lush et al., 1995). Genetic mapping of recombinant strains identified a Sac locus, named after saccharin, on the distal end of mouse chromosome 4 as the genetic determinant for this trait (Phillips et al., 1994; Lush et al., 1995; Blizard et al., 1999). In addition, the peripheral

Stimulation of taste cells by sweet taste compounds

9

nerve responses to sucrose were affected by the Sac locus as well (Bachmanov et al., 1997), suggesting that this locus encodes a sweet taste receptor or some other key sweet taste transduction element. In 1999, by random sequencing of subtracted rat taste papilla-enriched cDNA libraries, Hoon and colleagues isolated cDNAs that encoded two putative GPCRs: TR1 and TR2 (now referred to as T1R1 and T1R2) (Hoon et al., 1999). These two genes are expressed in a subset of taste bud cells and expression levels of each gene showed distinct topographical distributions across the tongue and palate that corresponds to the taste sensitivity map of the tongue. Based on its topographical expression pattern, T1R1 was suggested to be putative sweet receptor. However, high resolution genetic mapping eliminated T1R1 as a Sac-encoded sweet receptor (Li et al., 2001). With the availability of mouse and human genome sequences and bioinformatics tools, data mining of DNA sequences of the Sac locus on mouse chromosome 4 and a syntenous region on human chromosome 1 identified a novel putative 7-transmembrane domain receptor, T1R3 (Bachmanov et al., 2001; Kitagawa et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001). Subsequent analyses revealed that the allelic variations between sweet-sensitive and -insensitive mouse strains resulted in amino acid substitutions in T1R3 receptor that corresponded to the Sac phenotypes, suggesting that it is the sweet receptor that is encoded by this Sac locus. Introgression by serial backcrossing of a small T1R3 gene-containing chromosomal fragment from a sweet taster mouse strain (C57BL/6ByJ) to a sweet non-taster strain (129P3/J) conferred the latter high sensitivity to sweeteners (Bachmanov et al., 2001). Further confirmation of T1R3 gene as Sac is from a transgenic experiment that demonstrated the rescue of the sweet taste deficit by introduction of the T1R3 gene from a sweet taster strain into a sweet non-taster strain (Nelson et al., 2001). Based on the sequence similarity of the heptahelical domain, T1R3, as well as T1R1 and T1R2, is classified as a member of Class C GPCR family (Pin et al., 2003). Many members of this class including calcium sensing receptor (CaSRs), putative pheromone receptor V2Rs, neurotransmitter receptors mGluRs and GABABRs, as well as T1R1, T1R2 and T1R3 have a large N-terminal extracellular domain. Some of the receptors in this class form homo- or heterodimers. Double labeling in situ hybridization indicated some taste bud cells coexpressed T1R1 and T1R3, or T1R2 and T1R3 while a few cells express T1R3 alone. Thus T1R-expressing cells can be categorized into three types: cells coexpressing T1R1+T1R3; cells co-expressing T1R2+T1R3; cells expressing T1R3 alone (Montmayeur et al., 2001; Nelson et al., 2001). These expression patterns suggested that T1R3, like other members of Class C GPCRs, may form heterodimers with T1R1 and T1R2. Co-expression of T1Rs in a heterologous system in fact confirmed that the dimeric receptor of T1R2 and T1R3 functions as a sweet receptor and human T1R2/T1R3 can be activated by all sweet compounds tested, including sugars: fructose, galactose, glucose, lactose, sucrose and maltose; amino acids: glycine and D-tryptophan; sweet proteins: monellin and thaumatin; synthetic sweeteners: acesulfame K, aspartame,

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cyclamate, dulcin, neotame and saccharin. The order of the sensitivity of the receptors to these substances approximates that from animal behavioral tests. Interestingly, T1R3 can also form a heterodimeric receptor with T1R1 that can be activated by umami stimuli (Nelson et al., 2001, 2002; Li et al., 2002). Therefore, T1R3 appears to be a common monomer of sweet and umami receptors. Phylogenetic analysis showed that T1R3 is most closely similar to T1R1 and T1R2 with ~30% amino acid identity, while T1R1 and T1R2 are ~40% identical to each other (Hoon et al., 1999; Bachmanov et al., 2001; Kitagawa et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001; Nelson et al., 2001). Unlike most other GPCRs, which usually share high similarity across closely related species (e.g. another receptor of the Class C, calcium-sensing receptor has >90% amino acid identity among human, rodent and bovine sequences) (Chattopadhyay et al., 1996), taste receptors including T1R sweet, umami receptors and T2R bitter receptors display only about 70% sequence identity between human and rodents. This suggests that species-specific taste receptors may have different ligand interaction properties, and each may have evolutionarily tuned to their distinct ecological niches. Psychophysical, behavioral and electrophysiological studies showed that humans and old world primates can detect the sweetness of a subset of substances including monellin, brazzein, thaumatin, neotame, aspartame, cyclamate and neohesperidin dihydrochalcone that new world monkeys and rodents cannot. Furthermore, human sweet taste is subject to the suppression by lactisole while rodents are insensitive to this compound (Danilova et al., 1998, 2002; Hellekant et al., 1997; Sclafani and Perez, 1997; Brouwer et al., 1973; Naim et al., 1982; Schiffman et al., 1999; Johnson et al., 1994). In line with these observations, heterologously expressed human T1R2 and T1R3 dimeric receptors can respond to the stimulation of these sweeteners, and the responses are susceptible to lactisole inhibition while rodent receptors cannot be activated by these compounds and the response to other sweeteners are not suppressed by lactisole (Li et al., 2002). Unlike bitter T2R receptors, which have a short extracellular amino-terminus, T1R receptor proteins consist of several functional domains (Fig. 1.1): a large extracellular amino-terminal domain (ATD), followed by a cysteine rich domain (CRD), a heptahelical transmembrane domain (HD) and an intracellular carboxyl-terminal tail (Hoon et al., 1999; Bachmanov et al., 2001; Kitagawa et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001; Nelson et al., 2001; Adler et al., 2000). Based on the crystal structure of another member of Class C receptors, mGluR1, T1Rs are likely to have a clam shell-like `Venus flytrap module' (VFTM) in the ATD domain that is responsible for ligand binding. Mutagenesis and interspecies domain swapping between human and rodent T1R2 and T1R3 receptors have determined particular domains and specific amino acid residues to which the species-specific sensitivity to ligands and inhibitors are attributable. For example, human T1R2 VFTM but not rat T1R2 VFTM can be activated by aspartame and neotame; human T1R3 CRD domain and extracellular loops 2 and 3 of HD domain can interact with brazzein

Stimulation of taste cells by sweet taste compounds

11

Fig. 1.1 Schematic representation of a dimeric sweet receptor structure. Each monomer consists of a Venus flytrap module (VFTM), a cysteine rich domain (CRD), a heptahelical transmembrane domain (HD) and an intracellular carboxyl-terminal tail (C-tail) (Pin et al., 2003). Sweeteners including aspartame and neotame bind to the VFTM module of T1R2 while brazzein and cyclamate bind to the CRD domain and extracellular loops on T1R3, respectively (Jiang et al., 2004; Xu et al., 2004). Sweet inhibitor lactisole binds to the pocket comprised of T1R3 transmembrane helices (Jiang et al., 2005; Winnig et al., 2005; Xu et al., 2004).

and cyclamate, respectively; lactisole docks to the human T1R3 binding pocket comprised of the seven transmembrane helices, while rodent counterparts do not interact with these compounds (Xu et al., 2004; Jiang et al., 2004, 2005; Winnig et al., 2005). These data also demonstrate that multiple ligand binding sites exist in the two monomers of the dimeric sweet receptor T1R2/T1R3, which may explain the earlier observations indicative of multiple sweet receptors (Schiffman et al., 1981). Surprisingly but logically, lactisole and cyclamate that suppress/enhance sweet sensation, respectively, also exert the same effect on umami taste by interacting on the T1R3 moiety of the T1R1/T1R3 umami receptor (Xu et al., 2004). 1.3.3 Discrimination of taste modalities at taste bud cells In situ hybridization and immunohistochemistry showed that T1R3 is expressed in about 30% of taste bud cells from all types of taste papillae and palate while T1R1 in turn, is mostly expressed in fungiform and palate, less in foliate, and rarely in circumvallate (Fig. 1.2). In contrast, T1R2 is expressed mostly in circumvallate, less in palate and foliate, and rarely in fungiform (Montmayeur et al., 2001; Max et al., 2001; Nelson et al., 2001). Each of T1R1 and T1R2 is coexpressed with T1R3 in a taste bud cell. And some taste bud cells may express T1R3 alone. The bitter T2R receptors are expressed in a different subset of taste

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Optimising sweet taste in foods

Fig. 1.2 Expression of taste GPCRs in taste bud cells. Sweet receptor (T1R2/T1R3 or T1R3 alone), umami receptor (T1R1/T1R3) and bitter receptors (T2Rs) are expressed in discrete subsets of taste bud cells (Adler et al., 2000; Hoon et al., 1999; Nelson et al., 2001) . Sweet receptor cells are most abundant in circumvallate papilla, less in foliate and very few in fungiform. In contrast, umami receptor cells are rare in circumvallate, more in foliate and abundant in fungiform (Nelson et al., 2001). Bitter receptor cells, which can express multiple different bitter receptors, are more abundant in circumvallate and foliate than in fungiform (Adler et al., 2000).

bud cells (Adler et al., 2000; Nelson et al., 2001). This non-overlapping cellular expression profile suggests that sweet, umami and bitter taste modalities are discriminated at the taste bud cell level. Knockout of T1R3 receptors markedly reduced the sensitivity to sweet stimuli in behavioral tests but had no effect on bitter, sour and salty tastes (Damak et al., 2003; Zhao et al., 2003). The residual response to sweeteners in T1R3-null mice was abolished by double knockout of both T1R2 and T1R3 receptors. Transgenic introduction of human T1R2 into mice generated animals with humanized sweet taste preferences. These mice

Stimulation of taste cells by sweet taste compounds

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could detect substances that taste sweet to humans, but normally not to rodents. In addition, transgenic expression of a modified opioid receptor in T1R2expressing cells resulted in the attraction of animals to a synthetic opiate that is normally tasteless to wild type mice. And expression of a human receptor T2R16 for a bitter compound phenyl-B-D-glucopyranoside in mouse T1R2expressing sweet cells generated animals that exhibit strong attraction to this bitter compound (Mueller et al., 2005). Theses studies demonstrated that taste modalities are detected and segregated by the taste bud cells, rather than receptors themselves. 1.3.4 Could sweet tastants stimulate non-taste receptors in taste cells? In a recent study (Zubare-Samuelov et al., 2003) we found that some sweet and bitter tastants, e.g. the non-sugar sweeteners, saccharin, D-tryptophan and neohesperidin dihydrochalcone (NHD), and the bitter tastants cyclo(Leu-Trp) and limonin, stimulated pigment aggregation in a Xenopus laevis melanophore cell line as does the native hormone melatonin. Thus, these tastants were able to generate physiological responses independent of taste sensation. Furthermore, as the native melatonin, these tastants stimulated the pigment aggregation by stimulation of the inhibitory pathway of AC as pre-treatment of melanophores with pertussis toxin almost abolished the tastant-induced cAMP reduction. Importantly, the presence of luzindole (melatonin receptor antagonist) almost abolished the inhibition of cAMP formation induced by saccharin, D-tryptophan, and cyclo(Leu-Trp) and the presence of 2-adrenergic receptor antagonist, yohimbine, almost abolished the inhibition of cAMP formation induced by NHD. These data demonstrated that saccharin and D-tryptophan are agonists of the melatonin receptors and NHD is an agonist of the 2-adrenergic receptors under the experimental conditions. It becomes evident that these tastants may stimulate receptors that are not identified taste receptors. Interestingly, both the 2-adrenergic and the melatonin receptors were reported to be present in taste bud cells (Zubare-Samuelov et al., 2003; Herness et al., 2002). If such sweeteners, in addition to their ability to stimulate the T1R2/T1R3 sweet receptors, can also stimulate melatonin, 2-adrenergic receptors, and perhaps other non-taste receptors in taste cells, they may affect the signaling output in taste cells. Should these events occur within the subsecond time course of sweet taste sensation, they may affect taste sensation which may be related to the lack of pure sweetness when tasting non-sugar sweeteners, or even modify time-taste intensity relationships. Although the PLC 2 pathway appears to be essential for the cellular transduction of sweet taste (Zhang et al., 2003), to the best of our knowledge no biochemical data are yet available to show the direct activation of specific signaling pathways in taste cells by either the T1R2/R3 or the T1R3 homodimeric sweet taste receptors. In a recent study (Ozeck et al., 2004), sucrose and non-sugar sweeteners such as aspartame, cyclamate and saccharin stimulated the human T1R2/T1R3 sweet receptor expressed in HEK293 cells to

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Optimising sweet taste in foods

activate the inhibitory G-protein signaling pathways (in particular AC) in a pertussis toxin sensitive manner. These data suggest that either the T1R2/T1R3 GPCRs are coupled to the Gi subunits in taste cells to induce sweet taste transduction or these receptors can be coupled to more than one transduction pathway, further demonstrating a lack of specificity of the transduction output.

1.4

Downstream signaling components

In general, signaling pathways for GPCRs are characterized by the presence of a GTP binding protein (G-protein) which is a coupling component between GPCRs, effector enzymes and ionic channels (Simon et al., 1991; Kristiansen, 2004). There are two main classes of G-proteins. The first are heterotrimeric Gproteins that associate with GPCRs and are involved in signal transduction (Wong, 2003; Kristiansen, 2004). The second are small cytoplasmic G-proteins. The heterotrimeric G-proteins are composed of , and subunits, with molecular masses of about 39±45, 35±39 and 6±8 kDa, respectively. About 28 distinct G-protein  subunits, which are products of 16 different genes and splice variants have been identified. and subunits are tightly associated and can be regarded as one functional unit. Five different and 12 different subunits have been identified (Kristiansen, 2004). Although not all interactions are possible, many combinations occur which increased the diversity and specificity of signaling. The heterotrimeric G-proteins are divided into four families based on the degree of primary sequence similarities of their  subunits: Gs (Gs and Golf), Gi/o subfamily which inhibits AC and regulates ion channels (Gtr, Gtc, Ggust, Gi1-3, Go, and Gz), Gq/11 which activates PLC (Gq, G11, G14, and G15/16), and G12(G12 and G13) that activates Na+/H+ exchanger pathway (Wong, 2003; Kristiansen, 2004; Cabrera-Vera et al., 2003). The G-protein gustducin (Ggust) was first identified in taste cells along with some other Gproteins (McLaughlin et al., 1992). Upon activation by the GPCR, the -subunit binds GTP in exchange for GDP but both - and subunits interact specifically with various effectors in cells including ion channels in cell membranes and enzymes both in the cytosol and in cell membranes. In several signaling systems, specific `regulators of G-protein signaling' (RGS) proteins accelerate GTPase activity of the -subunits. Interestingly, RGS21, a novel RGS was recently found to be co-expressed with sweet T1R2/T1R3 and bitter T2R taste receptors in subpopulations of taste cells (von Buchholtz et al., 2004). Even though considerable research efforts have been made during the last 15 years trying to elucidate possible cellular mechanisms, the nature of the cellular signals responsible for sweet taste transduction is complex and to date is far from being fully clarified. Early studies indicated high AC (Kurihara and Koyama, 1972) and PDE (Kurihara, 1972) activities in gustatory epithelium, and the presence of these enzymes in the microvilli of cells in rabbit taste buds was also shown (Asanuma and Nomura, 1982). Furthermore, cAMP content could increase in intact bovine taste papillae in response to sucrose stimulation

Stimulation of taste cells by sweet taste compounds

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(Cagan, 1974). When the patch-clamp technique became available to record electrophysiological responses in single taste cells, intracellular administration of the cyclic nucleotides cAMP and cGMP into taste cells of frogs and mice (Avenet et al., 1988; Tonosaki and Funakoshi, 1988) decreased potassium conductance, leading to depolarization. In hamsters, fungiform taste buds responded to stimulation by sucrose and some non-sugar sweeteners by inducing action potentials. These responses were mimicked by stimulation with membranepermeant analogs of cAMP and cGMP (Cummings et al., 1993). Additional biochemical studies employing crude membrane preparations of taste tissue and isolated circumvallate (CV) taste bud sheets have suggested the involvement of the AC cascade in sugar taste transduction (Naim et al., 1991; Striem et al., 1989, 1991). However, additional biochemical experiments with isolated CV taste buds indicated that the non-sugar sweeteners SC45647 and saccharin did not induce cellular increase in cAMP, but rather an increase in IP3 and parallel elevation of intracellular Ca2+, as observed by confocal microscopy (Bernhardt et al., 1996). This indicates two different signaling pathways for sweet taste in a single cell responsive to sweet sucrose and SC45647 but not to the bitter quinine. In summary, the above data suggest that sweet taste transduction utilizes more than one pathway (Kinnamon, 1996; Lindemann, 1996, 2001). In the early studies, some of the controversial results may stem from a lack of appropriate methodology for monitoring post-receptor intracellular chemical signals in subsecond time course that parallels taste sensation. For example, our previous biochemical studies (Striem et al., 1989, 1991) (also supported by cellular electrophysiological studies (e.g., ref. (Avenet et al., 1988)) proposed that the AC is involved in sugar taste transduction. However, when we examined the same question on a subsecond time frame (Spielman et al., 1996; Breer et al., 1990), we found, following sucrose stimulation in intact taste bud cells that cGMP rather than cAMP increases in the first 100 ms. (Krizhanovsky et al., 2000). It was then proposed that cAMP does not appear to be involved in the early detection (ms time range) of sweet taste of sugar, but it is elevated later in the sequence of events (seconds time range or longer) and may involve adaptation and signal termination (Krizhanovsky et al., 2000; Kinnamon and Varkevisser, 1998; Varkevisser and Kinnamon, 2000). Controversial data also appeared in relation to how cAMP may be involved in Ca2+ inflow following stimulation by sugar. For example, an inhibitor of protein kinase A (PKA) was found to block the cAMP-induced decrease in potassium conductance in frog taste cells (Avenet et al., 1988) but not in hamster taste cells (Varkevisser and Kinnamon, 2000). Furthermore, a cyclic nucleotide-gated channel, the CNGgust was found in taste cells (Misaka et al., 1998) which may contribute to depolarization and Ca2+ inflow (Lindemann, 2001). It is still unclear how data from the above studies fit into the overall mechanism(s) of sweet taste transduction. The findings that -gustducin knockout mice exhibited reduced (though not completely eliminated) behavioral and electrophysiological responses to bitter-

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Optimising sweet taste in foods

and sweet, but not salty- or sour-taste stimulations (Wong et al., 1996) has led to the following hypothesis (Kinnamon, 1996). Gustducin may mediate bitter-taste transduction by its -subunit via activation of PDE and subsequent reduction of cAMP, while the associated -subunits may activate PLC 2 and induce an increase in IP3 following non-sugar stimulation. This can explain the involvement of both AC and PLC pathways in mediating sweet taste transduction induced by sugar and non-sugar sweeteners, respectively. This finding leads to the assumption that cells which respond to sweet are not the ones which respond to bitter stimulation (Lindemann, 1996, 2001). More recent studies have indicated that TRPM5 (Transient Receptor Potential ion channel), and PLC 2 are present in taste cells (Perez et al., 2002). TRPM5 appears to be a non-specific monovalent cation channel which is important to cell depolarization. It is activated by M concentrations of calcium ions (Hofmann et al., 2003; Liu and Liman, 2003; Prawitt et al., 2003). Most importantly, TRPM5- or PLC 2-knockout mice almost completely lose their sensitivity to sugar, non-sugar sweeteners, bitter and umami taste stimulation, as evidenced by behavioral and electrophysiological experiments (Zhang et al., 2003). TRPM5's function was evident following sweet and bitter stimulation in cells expressing a G-protein that couples a wide range of GPCRs to PLC; every T1R- and T2R-positive cell in the circumvallate (CV), foliate fungiform and palate taste buds co-expressed TRPM5, and the need for the co-existence of TRPM5 and PLC 2 in the taste cells was strongly suggested (Zhang et al., 2003). These new data led to the hypothesis that a common signaling pathway for the generation of these tastes exists, with PLC 2 potentially being one and TRPM5 being the last post-receptor component in the cellular-transduction chain needed to elicit these tastes. These data are in contrast, at least in part, with the aforementioned data suggesting the involvement of various post-receptor transduction pathways for sweet, bitter and umami tastes. The demonstration that the PLC 2±TRPM5 pathway is an essential part for taste signaling has led to the suggestion that other pathways may only modulate taste sensation but are not needed for the basic detection of sweet, bitter and umami tastes. 1.4.1 Could receptor-independent mechanisms be involved in sweet taste transduction? In contrast to sugars, the chemical structures of non-sugar sweeteners (as well as bitter tastants) are very diverse, all of these tastants are amphipathic, i.e., contain both hydrophobic and hydrophilic domains, and therefore they are putative membrane-permeant compounds. A variety of amphipathic neuropeptides, venom peptides and non-peptide substances have been found to be direct activators of G-proteins and are likely to activate transduction pathways due to their ability to permeate the plasma membrane, in addition to their action on specific receptors (Higashijima et al., 1988; Mousli et al., 1990). Furthermore, some of these taste compounds elicit taste and taste nerve responses (Bradley,

Stimulation of taste cells by sweet taste compounds

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1973; Hellekant et al., 1987; Fishberg et al., 1933) following intravenous or intralingual administration, independent of stimulation of putative receptors at the apical surface of the tongue. For example, already in the 1930s, Fishberg and colleagues (Fishberg et al., 1933) used intravenous injection of sodium saccharin to measure the blood circulation time in humans from the time of saccharin injection into the peripheral vein until subjects indicated that they could taste the compound. For the amphipathic tastants to interact directly with membrane transduction components located downstream to the GPCRs, they need to be able to permeate deeply into the membrane and/or translocate to the cytosolic side of the plasma membrane. Some amphipathic tastants were found to be able to permeate liposomes, isolated taste bud cells (Peri et al., 2000), and taste bud cells in vivo (Zubare-Samuelov et al., 2005) (see discussion below). In line with these results, some amphipathic non-sugar sweeteners and the bitter tastant quinine are direct activators of G-proteins in vitro (Spielman et al., 1992; Naim et al., 1994). However, the evidence that such a phenomenon exists in vivo remains to be proven.

1.5 Putative cellular mechanism(s) for the signal termination of sweet taste The efficiency of receptors signaling may be regulated by the receptor number and by their presence on the cell surface (Gainetdinov et al., 2004). It is known that often GPCR signaling desensitizes rapidly as a consequence of receptor phosphorylation. This phosphorylation is mediated by two families of protein kinases (Bohm et al., 1997; Hamm and Gilchrist, 1996; Pitcher et al., 1998). One family of kinases is second-messenger-dependent (heterologous desensitization). For example, cAMP-dependent protein kinase, PKA, phosphorylates the -adrenergic receptors. Similar second messenger-dependent regulation of receptor desensitization may occur via protein kinase C (PKC), mitogenactivated protein (MAP) and additional kinases. The same kinases can also phosphorylate effectors such as AC and PLC (Gainetdinov et al., 2004; Hamm and Gilchrist, 1996). The second family of kinases that desensitize GPCRs (homologous desensitization) are the GPCR kinases (GRKs). In contrast to the second messengers-dependent kinases, GRKs discriminate between the inactive and agonist-activated states of the receptor (catalytically activated by stimulated receptors) (Gainetdinov et al., 2004; Pitcher et al., 1998), and specifically phosphorylate the agonist-activated form of GPCRs. Thus, when ligands are involved, the phosphorylation of GPCRs by GRKs appears essential: deletion of GRK phosphorylation sites in the receptor results in stronger signaling responses to agonist stimulation in transfection studies (Mueller et al., 1997). GRKs promote the binding of the phosphorylated receptors to arrestin proteins which further uncouple the receptors from G-proteins and prevent further stimulation of G-proteins and downstream signaling pathways (Pitcher et al., 1998; Gainetdinov et al., 2004). Subsequently, desensitization of the receptors leads to

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Optimising sweet taste in foods

their removal from the cell surface (internalization). They may then be degraded in lysosomes (downregulation) or recycling to the cell surface (resensitization). There are seven known types of GRK subtypes which are classified based on their sequence and function. GRK1 and GRK7 are primarily expressed in the visual system, GRK4 is primarily expressed in testes. Therefore, it is believed that GRK2, GRK3, GRK5 and GRK6 subtypes account for the regulation of most of the GPCRs found throughout the body (Benovic et al., 1987; Gainetdinov et al., 2004). What GRK subtypes are present in the gustatory system? GRK5 and potentially GRK2 and GRK3 have been previously identified in bovine CV papilla (Premont et al., 1994), though there did not seem to be any evidence of their presence in taste cells. Recently, two studies were published on the presence of GRKs in taste bud cells (Masuho et al., 2005; Naim et al., 2004; Zubare-Samuelov et al., 2005). The presence of the bitter T2Rs (Adler et al., 2000) and the sweet (e.g., T1R3 and T1R2) (Max et al., 2001, Nelson et al., 2001) taste receptors in subpopulations of cells present in rodent CV and other taste papillae has been demonstrated. Evidently, for the GRKs to function in the signal termination of taste receptors they must be present in the same cell subpopulations that contain the above receptors. Our recent study (Naim et al., 2004; Zubare-Samuelov et al., 2005) using RT-PCR suggested the presence of T2R4 and T1R3 along with

Fig. 1.3 RT-PCR analysis of GRK1, GRK2, GRK3, GRK5, GRK6, T2R4 and T1R3 mRNA in circumvallate (CV) taste bud sheets and non-sensory epithelium (EP). cDNA was synthesized from CV and EP RNA and then amplified by PCR using specific primers for GRK1, GRK2, GRK3, GRK5, GRK6, T2R4 or T1R3; GAPDH was used as an internal reference gene. CON designates parallel PCR, omitting the RT step and using GRK2 primers. From Zubare-Samuelov et al. (2005). Used with permission by The American Physiological Society.

Stimulation of taste cells by sweet taste compounds

19

GRK2, GRK3, GRK5 and GRK6 in isolated taste bud cell sheets of rat CV papillae (Fig. 1.3). The same GRKs were found to be expressed in non-sensory epithelium but as expected, no PCR products for the T2R4 and T1R3 receptors were observed in non-sensory epithelial cells. Furthermore, antibodies against GRK2, GRK5 and GRK6 (with weak staining of GRK3) yielded clear, positive immunostaining in the 10 M frozen sections of the CV papilla. However, only GRK5 was clearly stained in the CV taste bud cells; GRK2 and GRK6 were notably stained in the surrounding epithelium but their presence in the taste cells remains questionable. These immunostaining experiments cannot provide evidence for T2R4 and T1R3 receptor expression in the same subpopulation of taste cells that contain the GRKs. However, since GRK5 appeared to be expressed in almost all taste bud cells, it is likely that it co-expresses in cells expressing T2R4 and T1R3. The study in the mouse (Masuho et al., 2005), using immunohistochemistry of the CV papilla, suggested the differential distribution of GRK2, GRK3 and GRK5 in the CV papilla while only GRK2 was present in taste bud cells. It remains to be determined whether the difference between the results in these two studies are related to species differences (rats vs. mice) or the different methodology for GRKs identification led to different observations. Nevertheless, these results call for further investigation to verify that GRK5 and possible other GRKs are indeed expressed in the same taste cells as the T1R2/T1R3 receptors, and to investigate whether they functionally coupled to these receptors. 1.5.1 Amphipathic tastants permeate taste cells in vivo and inhibit receptor-related kinases in vitro A few hypotheses may be proposed to explain the delay in taste-signal termination produced by many non-sugar sweeteners and bitter tastants. According to some studies (see Introduction), this phenomenon appears to be related to events occurring in taste cells and not in the central nervous system (Hellekant, 1994; Spielman et al., 2002). Interestingly, although sugar and nonsugar sweeteners appear to stimulate similar or identical sweet GPCRs, the `lingering' taste sensations are uniquely produced by non-sugar sweeteners (as well by bitter tastants). Because such amphipathic tastants, unlike sucrose, rapidly permeate isolated taste bud cells in situ (Peri et al., 2000), a recent study (Zubare-Samuelov et al., 2005) was initiated to investigate whether they may, after their permeation through the taste cell plasma membrane, interact with downstream transduction components which affect GPCR signal termination (e.g., GRKs, PKA), in addition to their extracellular interaction with GPCRs. For example, inhibition of receptor phosphorylation by GRKs can delay GPCRsignal termination, as shown in vision (Sitaramayya and Liebman, 1983; Gan et al., 2000, 2004) and other systems (Stadel et al., 1983; Pao and Benovic, 2005; Teli et al., 2005). The first step was undertaken to further investigate whether amphipathic tastants can indeed permeate taste cells via the apical oral route under

20

Optimising sweet taste in foods

physiological conditions and not only in isolated taste bud preparations (Peri et al., 2000). The auto-fluorescence that some sweet and bitter amphipathic tastants possess was used as before to monitor tastant permeation into taste cells using two different procedures (Zubare-Samuelov et al., 2005). First, in-situ confocal imaging of intact CV papilla surgically removed without collagenase treatment indicated evident dynamics of tastants permeation (the sweeteners saccharin and D-tryptophan and the bitter tastants caffeine, cyclo(Leu-Trp), naringin and quinine) into the CV cells located around the circular inner trench where most of the CV taste buds are located. The elimination of collagenase treatment in this procedure prevented the exposure of the basolateral side of taste bud cells to tastants and thus allowed permeation through the apical side of the membrane only, the route by which tastants interact with sensory taste cells under physiological conditions. In these experiments, the addition of the membrane-impermeant quencher, KI, had no effect on tastants fluorescence, indicating the presence of such tastants inside the cytosol or on the cytosolic side of the membrane rather than being adsorbed on the extracellular surface of the cells. In the second procedure, the oral cavity of anesthetized rats was stimulated on-and-off with tastant solutions for 90 s, followed by immediate animal sacrifice and an HPLC determination of the tastants' intracellular concentrations (Zubare-Samuelov et al., 2005): millimolar levels of these tastants were found inside the taste cells (Table 1.1). Together, the results provided evidence that these and perhaps additional (though not necessarily all) amphipathic tastants rapidly permeate taste cells under physiological conditions, either through the taste bud pore or through the tight junction, and that such permeation also occurs in non-sensory lingual epithelial cells. Subsequently, in the same study, the effect of tastants on GRK2and GRK5-phosphorylated rhodopsin, a well-studied model for GPCR phosphorylation in vitro (Pitcher et al., 1998), was investigated. The amphipathic non-sugar sweeteners cyclamate, saccharin, NHD and D-tryptophan, and the bitter tastants caffeine, quinine, limonin and naringin, with diverse chemical structures, were found to inhibit GRK2 and GRK5 phosphorylation of rhodopsin Table 1.1 Estimated permeation of amphipathic tastants into CV taste bud cells via the apical side Tastant

Extracellular Efflux (%) during conc. (mM) during 25 min 90 s of oral stimulation collagenase trt.

D-Tryptophan Quinine Cyclo(Leu-Trp) Caffeine

30 2 2 10

86* 75* 64* 70*

   

9.5 5.6 3.0 2.5

Intracellular conc. (mM) after 90 s of oral stimulation 6.4** 5.5** 0.96** 13.7*

   

1.0 0.8 0.1 3.6

Data are the means  SEM of four to nine replicates, each derived from one or two rats. * indicates significant values (P < 0.03) and ** (P < 0.001). From Zubare-Samuelov et al. (2005). Used with permission of The American Physiological Society.

Stimulation of taste cells by sweet taste compounds

21

Fig. 1.4 Tastants inhibit GRK2- (a) and (b) and GRK5- (c) and (d) induced phosphorylation of rhodopsin in a concentration-dependent manner. GRK2 and GRK5 were incubated with rhodopsin and one of the following compounds: NHD, saccharin, caffeine or naringin at the stated concentrations. Note that for saccharin phosphorylation, each concentration shows two bands, one for NaCl (control for sodium) and one for sodium saccharin. CON phosphorylation level when tastants were not present. Results are the means SEM of three independent experiments. From Zubare-Samuelov et al. (2005). Used with permission of The American Physiological Society.

(a GPCR) and PKA phosphorylation of casein in vitro. Their effects depended on the kinase being tested. A concentration-dependence, tested for NHD, saccharin, caffeine and naringin, was evident (Fig. 1.4) and depending on solubility, 90 to 100% inhibition could be shown. Although inhibition could already be observed at relatively low concentrations, e.g., 2.5 mM caffeine, 600 M NHD, 125 M naringin and 100 M limonin, the tastant concentrations needed for kinase inhibition under the experimental conditions were at the millimolar level, higher than the micromolar levels usually used with other kinase inhibitors in vitro and in vivo medically (Sasaki et al., 2002) or experimentally in non-taste systems (Vera et al., 2001). Moreover, these tastants do not appear to be very specific since they inhibited PKA as well as the GRKs, and may very well inhibit additional kinases. Nevertheless, the range of tastant concentrations applied in this study (Zubare-Samuelov et al., 2005) matches that used in taste stimulation during sensory and biochemical studies (Hellekant, 1994; Nelson et al., 2001; Rosenzweig et al., 1999; Schiffman et al., 1979; Wong et al., 1996; Zhang et al., 2003) and the tastant levels were found inside

22

Optimising sweet taste in foods

taste bud cells within seconds after their extracellular application (Table 1.1). The same tastants have also been recently demonstrated to permeate other epithelial cells in situ, e.g., Xenopus laevis melanophores (Zubare-Samuelov et al., 2003). Therefore, although these relatively high concentrations of amphipathic tastants suggest low potency, their physiological significance as kinase inhibitors in taste cells may result from their access to the cytosolic side of the taste cells, and thus to GRKs, which are present in taste tissue (Fig. 1.3), and to PKA or PKC which have been indirectly shown to be active in taste cells (Varkevisser and Kinnamon, 2000). Naringin (as well as some other flavonoids) has been found to inhibit phosphorylation, e.g., naringin inhibits AMPKactivating kinase (Larsen et al., 2002). Caffeine and theophylline have also been recently found to be phosphoinositide 3-kinase inhibitors (Foukas et al., 2002). However, nothing has been reported in relation to GRKs or PKA. The above results have led to a new hypothesis which may partly explain the lingering aftertaste produced by non-sugar sweetener and bitter tastants (Fig. 1.5). Amphipathic tastants stimulate sweet taste GPCRs located on the

Fig. 1.5 Hypothetical pathways for desensitization of sweet taste receptors (R). Nonsugar sweeteners (T) stimulate R from the extracellular side to activate G-proteins (, ,

). -Subunits, in turn, activate PLC 2. Receptor-mediated signaling may be desensitized by G-protein-coupled receptor kinases (GRKs) which phosphorylate R and promote the binding of arrestin proteins to the receptor, uncoupling the receptor from Gproteins. Alternatively, the receptor may be phosphorylated and desensitized by second messenger-dependent kinases, e.g. protein kinase A (PKA) or protein kinase C (PKC). Intracellular inhibition of these receptor phosphorylations, e.g. directly by the membranepermeant T, would expect to inhibit desensitization and therefore delay taste signaling termination. Modified from Pitcher et al. (1998) and based on Zubare-Samuelov et al. (2005).

Stimulation of taste cells by sweet taste compounds

23

extracellular surface of taste cells, and concomitantly also permeate to the cytosolic side of the cell membrane or to the cytosol under physiological conditions. Thus, such tastants have access to interact directly with GRKs or with other receptor-related kinases (e.g., PKA, PKC). Inhibition of GRK- and/or PKA/PKC-induced phosphorylation of GPCRs should lead to a delay in signal termination, and therefore may extend the taste response (e.g., lingering). In conclusion, results discussed in this chapter show the complexity of the cellular events occurring in taste cells upon stimulation by sweeteners. It is likely that such diverse cellular responses following the stimulation by different sweeteners, can explain the tremendous differences in sweet sensation of these sweeteners as implied to sweet taste quality.

1.6

Future trends

To further explore ways to optimize sweet taste quality, more studies at the cellular and molecular level are required. At the level of the sweet receptor(s), a better understanding of the interaction(s) with different sweet ligands is to be achieved. Each of these interactions may have a different affinity characteristic and, in view of the data on other GPCRs indicating their ability to activate more than a single transduction pathway, different sweet tastants may stimulate different signaling pathways which are related to different sweet taste qualities. More research is needed to investigate if some sweeteners can stimulate nontaste receptors that are present in taste cells. One may hypothesize that such effects, if occurring within the time course of taste sensation, may also be related to inferior sweet taste quality. Finally, investigations of peripheral taste signal termination mechanisms, and the possible interference with such mechanisms are imperative since they may explain the undesirable temporal properties produced by a variety of non-sugar sweeteners. To validate such mechanisms, it is necessary to further investigate the newly discovered taste GPCRs to verify their possible interactions with GRKs and other kinases, and to verify the inhibitory effect of taste stimulants in vivo. This hypothesis of signal termination is bold precisely because it involves a dual mechanism: first the ligand interacts extracellularly with GPCRs to initiate the transduction chain, second, the same ligand may interact intracellularly with downstream shut-off components to affect signal termination.

1.7

Acknowledgements

This research was supported by Grant 2003015 from The US-Israel Binational Science Foundation (BSF) and by Grant IS-3366 from The US-Israel Binational Agricultural Research and Development Fund (BARD).

24

1.8

Optimising sweet taste in foods

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2 Genetic differences in sweet taste perception V. B. Duffy, J. E. Hayes, M. E. Dinehart, University of Connecticut, USA

2.1

Introduction

Sweet taste serves as a sensory cue for energy as well as a source of pleasure. The ability to respond to and prefer sweet taste is observed in newborn infants (Mennella and Beauchamp, 1998; Steiner et al., 2001) and probably present before birth (El-Haddad et al., 2002). However, neither the stimulus±intensity relationship nor the intensity±hedonic relationship are uniform across individuals. The impact of these sensory relationships on human preference for and intake of sweet foods is of interest to supporting food enjoyment while promoting health and preventing obesity and chronic disease. High intakes of sugars have historically created concern for human nutrition, particularly those sugars added to foods in relationship with the quality of the diet (e.g., vitamins and minerals provided) and risk of overweight or obesity (Murphy and Johnson, 2003; WHO/FAO, 2003). Nutritionists make a distinction between those sugars naturally occurring in milk and fruits and those added to foods or beverages during processing or preparation. Added sugars are typically consumed in soft drinks, artificial fruit beverages, candy, cookies, cakes, and pastries (DHHS/USDA, 2005). Organizations such as the Institute of Medicine (IOM), the United States Department of Agriculture (USDA), the Department of Health and Human Services (DHHS) and the World Health Organization (WHO) have issued recommendations on intakes of added sugar. The Dietary Reference Intakes, established by the IOM from a critical review of the literature, recommend that intakes of added sugars not exceed 25 percent of total energy, beyond which, dietary quality suffers (IOM, 2002). Although no specific intake value is set, the 2005 Dietary

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Guidelines for Americans recommends that foods and beverages be selected with little added sugars or caloric sweeteners (DHHS/USDA, 2005). Added sugars, fats, and alcohol are combined and referred to as discretionary calories (i.e., energy). Individuals are advised to consume discretionary energy only after including the recommended servings of nutrient-dense foods from each of the food groups (DHHS/USDA, 2005). Based on the consensus of an expert panel, the WHO recommends intake not exceed 10 percent of total energy (WHO/ FAO, 2003). Most empirical evidence does not support that a sweet tooth causes obesity (Rodin, 1975; Thompson et al., 1977; Grinker, 1978; Drewnowski et al., 1985; Macdiarmid et al., 1998) although some studies disagree (Cabanac and Duclaux, 1970; Conner et al., 1988). Whether this myth persists because of an attribution error (Drewnowski et al., 1985, 1992) or vilification (Fischler, 1987; Rozin, 1987) is unclear. Nonetheless, high consumers of added sugar may overconsume total energy or, conversely, compensate for surplus energy by intakes of nutrient-dense foods (Kant, 2000). The public health recommendations about added sugars must be balanced with the reality that sweetness is an important component in the pleasure of eating. The American Dietetic Association (ADA), an organization of food and nutrition professionals, believes that all foods can be part of a healthy diet when eaten in the appropriate portion size and when combined with regular physical activity (Freeland-Graves and Nitzke, 2002). The ADA strives to avoid `good' and `bad' labels for food choices and instead looks at all foods in the context of the total diet (Freeland-Graves and Nitzke, 2002). Nutrition and health professionals can assist consumers in incorporating sweet tasting foods into diets that are enjoyable, healthful and meet individual health and nutrition needs (Duffy and Sigmand-Grant, 2004). Adding sweetness to foods with high nutrient quality may increase the palatability, the chance that they are consumed. To alter the traditional aphorism, `a spoonful of sugar helps the fruits, vegetables and fiber go down.' Although myriad psychosocial, physiological and cognitive factors influence sweet preference and intake in adults, this chapter will address oral sensory factors that affect preference for and intake of sweet foods and beverages. First is a review of the literature on human variation in sweet intensity and sweet preference, and how variability in these measures associates with differences in sweet intake. Following this is a review of genetic variation in taste, described by phenotypic markers, anatomical markers, and emerging gene markers, and how these markers explain some of the variability in sweet sensations, preference and intake. 2.1.1 Individual differences in sweet intensity Although genetic diversity in sweet response is well documented in animal models (Bachmanov et al., 2001) and reviewed elsewhere (Beauchamp et al., 2002), less is known about genetic diversity in humans. Under a basic taste

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paradigm (see Delwiche, 1996), sweetness is typically considered a unitary percept (Halpern, 2002), that is, a singular perceptual experience wherein all sweet sensations are qualitatively similar. Although the idea of a common structural motif (i.e., universal glycophore) (Shallenberger and Acree, 1967; Eggers et al., 2000) is parsimoniously elegant, a single binding site cannot entirely explain sweet taste (DuBois, 2004). Moreover, experiencing sweetness as a singular percept does not preclude multiple transduction mechanisms. Individual sweet receptors may contain distinct binding pockets that bind diverse classes of ligands (Xu et al., 2004) and individuals taste receptor cells (TRCs) may express diverse receptors, as occurs in some bitter TRCs (Glendinning and Hills, 1997; Adler et al., 2000). Broad tuning could also result if signals from narrowly tuned TRCs converge at some point prior to reaching the anatomical structures responsible for perception. While issues of TRC tuning (Amrein and Bray, 2003) and the implications for sensory coding (Erickson, 2000) are beyond the scope of this chapter, evidence supportive of multiple sweet receptor mechanisms in humans and variation between individuals and across sweeteners is discussed below. Evidence for multiple sweet mechanisms Three distinct lines of psychophysical evidence support the idea of multiple sweet mechanisms in humans. One measure of mechanism independence is the failure to cross-adapt; in a cross-adaptation paradigm, pretreatment with one tastant reduces the response to the test stimuli if the two tastants share a similar mechanism (McBurney et al., 1972; Keast and Breslin, 2002). McBurney (1972) found that sucrose cross-adapted all 13 sweet tastants tested whereas saccharin did not. Subsequently, Lawless and Stevens (1983) tested sucrose and three high intensity sweeteners for self- and cross-adaptation. All sweeteners self-adapted and sucrose cross-adapted aspartame, saccharin, and dihydrochalcone. Conversely, when one of the high intensity sweeteners was the adapting stimulus, cross-adaptation was incomplete. These findings were later extended by the demonstration that the ability of sugar sweeteners (sucrose, fructose) and high-intensity sweeteners (acesulfame-K, dulcin) to cross adapt is related to the number of shared binding motifs (Froloff et al., 1998). The second piece of evidence for multiple mechanisms is the existence of synergy; if two sweet compounds act via the same mechanism, mixtures of the two should behave in an additive manner. Instead, mixtures of acesulfame-K and aspartame exhibit synergy (Ayya and Lawless, 1992; Lawless, 1998; Keast et al., 2004), wherein the mixture elicits greater sweetness than that predicted via addition. Finally, in other studies, sweet blockers ± like sodium-PMP (Schiffman et al., 1999) and zinc sulfate (Keast et al., 2004) ± inhibit some but not all sweeteners. Collectively, these findings strongly support the hypothesis of multiple sweet receptors in humans. Variation across individuals In contrast to ample behavioral variation in animals (Beauchamp et al., 2002), reports of phenotypic variation in humans are scattered at best. In an early report

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by Richter and Campbell (1939), 13 of 45 young adults exhibited elevated sucrose recognition thresholds (>17.5 mM), well in excess of the population mode (5.8 mM) and the modern accepted value (3 mM). When matching sucrose solutions to the intensity of a 29 mM sodium chloride reference, Faurion and colleagues (1993) found the subjects (n ˆ 55) required solutions that ranged in concentration from 30 to 300 mM, an entire order of magnitude. From our laboratory, 87 adults (mean age = 26  4 yrs, 42 females, 45 males) reveal variability in intensity ratings of a single sucrose (0.32 M) and aspartame (1 mM) concentration. Both sweeteners produce a normal distribution with ratings ranging from near weak to near very strong (Fig. 2.1) on the general Labeled Magnitude Scale (gLMS) (Bartoshuk et al., 2004b). Ratings normalized

Fig. 2.1 Histograms of intensity ratings of sucrose and aspartame on the general Labeled Magnitude Scale (Bartoshuk et al., 2004b), where 6 = weak, 17 = moderate, 35 = strong, 53 = v. strong.

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to the intensity of tones have similar shaped distribution and degree of variability. The variability of intensity of these sweeteners does not appear to be due to idiosyncratic intensity ratings. Comparing the variance for each sweetener to a tone of equal mean intensity shows significantly greater variance for sucrose (F(86) ˆ 1.85, p < 0:01) and aspartame (F(81) ˆ 2.40, p < 0:01). Variation across sweeteners Even within individuals, not all sweeteners elicit equivalent responses. Among simple carbohydrates, glucose exhibits a markedly different recognition function from sucrose and fructose (Kennedy et al., 1997). Eylam and Kennedy (1998) subsequently identified some individuals (16 of 92 screened) who were hypogeusic to glucose but not fructose (n ˆ 12) or vice versa (n ˆ 4). In another report (Faurion, 1993), some subjects exhibited diverse responses to fructose and sucrose sweetness. Likewise, the relative sweetness of fructose to sucrose, expressed as a ratio, shows a large amount of inter-subject variability (Fontvieille et al., 1989). High-intensity sweeteners evoke sweet sensations at low concentrations and are diverse in chemical structure. Variability in the sweetness from high-intensity sweeteners has been attributed in part to the presence of bitter or metallic side tastes and to duration of sweetness (lingering). From our laboratory, we find a correlation between intensity ratings of sucrose and aspartame, a high-intensity sweetener that is purported to limit less desirable sensory characteristics (Fig. 2.2). Nonetheless, high residuals (circled) may represent distinct responses to these sweeteners, rather than measurement error.

Fig. 2.2 Joint distribution of intensity ratings from sucrose and aspartame on the general Labeled Magnitude Scale (Bartoshuk et al., 2004b). Circled are individuals who exceed the residual seen across the rest of the sample.

Genetic differences in sweet taste perception

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2.1.2 Individual differences in sweet hedonics Numerous researchers have noted great diversity in sweet liking and acceptance (Cameron, 1947; Pangborn, 1970; Thompson et al., 1977; Witherly et al., 1980; Meiselman, 1987; Booth et al., 1987). Histograms from our laboratory demonstrate this diversity in the same sample of adults shown in previous figures (Fig. 2.3). With increasing intensity, liking functions usually take the form of an inverted U-shape (Moskowitz, 1971, 2002). Merely averaging across a sample may obfuscate individual differences in liking (Lundgren et al., 1978; Moskowitz and Moskowitz, 2000). Thompson and colleagues (Thompson et al., 1977) identified Type I individuals, who showed an inverted U liking function with increasing sucrose concentration, in contrast to Type II individuals, who showed a monotonic increase with concentration. Pangborn and her students recapitulated these hedonic functions and noted two additional functions; individuals who showed a monotonic decrease in liking and those who showed a flat relationship between concentration and liking (Lundgren et al., 1978; Witherly et al., 1980), patterns which were later described (Drewnowski, 1987) as Type III and Type IV responses, respectively.

Fig. 2.3 Histograms of liking/disliking ratings of sucrose and aspartame on the general Labeled Magnitude Scale (Bartoshuk et al., 2004b), where +/-6 = weakly, 17 = moderately, 35 = strongly, 53 = v. strongly.

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Fig. 2.4 Individuals categorized as dislikers and likers based on hedonic ratings on the general Labeled Magnitude Scale (Bartoshuk et al., 2004b) to increasing concentration of sucrose.

Alternatively, individuals have been classified as sweet likers or dislikers based on a positive or negative slope for that individual's hedonic-intensity function (Looy and Weingarten, 1992); a classification scheme which would collapse Type I (inverted U) and Type III (monotonic decrease) into dislikers, with Type II representing likers. Figure 2.4 shows adults classified by this method: 21 individuals could be classified as dislikers (type I and type III responders), and 42 as likers.

2.2

PTC/PROP tasting

The ability to taste the bitterness of the thiourea compounds phenylthiocarbamide (PTC) and PROP is a well documented phenotypic polymorphism (Richter and Clisby, 1941; Hall et al., 1975; Lawless, 1980; Bartoshuk et al., 1994). The existence of this taste blindness was discovered serendipitously in 1931 when, during the synthesis of PTC, Fox (1931) accidentally released some into the air. A colleague commented on the intense bitter taste of the dust while Fox tasted nothing. The distribution of thresholds to PTC or PROP is bimodal; nontasters exhibit elevated thresholds (low sensitivity) while tasters show lower thresholds (higher sensitivity) (see Fox, 1931; Harris and Kalmus, 1949). Early family studies suggested that PTC nontasters carried two recessive alleles and tasters carried either one or both dominant alleles (Snyder, 1931; Blakeslee, 1932). Following the lead of Fischer and Griffen (1961), PROP eventually replaced PTC as the marker of choice because of concerns with the safety of PTC and because the sulfurous odor of PTC could provide a nontaste cue in a detection task (Lawless, 1980).

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2.2.1 Supertasting and PROP Using scaling, Bartoshuk and colleagues (1994) found that tasters could be further separated into medium tasters, for whom concentrated PROP solutions are mildly bitter, and supertasters, for whom it is intensely bitter. Supertasters cannot be identified via thresholds as the distributions between those who are sensitive and extremely sensitive to PROP overlap (Reed et al., 1995). The underlying reason behind supertasting is unclear, although a `general taste function' (Lawless, 1979a), differences in fungiform papillae density (Bartoshuk et al., 1994; Delwiche et al., 2001) or some sort of central gain (Green and George, 2004; Green and Hayes, 2004) remain as possible explanations. The fungiform papillae hypothesis is supported by observations that supertasters have, on average, the greatest number of fungiform papillae and taste buds as assessed with videomicroscopy (Bartoshuk et al., 1994) and minimal magnification (Tepper and Nurse, 1997; Delwiche et al., 2001). Early work on the genetics of PTC tasting suggested a two-locus model where one locus controls PTC tasting and the other, a more general taste ability (Olson et al., 1989). The genetic control of fungiform papillae density is unknown at present. More recently, the response to PTC (Kim et al., 2003) and PROP (Duffy et al., 2004b; Bufe et al., 2005) associates with the T2R38 receptor gene, which is located on chromosome 7q36. Three SNPs (single nucleotide polymorphisms) result in five distinct haplotypes for this gene, two of which are common (Kim et al., 2003). The two common molecular haplotypes (PAV and AVI) are named for their respective three amino acid substitutions: Pro49Ala, Ala262Val, and Val296Ile. From non-human primates (Kim et al., 2003; Wooding et al., 2004), the ancestral haplotype is PAV, which associates with tasting, while the other common form is AVI, which associates with nontasting. The polymorphism of the T2R38 gene across the globe could result from balancing selection (Wooding et al., 2004) but also purifying selection and random genetic drift (Kidd et al., 2004). Although the T2R38 gene explains a large proportion of the variability in threshold response to PTC (Wooding et al., 2004; Prodi et al., 2004) and PROP (Duffy et al., 2004c; Mennella et al., 2005), this gene cannot explain supertasting (Bartoshuk et al., 2004a; Duffy et al., 2004c; Hayes et al., 2005).

2.3 Sweetness: relation to PROP, fungiform papillae density, T2R38 Bartoshuk (1979) noted that magnitude estimates of the sweetness of both saccharin and sucrose were lower in subjects who exhibited elevated PROP thresholds (i.e., nontasters). Subsequently, a magnitude matching protocol confirmed that PROP nontasters tasted sucrose, saccharin, and dihydrochalcone as less sweet than did tasters (Gent and Bartoshuk, 1983). Similarly, subjects with lower PROP thresholds exhibit lower recognition thresholds for both sucrose and fructose (Pasquet et al., 2002). Since threshold is often a poor

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Fig. 2.5 Associations between PROP threshold and intensity of sucrose (top) and aspartame (bottom).

predictor of suprathreshold response (Bartoshuk, 1978; Pangborn, 1980) and does not identify supertasters (Bartoshuk et al., 1994), PROP effects on sweetness are attenuated when classifying PROP tasting with thresholds. Figure 2.5 shows data from adults in our laboratory to demonstrate this statement. Most studies report an association between PROP bitterness and the sweetness of sucrose solutions (Duffy et al., 2003; Bartoshuk, 2000; Bartoshuk et al., 2003; Marks et al., 1992), although the effect size may be underestimated in some because of context and ceiling effects (Bartoshuk, 2000; Bartoshuk et al., 2003; Lawless, 1983; Lawless et al., 2000). Conversely, studies that fail to find an association may have context (Lawless, 1979b) or ceiling effects (Drewnowski et al., 1997b, 1998; Smagghe and Louis-Sylvestre, 1998; Ly and Drewnowski, 2001), which emphasizes the importance of psychophysical

Genetic differences in sweet taste perception

Fig. 2.6

39

Associations between the bitterness of concentrated PROP and intensities of sucrose (top) and aspartame (bottom).

methods (Bartoshuk et al., 2003). Data from our laboratory (Fig. 2.6) show PROP effects on sucrose and aspartame intensity. Partial coefficients are reported in a model that included the intensity of 1000 Hz tones at 74 dB as a non-oral sensory standard. PROP effects on sweetness are blunted or nonexistent when using category scales that do not permit valid comparisons of intensity ratings across subjects (Prutkin et al., 2000). The relationship between PROP bitterness and sweetness is mediated in part by the number of fungiform papillae. This point is demonstrated in multiple regression analyses to explain variability in sweetness of 1 M sucrose across 85 adults (20 to 59 years of age) from our laboratory. Those with more fungiform papillae (sr = 0.32, p ˆ 0:001) and who were younger (sr ˆ ÿ0.38, p ˆ 0:001)

40

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tasted greater sweetness. Sweetness also varied by T2R38 gene (sr ˆ 0.23, p ˆ 0:02). In analysis of variance with planned comparisons and using t-tests with the error term generated by the ANOVA, the AVI/AVI nontasters reported significantly greater intensity than did those who were PAV/PAV tasters (p < 0:05). A similar finding was seen for aspartame intensity across a cohort of 55 adults (20 to 40 years) from our laboratory. Greater sweetness of 3.2 mM aspartame was tasted by those with greater numbers of fungiform papillae (sr ˆ 0.32, p < 0:05) and influenced by the T2R38 genotype (sr = 0.26, p ˆ 0:05). In pairwise comparisons, AVI/AVI nontasters reported significantly greater intensity than did the PAV/PAV tasters (p ˆ 0:05). Mixtures can blunt or even reverse PROP effects on sweetness. In mixtures of simple tastants, sucrose sweetness is attenuated in medium and supertasters in two-component mixtures by high concentrations of quinine (240 to 360 mM) (Prescott et al., 2001; Yiee et al., 2002, 2003) and citric acid (14 mM) (Yiee et al., 2002, 2003); the suppression is seen also in three- and four-component mixtures (Yiee et al., 2002, 2003). Sucrose added to strong coffee mostly blunts the bitterness for tasters (Ly and Drewnowski, 2001) and supertasters (Yiee et

Fig. 2.7

Associations between PROP bitterness and sweetness (top) and bitterness (bottom) from DewarsÕ Scotch (Lanier et al., 2005).

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al., 2002, 2003). Within other non-alcoholic beverages, a positive relationship is seen between PROP and sweetness in a commercial lemonade, which was tasted less sour than commercial grapefruit juice (Yiee et al., 2002, 2003). In contrast, sour/bitter or irritating qualities in beverages may conceal PROP effects on sweetness, particularly for supertasters, as seen with grapefruit juice and tonic water (Yiee et al., 2002, 2003), orange juice or citric acid/carbonated mixtures (Prescott et al., 2004). Components of a mixture can also completely reverse PROP effects on sweetness as seen in perceptually complex beverages such as scotch (Fig. 2.7); greater bitterness and possibly irritation, suppresses the sweetness in supertasters (Lanier et al., 2005). High-intensity sweeteners with bitter tastes act perceptually as mixtures as some are known for having bitter or metallic side tastes (Ayya and Lawless, 1992; Schiffman et al., 1995). One study, which assessed PROP tasting by threshold, found that nontasters tasted less suprathreshold bitterness from saccharin (Bartoshuk, 1979). Subsequent work suggests that these PROP effects were due to differences in general taste sensitivity (Lawless, 1979a; Olson et al., 1989) rather than a shared receptor mechanism. Modern scaling methods found that while acesulfame K and saccharin bitterness covary within subjects, they were independent of PROP bitterness (Horne et al., 2002). The independence of PROP tasting from acesulfame K and saccharin bitterness was subsequently confirmed by evidence in humans that PROP tasting associates with the T2R38 genotype (Duffy et al., 2004c) while saccharin and acesulfame K bitterness occur via T2R43/44 (Kuhn et al., 2004). Thus, product developers and sensory specialists are cautioned that screening panelists for PROP tasting may not identify individuals sensitive to the side tastes of some high intensity sweeteners.

2.4 Relating genetic taste markers with dietary sweet behaviors The idea that individual differences in taste perception may influence preference and intake dates back over 40 years (Fischer et al., 1963; Kang et al., 1967), though work in this area has typically focused on associations between PROP/ PTC tasting and foods/beverages thought to be bitter (see Mattes and Labov, 1989; Mattes, 1994; Drewnowski et al., 1999, 2000; Kaminski et al., 2000; Duffy et al., 2004c). Recent advances in molecular genetics have allowed the direct associations between T2R38 haplotype and dietary behaviors (Duffy et al., 2004a, 2004c; Mennella et al., 2005). The general hypothesis for these studies has been that genetic variation in taste and oral sensation influences food and beverage sensations and thus preference. Since we consume what we prefer and avoid what we do not, genetic variation in taste and oral sensation ultimately affects dietary selection. Below we explore this general hypothesis applied to sweet preference and intake by utilizing a number of markers of genetic variation in taste and oral sensation.

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2.5 Sweet liking: relation to PROP, fungiform papillae density, T2R38 Classification of sucrose likers and dislikers by PROP threshold reveals that dislikers are almost exclusively tasters while likers are evenly split between tasters and nontasters (Looy and Weingarten, 1992). In agreement, data from our laboratory shows that dislikers and likers (as shown in Fig. 2.4) differ in PROP bitterness. Dislikers (n ˆ 21) were significantly more likely to be PROP tasters, both medium and supertasters, whereas likers (n ˆ 42) were significantly more likely to be PROP nontasters (Fig. 2.8). A number of studies do not find differences in PROP tasting among sweet liker and disliker groups (Drewnowski et al., 1997a, 1997b, 1998). The PROP relationship with sweet liker/disliker classification is probably not unique to PROP per se, but rather due to an overall difference in oral sensation. Genotyping for the T2R38 receptor was available in a sub-sample of those classified as dislikers (10 of 21) and likers (29 of 42). A chi-square statistic close to zero indicates that the observed distribution equaled expected for T2R38 genotype across dislikers (4 of 10 were AVI/AVI nontasters; 6 of 10 were AVI/PAV or PAV/PAV tasters) and likers (13 of 29 were AVI/AVI nontasters; 16 of 29 were AVI/PAV or PAV/PAV tasters). Thus, the liker/disliker distribution difference in PROP tasting probably is less likely to result from the presence or absence of a functional PTC/PROP receptor and more likely to result from factors such as density of fungiform papillae or central response to sweetness. The inability to uncover phenotypic PROP effects in some studies likely results from misclassification bias that attenuates measurement of the underlying relationship. Use of psychophysical methods that do not accurately characterize individual differences in oral sensations will result in the failure to uncover sensory differences that associate with liking and disliking.

Fig. 2.8 The joint distribution of PROP nontasters (PROP=moderate) and tasters by sucrose liking/disliking categories. Two of 21 dislikers were nontasters, 19 were tasters; 30 of the likers were nontasters, 12 were tasters.

Genetic differences in sweet taste perception

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Studies from our laboratory have seen a relationship between PROP bitterness and preference for sweet foods in adults of normal adiposity (body mass index  25) assessed by questionnaire and by ratings for sampled sweet foods. Since high cognitive restraint may influence preference ratings for sweet and high-energy foods (e.g., Lahteenmaki and Tuorila, 1993; Kanarek et al., 1995), we used validated instruments to exclude individuals with high levels of cognitive restraint from participation (e.g., Herman and Polivy, 1980; Polivy et al., 1994; Stunkard and Messick, 1985). Liking of sweet foods (ice cream, doughnuts, cookies, cake, pie, milk chocolate) assessed via questionnaire decreased with increasing PROP bitterness in women (n ˆ 24), but showed a flat association in men (n ˆ 22) (Duffy and Bartoshuk, 2000). Subsequent analyses reveals that individuals with low fungiform papillae and low PROP tasting reported greater liking for sweet foods than those with high papillae and high PROP tasting (t(34) ˆ 2.05, p < 0:05). Thus, the sex effect on sweet preference could have resulted from differences in density fungiform papillae. In a separate study of 38 females and 44 males (mean age 26  4 years) (Duffy et al., 2003), we examined associations between sweet preference (questionnaire and sampled sweet foods) and PROP as well as quinine, another bitter marker of variability in taste (see below for more discussion about quinine and sweet behaviors). PROP only tended to explain variability in preference across sweet foods from questionnaire, yet was a significant predictor of preference ratings for sampled sweet foods (Fig. 2.9). Those who tasted PROP as least bitter reported significantly greater preference for sampled foods (white icing, milk chocolate, marshmallow, grape jelly), independent of sex, adiposity, level of hunger and sweetness of these foods. Additionally, those who tasted PROP as least bitter consumed significantly more energy from added sugars (assessed via nonconsecutive food records) and tended to consume more sweet foods (assessed by food frequency survey). These findings show that PROP can explain variability in sweet liking and intake among younger adults (mean age below 30), who are primarily of European ancestry, of normal adiposity and have low levels of dietary restraint. Other studies have not seen PROP effects on sweet hedonics. Conceivably, the failure of some (Drewnowski et al., 1997b, 1998; Ly and Drewnowski, 2001) to find an effect could result from either scaling issues (Duffy and Bartoshuk, 2000; Bartoshuk et al., 2003) or the inclusion of samples with diversity in ancestry, body weight, and control over eating. Recent work from the Mennella laboratory shows that the T2R38 genotype explains variability in sweet preference in 143 children (ages 5 to 10 years) and their mothers (mean age 35 years), who were African American or non-Hispanic white (Mennella et al., 2005). Children and mothers provided preference ratings for sampled sucrose solutions, recalled their favorite cereals, and, for mothers, how much sugar they added to coffee. Children who were tasters by genotype preferred higher sucrose concentration than children who had nontaster genotypes, findings that disagree PROP phenotype-sweet relationships found in our laboratory and others (Looy and Weingarten, 1992; Lin, 2003). Genotype did not predict sweet preference in mothers because of the influences of ancestry

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Fig. 2.9 Preference for sampled sweet foods (white icing, milk chocolate, marshmallow, grape jelly) by PROP bitterness (top) and T2R38 genotype (bottom).

on sweet preferences ± individuals of African ancestry report greater preferences for sweets than those of European ancestry (Mennella et al., 2005; Greene et al., 1975; Schiffman et al., 2000). We were able to analyze the relationship between T2R38 genotype and dietary sweet behaviors in a subset of individuals (n ˆ 55) from our laboratory for whom this genotyping was available. In multiple regression equations controlling for sex, age, and adiposity as described (Duffy et al., 2003), the T2R38 genotype did not explain significant variance in preference for sweet foods from questionnaire (p ˆ 0:66), intake of energy from added sugars

Genetic differences in sweet taste perception

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(p ˆ 0:72), or frequency of consuming sweet foods (p ˆ 0:48). The T2R38 genotype did predict significant variance in preference for sampled sweet foods such that those who had the nontaster genotype (AVI/AVI) reported significantly greater preference for these foods than did the homozygote taster (PAV/PAV) as shown in Fig. 2.9. Interestingly, this agrees with findings of Mennella et al. (2005), who found an interaction between genotype and ancestry for sucrose preference in the mothers. For African American mothers, tasters by genotype showed a higher sucrose preference than did nontasters by genotype, although not significant because the means plus variances overlapped. However, for mothers of European ancestry, nontasters by genotype reported a significantly higher sucrose preference than did tasters by genotype. Thus, when comparing groups with similar ancestry and age, our findings and those from Mennella's laboratory (2005) are consistent ± nontasters have greater sweet preference than do tasters by genotype or supertasters by phenotype. However, the finding is not robust across all measures of sweet preference and intake and across groups that vary in age and ancestry, suggesting that other measures of variance in taste must be considered when trying to explore sensory factors driving sweet behaviors. In summary, the work described in the previous section raises more questions than it answers. It appears that individuals who vary in sweet preference do have differences in oral sensation that may or may not be dependent on the presence or absence of a single bitter receptor. Individuals who taste PROP as more bitter show less liking for foods or beverages that are very sweet ± possibly because of greater innervation density (e.g., numbers of fungiform papillae) or differences in the manner in which tastes, nontaste oral sensations (creaminess, viscosity) and flavor (including retronasal olfaction) are perceived. This section also suggests the need to find additional markers of oral sensory variation that can explain differences in sweet preference.

2.6

Variation in other bitter markers

Fischer and colleagues (1961) were the first to examine variability in PROP and quinine tasting pertaining to food preferences. They compared the characteristic bimodal threshold distribution for PROP with the normal distributions produced by stimuli representing all four taste qualities, with particular emphasis on quinine thresholds (Fischer and Griffin, 1961). Fischer believed that his quinine measure reflected a general ability to taste; combining this general measure with the specific ability to taste PTC/PROP compounds produced a group of `acute tasters' who had more food dislikes, tended to avoid smoking (Fischer et al., 1963) and were thinner (Fischer et al., 1966). Using PROP and quinine as markers of variation in taste has been shown to enhance explanation of alcohol (Lanier et al., 2005) and sweet (Duffy et al., 2003) preference and intake. A subset of individuals exists who are discordant in PROP and quinine bitterness. These individuals taste greater bitterness from one marker relative to

46

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the other and, more interestingly, differ in dietary behaviors. Those tasting PROP as less than `strongly bitter' but quinine as above `strongly bitter' have greater preference for and intake of sweet foods than those with the opposite bitter taste profile (Duffy et al., 2003). Recent work in our laboratory has produced similar results; those tasting quinine as relatively more bitter than PROP also consume alcoholic beverages most frequently (Dinehart et al., 2005). Quinine was found to explain variability in a number of measures of sweet preference and intake while PROP only explained variability in some of these behaviors, particularly in younger participants (Dinehart et al., 2005; Duffy et al., 2003). Discordance between PROP and quinine bitterness may result from damage to the chorda tympani nerve (Bartoshuk et al., 1995), a branch of cranial nerve VII that supplies taste to the anterior two-thirds of the tongue. Taste damage from this nerve releases inhibition on cranial nerves IX and X and can result in increases in quinine bitterness as perceived with the whole mouth (Kveton and Bartoshuk, 1994; Halpern and Nelson, 1965; Yanagisawa et al., 1998). Another explanation for discordance between PROP and quinine bitterness may be that bitterness of these compounds results from differential expression of multiple bitter receptors (Margolskee, 2002). Since PROP and quinine do not totally cross-adapt they may stimulate different taste receptors, which would explain differences in bitter perception between the two (McBurney, 1972). Supportive of the independence of quinine from PROP and PTC is the observation that, at least among Japanese dental students, PTC nontasters are more quinine sensitive than are PTC tasters (Sato et al., 1997). It is uncertain whether quinine represents another genetic taste variant, a measure of overall taste sensation, or an environmentally mediated pattern of sensation. Nonetheless, we believe that using multiple markers of variation in taste and oral sensation improves the ability to explain differences in preference for and intake of sweets.

2.7 SAC Gene: human homolog and hedonic response to sweeteners In mice, varied strains differ as to whether they are `taster' mice that prefer sweet solutions or `nontaster' strains that failed to prefer sweet solutions to water. These phenotypic differences were linked to allelic variation in the saccharin preference (Sac) locus, the T1R3 taste receptor, which forms a heterodimer with T1R1 (Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001; Nelson et al., 2001; Bachmanov et al., 2001). The T1R3 gene from a region on chromosome 1 (1p36) appears to be the human homolog of the mouse SAC, a chromosomal region that also contains T1R1, T1R2 genes (Liao and Schultz, 2003). T1R2 and T1R3 have complementary functional roles to form a heteromeric taste receptor complex that responds to structurally diverse sweeteners (Xu et al., 2004).

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Table 2.1 Description of SAC gene (Liao and Schultz, 2003) groups (mean  SD) Heterozygote Sex Age* BMIy FP

2 F; 4 29  26.3  25 

M 4 4.9 9

Homozygote 15 F; 19 M 25  4 23.4  2.8 23  7

* p < 0:1 y p < 0:05

Reed and colleagues (2002) presented preliminary data on behaviors toward sucrose and aspartame in humans who varied on the human homolog of the mouse SAC, T1R3. The psychophysical data were from our laboratory, the Reed laboratory sequenced DNA from whole bloods. The sample was from adults previous described (Duffy et al., 2003); only those who reported being of European ancestry are shown (Table 2.1). Six heterozygous and 34 homozygous subjects were identified. The heterozygotes tended to be older and averaged significantly heavier, although the BMI distribution between the two groups was not significantly different (Fisher's Exact Test, p ˆ 0:16). The subjects rated the intensity and liking/disliking for a sucrose concentration series (Fig. 2.10), and intensity and liking/disliking for a single concentration of aspartame (3.2 mM). A group by concentration ANCOVA that controlled for age and BMI showed significant main effects of group (F(1, 35) ˆ 6.09, p ˆ 0:02) and concentration (F(2,76) ˆ 6.58, p ˆ 0:002): the heterozygotes showed a steeper rise in hedonic response to increasing concentrations of sucrose. The sucrose intensities between the two groups were overlapping. A similar trend was seen for aspartame. In ANCOVA controlling for covariance from age and BMI, the heterozygotes tended to rate the 3.2 mM aspartame as more pleasant (p < 0:1) but not vary in its intensity (p ˆ 0:65). These data show that another gene marker may explain some variability in sweet liking in humans and, that according to preliminary analyses, was not associated with perceived sweetness. 2.7.1 Implication for designing foods that meet consumers' sweet taste Sweetness is a pleasurable sensation yet the consumption of sweet foods and beverages has been under increasing scrutiny by nutrition professionals as well as the lay public because of its potential connection with weight management and dietary quality. Our obesiogenic environment provides ready access to good tasting, high-energy foods and surrounds us with technology that minimizes energy expenditure. The food supply needs to offer foods and beverages that taste sweet but, when ingested, do not displace healthful, nutrient-rich foods. Additionally, adding sweeteners to the food supply should ultimately aim to improve dietary quality while promoting food enjoyment (Duffy and Sigmand-Grant, 2004).

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Fig. 2.10 Intensity (top) and hedonic (bottom) functions of sucrose in individuals who vary in the human SAC gene (Liao and Schultz, 2003).

The present chapter has reviewed genetic variation in taste and oral sensation and how markers of this variation have been shown to associate with dietary sweet behaviors, primarily in studies that employ convenience samples of adults recruited for academic research in contrast to efforts by industry that use consumer or trained panels for product acceptability testing or market research. The studies support that markers of variability in taste do explain some of the variability in sweet behaviors. Although the ability of laboratory tests to predict consumption has been questioned (Lucas and Bellisle, 1987), and the failure of taste tests to predict market performance has been discussed (Garber et al.,

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2003), we contend that laboratory testing can, and does predict real-world consumption. However, the observation that `for hedonic measurements, group averages may be misleading or even completely artifactual (Lundgren et al., 1978)' emphasizes the criticality in identifying different market segments (Garber et al., 2003; Wansink, 2003) for whom the product is intended. As noted by Buck (2003), consumers vary widely in their acceptance of commercially available products. Some suggest that identifying potential segmentation variables (usage frequency, personality and lifestyle factors) can enhance the value of sensory testing (Wansink, 2003). At a minimum, the decision as to whether a trained panel should be according to specific markers of taste, or show a representational cross-section is entirely dependent on the goals of the research, whether market or sensory research. While we do not anticipate the appearance of products tailored to specific genotypes, both sensory practitioners and basic researchers (Cardello, 2003) can benefit from an increased understanding of how genetic variation in taste and oral sensation can influence liking.

2.8

Acknowledgements

The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003-35200-12943 and NIH Institutes of Deafness and Communication Disorders grant number DC00283.

2.9

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and DUFFY, V. B. (2002) Obes Res, 10, 67 (abstract). RICHTER, C. P. and CAMPBELL, K. H. (1939) Am J Physiol ± Legacy Content, 128, 291±297. RICHTER, C. P. and CLISBY, K. H. (1941) Am J Physiol, 134, 157±164. RODIN, J. (1975) J Comp Physiol Psychol, 89, 1003±1009. ROZIN, P. (1987) In Sweetness (Eds, Dobbing, J. and International Life Sciences Institute) Springer-Verlag, London, New York. SAINZ, E., KORLEY, J. N., BATTEY, J. F. and SULLIVAN, S. L. (2001) J Neurochem, 77, 896±903. SATO, T., OKADA, Y., MIYAMOTO, T. and FUJIYAMA, R. (1997) Chem Senses, 22, 547±551. SCHIFFMAN, S. S., BOOTH, B. J., LOSEE, M. L., PECORE, S. D. and WARWICK, Z. S. (1995) Brain Res Bull, 36, 505±513. SCHIFFMAN, S. S., BOOTH, B. J., SATTELY-MILLER, E. A., GRAHAM, B. G. and GIBES, K. M. (1999) Chem Senses, 24, 439±447. SCHIFFMAN, S. S., GRAHAM, B. G., SATTELY-MILLER, E. A. and PETERSON-DANCY, M. (2000) Nutrition, 16, 886±893. SHALLENBERGER, R. S. and ACREE, T. E. (1967) Nature, 216, 480±482. SMAGGHE, K. and LOUIS-SYLVESTRE, J. (1998) Appetite, 30, 325±339. SNYDER, L. H. (1931) Science, 74, 151±152. STEINER, J. E., GLASER, D., HAWILO, M. E. and BERRIDGE, K. C. (2001) Neurosci Biobehav Rev, 25, 53±74. STUNKARD, A. and MESSICK, S. (1985) Journal of Psychosomatic Research, 29, 71±83. TEPPER, B. J. and NURSE, R. J. (1997) Physiol Behav, 61, 949±954. THOMPSON, D. A., MOSKOWITZ, H. R. and CAMPBELL, R. G. (1977) Physiol Behav, 19, 335± 337. WANSINK, B. (2003) Food Qual Pref, 14, 23±26. WHO/FAO (2003) Food and Agriculture Organization of the United Nations, Geneva, Switzerland, pp. 54±71. WITHERLY, S. A., PANGBORN, R. M. and STERN, J. S. (1980) Appetite, 1, 53±63. WOODING, S., KIM, U. K., BAMSHAD, M. J., LARSEN, J., JORDE, L. B. and DRAYNA, D. (2004) Am J Hum Genet, 74, 637±646. XU, H., STASZEWSKI, L., TANG, H., ADLER, E., ZOLLER, M. and LI, X. (2004) Proc Natl Acad Sci USA, 101, 14258±14263. YANAGISAWA, K., BARTOSHUK, L. M., CATALANOTTO, F. A., KARRER, T. A. and KVETON, J. F. (1998) Physiol Behav, 63, 329±335. YIEE, J., DUFFY, V. and BARTOSHUK, L. (2002) Appetite, 39, 108 (abstract). YIEE, J. H., DUFFY, V. B. and BARTOSHUK, L. M. (2003) Chem Senses, 28, 134 (abstract). MARGOLSKEE, R., BARTOSHUK, L. M.

3 Children's liking of sweet tastes and its biological basis M. Y. Pepino and J. A. Mennella, Monell Chemical Senses Center, USA

3.1

Introduction

The liking for things that taste sweet is universal and a hallmark of development. At birth, sweet liking ensures the acceptance of that which infants need to survive ± the sweet taste of their mothers' milk. Heightened preferences for sweet tastes, which persist throughout childhood and adolescence, may have an ecological basis since, in nature, sweet tasting foods, such as fruits, are associated with energy-producing sugars, minerals, and vitamins (Beauchamp, 1999). Although strong evidence is lacking, it has been suggested that such preferences evolved to solve a basic nutritional problem of attracting children to sources of high energy during periods of maximal growth (Simmen and Hladik, 1998; Drewnowski, 2000; Coldwell and Oswald, 2004). In more modern times, children express their liking for sweets by consuming foods other than nature's fruits. Since the 19th century, sweet candies have been the first material goods that children spend their own money on (Woloson, 2002). In this chapter, we review the scientific literature that suggests that children's liking for all that is sweet is not solely a product of modern-day technology and advertising, but reflects their basic biology. To this end, we summarize the insights gleaned from scientific research on the ontogeny of human behavioral responses to sweet tastes. We review the scientific literature that documents the innate heightened preferences for sweet tastes and how this innate preference can be modified by experiences early in life. This review serves as a foundation for a discussion on the physiological and motivational properties of sugars.

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3.2 Ontogeny of sweet taste preferences: from fetal life to adolescence 3.2.1 Fetus Although the response of human fetuses to sweet taste has never been directly investigated, indirect evidence suggests that changes in the chemosensory properties of amniotic fluid may modulate intrauterine behaviors. Athough there were a number of methodological flaws (see Mistretta and Bradley, 1977), two studies reported increased swallowing following the injection of sweet stimuli such as saccharin (Liley, 1972; De Snoo, 1937) in the amniotic fluid of pregnant women and decreased swallowing following the injection of a bitter-tasting substance (Liley, 1972) thus suggesting that prenatal monitoring of taste stimuli dissolved in amniotic fluid is possible during late gestation. Perhaps stronger evidence that the fetus can respond to taste stimuli comes from research on premature infants who were six to nine months' gestational age. To overcome methodological limitations and because premature infants often have immature suck-swallow coordination, innovative methods were developed that avoided the risk of fluid aspiration by embedding the taste substance in a nippleshaped gelatin medium that released small amounts of the sweet substance when mouthed or sucked. Infants born pre-term and tested between 33 and 40 weeks postconception produced stronger and more frequent sucking responses when offered the sucrose-sweetened nipple compared with a latex nipple (Maone et al., 1990). Pre-term infants, who had been fed exclusively via gastric tubes, exhibited more non-nutritive sucking in response to glucose than to water (Tatzer et al., 1985). Taken together, these data suggest that the ability to detect sweet tastes and, in turn, modulate a variety of behaviors is evidenced during fetal life. The preference for sweet tastes is not only present early in ontogeny, but research on other infant primates revealed that it is remarkably well conserved phylogenetically (Steiner et al., 2001). 3.2.2 Infants Within hours after birth, the liking for sweet tastes is unequivocal. In fact, newborns are sweet connoisseurs. They respond to even dilute sweet tastes, differentiate varying degrees of sweetness and will consume more of a sugar solution when compared to water (Nowlis and Kessen, 1976; Desor et al., 1973). When a sweet solution is placed in the oral cavity, the baby's face relaxes, resembling an expression of satisfaction which is often followed by a smile (Steiner, 1977; Rosenstein and Oster, 1988). That the positive facial expressions elicited by sweet tasting sugars are reflex-like is supported by several findings. First, single response components, such as tongue movements, can be reliably elicited in newborns by sweet tastes in a concentration-dependent manner (e.g., Nowlis and Kessen, 1976; Weiffenbach, 1977). Second, neonates born with severe developmental malformations of the central nervous system (e.g., anencephalic infants) react to the sweet taste (and other tastes) like a normal term born neonate (Steiner, 1977).

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Consistent with that observed for intake and facial expression, infants can express that they perceive and respond positively to sweet stimuli via a variety of other behaviors. Sweet taste solutions of sucrose and aspartame in the oral cavity reliably elicits mouthing and sucking movements and hand-to-mouth contacts (e.g., Miller et al., 1994; Barr et al., 1999; Blass et al., 1989), both of which are feeding-related behaviors. Newborns also respond to sweet stimulation with changes in autonomic response (e.g., Blass and Watt, 1999; Crook and Lipsitt, 1976; Haouari et al., 1995; Lipsitt, 1977; Lipsitt et al., 1976; Rao et al., 1997). However, the behavioral state of the baby modified the direction of the change. For example, heart rates increased proportionally when increasing concentration of sucrose were placed in the mouths of non-agitated infants (Ashmead et al., 1980). Conversely, heart rate decreased when sweet tastes were introduced to infants who were agitated, resulting in overall calmness (e.g., Blass and Watt, 1999). Furthermore, electroencephalographic recordings of 2- to 3-day-old newborns revealed that infants respond to sucrose administration with an asymmetry of brain electrical activity, a response which is usually associated with hedonically positive emotional reactions or approach behavior (Fox and Davidson, 1986). Although each measure has its limitations, the convergence of findings obtained from different methodologies suggested that from birth infants are quite sensitive to and prefer sweet tastes. 3.2.3 Children and adolescents The heightened preference for sweet taste is universal and evident among children around the world (e.g., Brazil: Tomita et al., 1999; France: Bellisle et al., 1990; Iraq: Jamel et al., 1997; Israel: Steiner et al., 1984; Mexico: Vazquez et al., 1982; Netherlands: De Graaf and Zandstra, 1999; and North America: Beauchamp and Cowart, 1987; Desor et al., 1975). Both cross-sectional and longitudinal studies revealed that the preference for sweets remains heightened throughout childhood (Pepino and Mennella, 2005; Mennella et al., 2005a) and early adolescence (Desor et al., 1975) and declines to adult levels during late adolescence (Desor and Beauchamp, 1987). In a cross-sectional study that measured sweet preference in more than 750 participants, 50% of the children and adolescents, but only 25% of the adults, selected the 0.60 M sucrose concentration as their favorite solution (Desor et al., 1975). To put this in perspective, a 0.60 M sucrose concentration is equivalent to approximately 12 spoonfuls of sugar in 230 ml of water (an 8 oz glass), whereas a typical cola has a 0.34 M sugar concentration. The level of sucrose that children prefer, as measured in the laboratory, is significantly related to children's preferences for sweet tasting foods such as cereals (Liem and Mennella, 2002; Mennella et al., 2005a) and beverages (Olson and Gemmill, 1981; Mennella et al., 2005a). Sweet taste can clearly drive consumption in the young. Adding sugar to beverages such as Kool Aid (Beauchamp and Moran, 1984) and solid foods such as spaghetti (Filer, 1978),

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ricotta and jicama (Sullivan and Birch, 1990) increased both liking and acceptance by children. Findings from the USDA Department of Agriculture 1994±1996 Continuing Survey of Food Intakes by Individuals of more than 15,000 Americans who were two years of age or older (Guthrie and Morton, 2000) paralleled the findings from basic research of an age-related decline in sweet preferences. That is, the proportion of energy obtained from added sweeteners peaks in adolescents, with approximately 20% of energy derived from added sugars, and declines to approximately 12.4% of energy for those 65 years and older (Guthrie and Morton, 2000). What causes the developmental shift in sweet preferences and consumption remains a mystery, but this age-related decline in sweet preference has been observed in other mammals (Wurtman and Wurtman, 1979; Marlin, 1983). One explanation may be that children are less sensitive to sweet tastes, thereby requiring larger amounts of sugars. However, there is no strong evidence that the heightened sweet preference evidenced during infancy and childhood is due to decreased sensitivity to sweet tastes (see Cowart, 1981 for review). Whereas some investigators may report that children are more sensitive than adults when small regions of the anterior tongue are stimulated with sucrose (Stein et al., 1994), others found that taste sensitivity during childhood does not differ from that during adulthood (Cowart, 1981). Another explanation is that the heightened sweet preferences early in life are linked to the growing child's need for calories (Drewnowski, 2000). Recent findings by Coldwell and Oswald (2004) lend support to this hypothesis. Elevento 15-year-old children were divided into likers and dislikers based on their sucrose preferences. Although there were no differences between these two groups in sucrose detection thresholds, age, body mass index, percent of body fat, plasma leptin, dietary restraint, pubertal development or gonadal hormone levels, growth rate was significantly lower in dislikers when compared to likers. These data suggest that the age-related decline in sucrose preferences may be related to the cessation of growth (Coldwell and Oswald, 2004). 3.2.4 Individual differences Despite such age-related changes in sweet preferences, individual variations exist at both ends of the age spectrum (Desor et al., 1977; Beauchamp and Moran, 1982; Enns et al., 1979; Drewnowski et al., 1985; Pliner and Fleming, 1983; Mennella et al., 2005a). Genetic variation may contribute to some differences in sweet taste preferences (see McDaniel and Reed, 2004). We recently demonstrated that alleles of the bitter taste receptor TAS2R38 gene could explain much of the phenotypic variation in the sensitivity to the bitter compound, propylthiouracil and partially explained individual differences in children's sweet preferences (Mennella et al., 2005a; see also Keller and Tepper, 2004). Here individuals homozygous for the bitter-insensitive allele are referred to as AA, heterozygotes as AP, and those homozygous for the bitter-sensitive allele as PP. The data

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revealed that AP and PP children preferred significantly higher concentrations of sugars in liquids when compared with children homozygous for the bitterinsensitive allele (AA). Similar findings were observed for foods such as cereals. Future research is needed to determine how variation in other taste receptor genes, particularly those involved in sweet taste perception, contribute to individual differences in preferences and food habits. Unlike children, there was no correspondence between TAS2R38 genotypes and sweet preference in adults. Here the effects of race/ethnicity were the strongest determinants, thus suggesting that cultural forces and experience may override this genotype effect on sweet preferences. To the point, several studies demonstrated that African-American, infants, children, teenagers as well as adults preferred higher concentrations of sucrose when compare to similarly aged Caucasians (Beauchamp and Moran, 1982; Desor et al., 1975; Bacon et al., 1994; Pepino and Mennella, 2005). Differential exposure to sweets foods, as will be discussed in the following section, may contribute to these race/ethnicity differences since the tradition of feeding sweetened water or adding sugar to the children's food is practiced significantly more among African American (Pepino and Mennella, 2005; Liem and Mennella, 2002) and Hispanic (Mennella et al., 2005b) mothers than Caucasians. Nevertheless, although there are individual differences in sweet preferences that are determined in part by our genetics and are shaped by the culture in which we live, there is no doubt that children, as a group, prefer significantly higher concentrations of sugars in foods and liquids than do adults.

3.3

Role of experiential factors on sweet taste preferences

Considering the strong innate preference that humans have for sweet tastes, it is not surprising that a review of ancient and current-day customs of the first liquids or foods given to infants prior to the first breast feed, hence the term prelacteal feed, (Morse et al., 1990) revealed a striking commonality in their sweet taste (Jerome, 1977; Mennella, 1997). Sugared waters or teas are perhaps the most popular pre-lacteals given to infants throughout the world (Morse et al., 1990; Mennella, 1997; Mennella et al., 2005b). To what extent do these early experiences alter or modulate the development of sweet preferences later in life? Longitudinal studies (Beauchamp and Moran, 1982, 1984) revealed that babies who were routinely fed sweetened water (e.g., water sweetened with table sugar, Karo syrup or honey) during the first months of life exhibited a greater preference for sweetened water when tested at six months (Beauchamp and Moran, 1982) and then again at two years of age (Beauchamp and Moran, 1984) when compared to those who had little or no experience with sweetened water. A more recent cross-sectional study on 6- to 10-year-old children supported these findings and revealed that such feeding practices may have longer term effects on the preference for sweetened water than previously realized (Pepino and Mennella, 2005).

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Whether early experience with sweetened water modifies overall liking for sweetened foods is less uncertain. In fact, there is no compelling data suggesting that repeated exposure to sugar water results in a generalized heightened hedonic response to sweetness. Being fed sugar water during infancy did not heighten 2year-old children's preference for sweetened versus the unsweetened Kool-Aid. All children, regardless of whether they were fed sugar water, ingested significantly more of the sweetened when compared to the plain Kool-Aid (Beauchamp and Moran, 1984). Moreover, 4- to 7-year-old children whose mothers reported adding sugar to their foods on a routine basis were significantly more likely to prefer apple juices with added sugar and cereals with higher sugar contents than similar age children whose mothers reported never adding sugar to the foods at home (Liem and Mennella, 2002). These data support the hypothesis that the context in which the taste experience occurred is an important factor and through familiarization, children develop a sense of what should, or should not, taste sweet (Beauchamp and Cowart, 1985).

3.4

Physiological properties of sweet tastes

Tasting a sweet substance not only elicits feeding-related behaviors but also modifies crying behaviors and physiological responses. A small amount of a sweet solution placed on the tongue of a crying newborn exerts a rapid, calming effect which persists for several minutes (Blass and Hoffmeyer, 1991; Barr et al., 1999). Sweet tastes can also attenuate heart rate increases, blunt expressions of pain, and calm both preterm and full-term infants who have been subjected to painful events such as heel stick or circumcision (Stevens et al., 2004 for review). That sucrose attenuated a negative electroencephalographic response to a painful procedure (Fernandez et al., 2003) suggests that sucrose blocks pain afferents which, in turn, diminishes stress and cardiac changes. Because noncaloric sweet substances such as aspartame mimic the calming effects of sucrose (Barr et al., 1999; Bucher et al., 2000) and because the administration of sucrose by direct stomach loading is not effective (Ramenghi et al., 1999), afferent signals from the mouth, rather than gastric or metabolic changes, appear to be responsible for the analgesic properties of sweet tastes. Other flavors and orosensory components also reduce pain in infants and may act synergistically with sweet tastes. These include the flavor of mothers' milk (Upadhyay et al., 2004) and some of its constituents (i.e., fat, protein) (Barr et al., 1996; Blass, 1997). Orotactile stimulation from a pacifier (Blass and Hoffmeyer, 1991; Blass and Watt, 1999; Carbajal et al., 1999), maternal skin-toskin contact (Gray et al., 2000) or being held before and during the painful procedure (Gormally et al., 2001) also produce the analgesic effect, but the effects are transient when compared to when sweet taste is in the oral cavity (Smith et al., 1990). Using sterile sugar water to reduce pain in infants is now routine practice in several hospital nurseries because of its natural simplicity, viability and efficacy (Anand, 2001). However, more research is needed to

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determine sweet taste's efficacy in reducing pain after repeated administrations (Stevens et al., 2004; Mennella et al., 2003; Vazquez et al., 1982). Sweet tastes continue to have analgesic properties during childhood. The presence of sucrose, but not water, in the oral cavity delayed 8- to 11-year-old children's reporting of pain onset when undergoing a cold-induced pain stimulus test, the Cold Pressor Test (Miller et al., 1994). Research from our laboratory recently confirmed these findings and revealed that sucrose's efficacy in reducing pain is related to the hedonic value of sweet taste for the child (Pepino et al., 2004). The more children like sucrose, the better it works in increasing pain tolerance.

3.5

Conclusions

Liking sweet tasting foods and liquids reflects the child's basic biology. When given the choice, it is not surprising that children will eat foods and beverages that are high in sugar content. The heightened preference for sweet tastes during development may have ensured the acceptance of nature's first food ± mothers' milk, as well as nature's sweet tasting foods such as fruits which are high in energy, minerals and vitamins. However, we are now living in an age of abundance with a diet that contains highly concentrated sugars in foods and beverages, in part, because of modern technological means of refining sugars. In other words, there is great mismatch between the environment in which humans evolved and the one we currently reside in. Organizations worldwide (e.g., Joint World Health Organization/Food and Agriculture Organization Expert Consultation on Diet, Nutrition and Prevention of Chronic Disease and United States Department of Agriculture) recommend limiting the intake of free sugars to less than 10% of total energy (WHO/Food and Agriculture Organization of the United Nations Expert Consultation, 2003; Welsh et al., 1993). However, at least in the United States, estimates suggest that the levels of consumption far exceed recommended levels with adolescents consuming around 20% of total energies from added sweeteners (Guthrie and Morton, 2000). Whether the food habits of children are determined by genetics or shaped by the culture in which we live or both, there is no doubt that children, as a group, prefer significantly higher concentrations of sugars in foods and liquids than do adults. However, there are striking individual and group differences in the levels of sweetness preferred. For example, across all age groups, African-American non-Hispanics prefer significantly higher levels of sweetness than Caucasians (Beauchamp and Moran, 1982; Desor et al., 1975; Bacon et al., 1994; Pepino and Mennella, 2005; Krebs-Smith, 2001). Therefore, it is important to realize that trying to limit sweet food /beverages consumption may be more difficult for some children or certain ethnic groups because of the inherent hedonic value of sweet tastes and how sweets make them feel. More knowledge about the factors that contribute to preferences for sweet-tasting foods and beverages in children,

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a generation who will struggle with obesity and diabetes, may suggest population-based strategies to overcome diet-induced disease and promote healthy eating habits.

3.6

Acknowledgements

Preparation of this manuscript was supported in part by NIH Grants AA09523 and HD37119.

3.7

References

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sweetener reduces nociceptive reaction in term newborn infants', Early Hum Dev, 59 (1), 51±60. CARBAJAL R, CHAUVET X, COUDERC S and OLIVIER-MARTIN M (1999), `Randomised trial of analgesic effects of sucrose, glucose, and pacifiers in term neonates', BMJ, 319 (7222), 1393±7. COLDWELL S E and OSWALD T K (2004), `Growth-rate differs between children with adultlike versus child-like sugar preference', J Dent Res, 83 (Spec Iss A), Abst 0740. COWART B J (1981), `Development of taste perception in humans: sensitivity and preference throughout the life span', Psychol Bull, 90 (1), 43±73. CROOK C K and LIPSITT L P (1976), `Neonatal nutritive sucking: effects of taste stimulation upon sucking rhythm and heart rate', Child Dev, 47 (2), 518±22. DE GRAAF C and ZANDSTRA E H (1999), `Sweetness intensity and pleasantness in children, adolescents, and adults', Physiol Behav, 67 (4), 513±20. DE SNOO K (1937), `Das trinkende Kind im Uterus' [The drinking child in-utero], Monatsschr Geburtsh Gynaekol [International Monthly Review of Obstetrics and Gynaecology], 105, 88±97. DESOR J A and BEAUCHAMP G K (1987), `Longitudinal changes in sweet preferences in humans', Physiol Behav, 39 (5), 639±41. DESOR J A, MALLER O and TURNER R E (1973), `Taste in acceptance of sugars by human infants', J Comp Phys Psychol, 84, 496±501. DESOR J A, GREENE L S and MALLER O (1975), `Preferences for sweet and salty in 9- to 15year-old and adult humans', Science, 190 (4215), 686±7. DESOR J A, MALLER O and GREENE L S (1977), `Preference for sweet in humans: Infants, children, and adults', in Weiffenbach J M, Taste and Development: the genesis of sweet preference, Wahington, D.C, U.S. Government Printing Office, 161±72. DREWNOWSKI A (2000), `Sensory control of energy density at different life stages', Proc Nutr Soc, 59 (2), 239±44. DREWNOWSKI A, BRUNZELL J D, SANDE K, IVERIUS P H and GREENWOOD M R (1985), `Sweet tooth reconsidered: taste responsiveness in human obesity', Physiol Behav, 35 (4), 617±22. ENNS M P, VAN ITALLIE T B and GRINKER J A (1979), `Contributions of age, sex and degree of fatness on preferences and magnitude estimations for sucrose in humans', Physiol Behav, 22 (5), 999±1003. FERNANDEZ M, BLASS E M, HERNANDEZ-REIF M, FIELD T, DIEGO M and SANDERS C (2003), `Sucrose attenuates a negative electroencephalographic response to an aversive stimulus for newborns', J Dev Behav Pediatr, 24 (4), 261±6. FILER L J (1978), `Studies of taste perception in infancy and childhood', Ped Bas, 5±9. FOX N A and DAVIDSON R J (1986), `Taste-elicited changes in facial signs of emotion and the asymmetry of brain electrical activity in human newborns', Neuropsychologia, 24 (3), 417±22. GORMALLY S, BARR R G, WERTHEIM L, ALKAWAF R, CALINOIU N and YOUNG S N (2001), `Contact and nutrient care giving effects on newborn infant pain responses', Dev Med Child Neurol, 43 (1), 28±38. GRAY L, WATT L and BLASS E M (2000), `Skin-to-skin contact is analgesic in healthy newborns', Pediatrics, 105 (1), e14. GUTHRIE J F and MORTON J F (2000), `Food sources of added sweeteners in the diets of Americans', J Am Diet Assoc, 100 (1), 43±51. HAOUARI N, WOOD C, GRIFFITHS G and LEVENE M (1995). `The analgesic effect of sucrose in full term infants: a randomised controlled trial', BMJ, 310 (6993), 1498±500.

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4 Taste±odour interactions in sweet taste perception D. Valentin, C. Chrea and D. H. Nguyen, CSEG, France

4.1

Introduction

The sense of smell plays a major role in food flavour perception: individuals who lose their sense of smell often report that food has no taste anymore. Yet, from a neuroanatomical point of view taste and smell are very different senses. Taste is perceived primarily on the tongue whereas odours are perceived in the upper part of the nasal cavity either directly or via the back of the mouth. But does this neuroanatomical dissociation imply that taste and odour perception are independent? Probably not! Indeed, information coming from the gustatory and the olfactory systems are likely to be combined at a higher level of processing in the brain to give rise to a unique perception referred to as `flavour' (Abdi, 2002; Prescott, 1999). The question thus is: What is the nature of this combination? Is it an additive combination whereby taste and odour perception are simply added to form an overall perception or can we observe interactions between taste and odours? In this context, taste odour interaction refers to a modification in perceived taste intensity in the presence of an odour. For example, a sweet solution will taste sweeter in the presence of a vanilla aroma even though the vanilla aroma possesses no taste properties. The aim of this chapter is to examine whether such interactions exist for sweet taste perception. We begin with an overview of the literature on the effect of odour on sweet taste perception. Then we present the factors that might affect taste±odour interactions and the different underlying mechanisms proposed in the literature. Finally we highlight some implications of taste±odour interactions for food product development.

Taste±odour interactions in sweet taste perception

4.2

67

Overview of the literature

Early work suggested that odour and taste were independent. Murphy et al. (1977), for example, examined the perceived odour intensity, taste intensity and overall intensity of mixtures of sodium saccharin and ethyl butyrate. They found that the overall intensity of the mixtures approximate the simple sum of the intensities of the unmixed components (90%). The same pattern of results was also obtained by Murphy and Cain (1980) for sucrose-citral mixtures. However, in both studies, the authors indicate that, despite this apparent independency between taste and aroma, participants tend to perceive olfactory stimulation as taste. For example, Murphy and Cain (1980) report that whereas citral had no effect on sweet perception, it was judged to `have taste magnitude when presented alone'. This effect, referred to as `olfactory referral' by the authors, does not result from the stimulation of receptors in the oral cavity since it can be abolished by pinching the nostrils (Murphy and Cain, 1980; Schifferstein and Verlegh, 1996). It was first attributed to trigeminal stimulations (Murphy and Cain, 1980). According to these authors `the trigeminal system may serve to bind the anatomically and physiologically distinct olfactory and taste systems into a single perceptual system during eating' (see Murphy and Cain, 1980, p. 605). As we shall see in Section 4.3, other explanations are proposed to account for this effect. Since these first demonstrations of taste±odour confusion many studies have investigated the effect of odours on sweet taste perception over the past 20 years. Although those studies use different experimental paradigms, the general principle remains the same. Participants are presented with a series of solutions made of a sweetener alone and a sweetener plus an odorant. Their task is to estimate the sweet (and aroma) intensity of each solution. An odour-induced taste enhancement is said to occur if the perceived sweet intensity of the mixture is greater than the perceived sweet intensity of the sweetener alone. And inversely an odour-induced taste suppression is said to occur if the perceived sweet intensity of the mixture is smaller than the perceived sweet intensity of the sweetener alone. The picture emerging from these studies is somewhat confusing (cf. Table 4.1). However, despite some divergence in the results, most studies showed that sweet intensity can be enhanced by odour. For example, Frank and Byram (1988) reported that the perceived sweetness of whip cream samples increased when a strawberry aroma was added. The same type of effect was obtained by other authors in presence of several aromas including almond, caramel, coffee, lemon, peach and vanilla and for several sweeteners such as sucrose, fructose, aspartame and saccharine. This effect occurs both when subjects swallow the test solutions as well as when they spit it (Frank et al., 1989). In addition, Cliff and Noble (1990) using a peach aroma showed that increasing the concentration of aroma increased not only the intensity of perceived sweetness of a glucose solution but also its duration. More striking, Algom et al. (1993) indicated that both perceived and mentally imagined mixtures of different concentrations of glucose and orange aroma showed the

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Table 4.1 Overview of the impact of odours on sweet taste perception. SS ˆ Single scale ratings, MS ˆ multiple scale ratings D ˆ detection, M ˆ matching. ‡ indicates an enhancement effect, ÿ a suppression effect, ˆ no effect,


not applicable Authors

Odorant

Tastant

SS

M

D

De Araujo et al. (2003) Djordjevic et al. (2004) Frank and Byram (1988) Frank et al. (1989) Frank et al. (1990) Frank et al. (1993) Nguyen (2000) Schifferstein and Verlegh (1996) Stevenson (2001) Stevenson et al. (1999) van der Klaauw and Frank (1994) van der Klaauw and Frank (1996) van der Klaauw and Frank (1996) van der Klaauw and Frank (1993) van der Klaauw and Frank (1993) Clark and Lawless (1994) Frank et al. (1991) van der Klaauw and Frank (1993) Frank et al. (1993) van der Klaauw and Frank (1993) van der Klaauw and Frank (1993) Frank et al. (1991) van der Klaauw and Frank (1993) Lawless and Schlegel (1984) Nguyen (2000) Schifferstein and Verlegh (1996) Frank et al. (1991) Clark and Lawless (1994) Nguyen (2000) Small et al. (2004) Lavin and Lawless (1998) Prescott (1999) Prescott et al. (2004) Stevenson et al. (1995) Stevenson et al. (1999) Djordjevic et al. (2004) Schifferstein and Verlegh (1996) Stevenson et al. (1995) Stevenson et al. (1999) Bingham et al. (1990) Stevenson et al. (1999) Frank and Byram (1988) Prescott (1999) Frank et al. (1991) Stevenson et al. (1999)

Strawberry

Sucrose




ˆ
































ÿ


























‡
































ÿ































Frank et al. (1993)

MS

Lemon

Sucrose Fructose Saccharine Aspartame Aspartame Sucrose

Vanilla

Aspartame Sucrose

Waterchestnut

Milk Sucrose

Ham

Sucrose

Lychee

Sucrose

Maltol

Sucrose

Peanut butter

Sucrose

Peanut Acetyl methyl carbamol Almond

Aspartame Sucrose




‡











‡





‡





‡ ˆ


‡ ˆ,ÿ


ˆ





‡





‡


‡


‡


‡


‡ ‡ ‡


ˆ ˆ


‡ ÿ ÿ ‡ ÿ


‡ ˆ


‡





‡ ÿ


ˆ ˆ


ˆ ˆ


ˆ ˆ





ˆ


ˆ ˆ


ˆ





‡





‡








‡


‡





‡





‡








‡


ˆ





ˆ








ˆ





ˆ











ˆ








ˆ





ˆ


‡ ˆ





ÿ


ˆ ˆ


ÿ








ˆ





ˆ




Sucrose

‡

Fructose Saccharine Aspartame Wintergreen

ˆ,ÿ




Taste±odour interactions in sweet taste perception Table 4.1

69

Continued

Authors

Odorant

Tastant

SS

MS

M

D

Stevenson et al. (1999) Stevenson et al. (1999) Frank et al. (1991) van der Klaauw and Frank (1994) Stevenson et al. (1999) Stevenson et al. (1999) Stevenson et al. (1999) Stevenson et al. (1999) Prescott (1999) Algom et al. (1993) Cliff and Noble (1990) Prescott et al. (2004) Prescott (1999)

Angelica oil Caramel Chocolate Coffee Damascene Eucalyptol Mango Maracuja Oolong tea Orange Peach Prune Raspberry

Sucrose Sucrose Aspartame Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose










‡











ÿ ‡ ‡ ˆ ‡

ÿ ‡ ˆ


ÿ ˆ ˆ ‡

























‡





































































same patterns of interaction. In other words imagined odours interact with perceived sweetness the same way as perceived odours (Djordjevic et al., 2004). Schifferstein (1997), however, indicates that in the case of mentally imagined mixtures participants might not base their response on mental images but rather on their knowledge of sensory interaction. Finally, Sakai et al. (2001) found that sweetness enhancement of aspartame by vanilla persists when vanilla is presented simultaneously with, but not dissolved in, the taste stimuli. So, to summarise it seems that sweet taste perception might be modified in the presence of an odour and that this effect is not due to changes in the physicochemical properties of tastants in the presence of odorants since it can be observed when odorants and tastants are presented separately. Yet, despite the many studies demonstrating an enhancement of sweet taste in the presence of an odour, the conditions of appearance of such an effect are far from being clear. Indeed, as can be noted on Table 4.1, all odours do not have the same effect on sweetness perception. Some odours such as strawberry, vanilla, lemon, almond, caramel, maracuja and lychee tend to increase sweetness intensity of sucrose or aspartame. Other odours tend to have no effect (peanut butter, ham, chocolate, mango and wintergreen) or tend to decrease sweetness intensity of sucrose or aspartame (liquorice, almond, damascene, and angelica oil). Moreover, the same odour does not seem to always induce a similar effect. For example, lemon seems sometimes to enhance sweet taste intensity, sometimes to suppress it and other times to have no effect.

4.3

Factors affecting taste±odour interactions

Three major classes of factors that we refer to as `task-driven', `stimulus-driven' and `subject-driven' factors have been proposed to explain the variability of the effect of odour on sweet taste.

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4.3.1 Task-driven factors A first explanation for the variability of the effects observed in Table 4.1 might be found in differences in the methodology used by the authors. For example, Lawless and Schlegel (1984) found a taste odour interaction in mixtures with variable sucrose and citral concentrations using Thurstonian scale values derived from triangle tests but not using attribute ratings. Inversely, Djordjevic et al. (2004) failed to find an effect of strawberry odour on sweet taste perception with a forced choice detection task whereas other authors found an effect with intensity scaling. These results indicate that the instructions given to the participants as well as the strategy implied by the task performed play an influential role in taste±odour interactions. Instructions given to the subjects Most studies of taste±odour interaction use intensity ratings as measurements. Several studies showed that, in this type of experimental set up, the instructions given to the subjects are crucial. In particular, Frank and his collaborators (Frank et al., 1990; van der Klaauw and Frank, 1996) and Clark and Lawless (1994) showed that sweetness enhancement depends upon the response alternatives given to the subjects. For example, in Frank et al. (1993), participants were asked to evaluate either the sweetness of sucrose and sucrose-strawberry mixtures or the sweetness, sourness and fruitiness of the same mixtures. When sweetness was the only alternative, an enhancement was observed: the sucrosestrawberry mixture was rated as sweeter than the sucrose solution. No such enhancement was observed when participants had to rate simultaneously the sweetness, the fruitiness, sourness and fruitiness of the mixtures. In another condition, participants were asked to first rate the global intensity of the mixture and then separate this global intensity in six ratings (sweet, salty, sour, bitter, fruity and other). A suppression effect was observed: The sucrose-strawberry mixture was rated as less sweet than the sucrose solution. According to Frank et al. (1993), providing participants with alternative scales helps them separate their perception in taste and odour perception and thus reduces olfactory referral. Further work by van der Klaauw and Frank (1996) examined if the effect of the instructions given to the subjects was due to the number of alternatives proposed to the participants or to the appropriateness of these alternatives. Six rating conditions were used: 1) sweet only, 2) sweet and bitter, 3) sweet, bitter, and floral, 4) sweet and floral, 5) sweet and fruity, 6) sweet, or fruity or bitter (participants in this condition had to make only one rating per stimulus but were not told before the presentation of the stimulus if this rating was sweet, fruity or bitter). As shown in Fig. 4.1, the number of alternatives did not play an important role in determining sweet taste enhancement: no decrease in odour-induced enhancement was observed when the number of alternatives increased. In contrast, the appropriateness of alternatives seems to be crucial: inappropriate alternatives such as bitter did not reduce taste enhancement. Finally, no enhancement was observed in the last condition thus indicating that attending to different

Taste±odour interactions in sweet taste perception

71

* p>0.05 (adapted from van der Klaauw and Frank (1996).

Fig. 4.1 Changes in sweetness perception (sweetness rating for the mixture minus sweetness rating for sucrose alone) for strawberry sucrose mixtures under six ratings condition.

components of the overall sensation is more important than actually making multiple ratings. Strategy implied by the task As we just saw the number of rating scales available to describe a mixture might affect the appearance of taste±odour interactions. According to Frank and his collaborators this effect can be explained in terms of attentional strategies. Providing participants with multiple scales encourages them to adopt an analytical approach whereas providing them with a single scale encourages them to adopt an integrative or synthetic approach. More specifically Frank (2002) indicates that `When subjects are asked to attend to multiple stimulus attributes, the instructions encourage them to disentangle the concepts for each of the attributes and this constricts the conceptual boundaries of the attributes and minimizes dimensional interactions' (see Frank, 2002, p. 142). For example, providing only a sweetness scale to evaluate a sucrose-strawberry mixture might lead to a wider interpretation of the sweetness concept including sensation such as fruitiness. By contrast providing both a sweetness and a fruitiness scale might lead to a more narrow sweetness concept excluding fruitiness sensation. An implication of this interpretation is that flavour perception (i.e., the combination of taste and odour) should be analysable into its taste and odour components by individuals trained to focus their attention on these components but not by untrained consumers. In agreement with this hypothesis, Bingham et al. (1990) found that untrained participants rated a mixture of maltol and sucrose as sweeter than an equivalent concentration of sucrose alone but not participants who have been trained to use an analytical tasting strategy. Such effects of

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training, however, are not always found. Indeed, Stevenson (2001) and Stevenson and Case (2003) found no difference in the ability of trained and untrained participants to separate an odour-sucrose mixture into its components. In other words, as Stevenson (2001) put it, `enhancement effects, and thus confusion between odours and tastes, may be more cognitively impenetrable than has sometimes been assumed' (see Stevenson, 2001, p. 242). 4.3.2 Stimuli-driven factors A second explanation for the variability of the effects observed in Table 4.1 might be linked to the nature of the odorant and tastant mixtures. Indeed Frank and Byram (1988), for example, found sweetness to be enhanced by strawberry odour, but not by peanut butter odour. According to Stevenson et al. (1995; see also Stevenson et al., 1998) the degree of association between tastant and odorant is crucial in taste±odour interactions. More specifically, these authors suggest that an interaction will occur when the odour and the taste have previously been experienced conjointly. Other authors mention as a determining factor the nature of the relationship between tastant and odorant. The harmony between tastant and odorant was first studied by Murphy and Cain (1980) who failed to show an effect of this factor on taste±odour interaction. Their results reveal the same pattern of additivity for both a harmonious (citral and sucrose) and an inharmonious (citral and sodium chloride) mixture. However, this result has to be interpreted with some caution because the degree of `harmony' between tastant and odorant was not estimated by the participants but was based on assumptions from the authors. Later, Frank et al. (1991) asked subjects to evaluate the similarity of pairs of odorants and tastants. They found that the more similar tastant and odorant pairs yielded the stronger effect of odour on taste. Using a similar method, Schifferstein and Verlegh (1996) examined the effect of the congruency between taste and odour. They found a taste enhancement for congruent mixtures (sucrose-strawberry, sucrose-lemon) but not for incongruent mixtures (sucroseham). In agreement with this result, a single cell recording study (Rolls and Bayliss, 1994) indicates that some cells of monkey orbitofrontal cortex respond to both olfactory and gustatory stimuli but mostly for congruent stimuli such as glucose and fruity odours. However, Schifferstein and Verlegh reported that the degree of congruence was not linearly correlated with the amount of odourinduced taste enhancement. They suggested that `the congruency judgements are not effective in explaining the enhancement because they do not reflect the degree of association between two elements but they reflect how much somebody likes the combination of two elements' (see Schifferstein and Verlegh, 1996, p. 102). A better predictor might be the smelled taste of the odorants (e.g., the sweetness rating of a strawberry aroma) as proposed by Stevenson et al. (1999). Indeed these authors reported that the degree to which an odour smelled sweet predicted about 60% of odour-induced sweetness enhancement or suppression. In this study caramel, maracuja, strawberry and lychee were found to enhance sweet perception whereas angelica oil and damascene suppressed it.

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To evaluate if the different concepts used in the literature tapped the same aspect of the relationship between odorant and tastant, Nguyen (2000) asked four groups of participants to rate the harmony, the congruency, the similarity and the smelled taste of four mixtures. The four mixtures were chosen based on a free association task whose goal was to evaluate the cognitive association between odours and tastes (Sauvageot et al., 2000). Two of them were associated: sucrose-vanilla and acid-lemon. That is some subjects spontaneously answered acid when prompted with the word `lemon' and some sweet when prompted with the word `vanilla'. The two others were not associated. That is no subjects answered acid to vanilla or sweet to lemon. A fifth group of participants had to rate the sweetness or the sourness of the mixtures and two control mixtures made of pure sucrose or acid. Contrast analyses showed that, although the four rating scales were positively correlated, only smelled taste (40%), and similarity (29%), predicted the observed odourinduced taste enhancement. Moreover, a very high correlation was observed between similarity and smelled taste indicating that these two scales measure the same aspect of the odorant±tastant relationship, probably what Stevenson (2001) calls the confusion between odour and taste. The fact that harmony and congruence ratings did not predict taste enhancement is in agreement with previous data by Murphy and Cain (1980) and Schifferstein and Verlegh (1996). These aspects of the relationship between tastants and odorants might be more difficult to estimate and more prone to individual variations than smelled taste and similarity. Yet, the most surprising aspect of this study is that an odour-induced taste enhancement effect was observed for the acid-vanilla mixture. This mixture was considered as being incongruent, not harmonious and not similar. Furthermore, Sauvageot et al.'s (2000) study showed that no subjects associated the term `sour' with the term `vanilla' even in a forced choice association task in which the possible choices corresponded to the four fundamental tastes (sweet, salty, sour, bitter). Finally, the vanilla aroma was not perceived to smell `sour'. Thus, the positive effect of vanilla on sourness cannot be explained in terms of relationship, cognitive association, or perceptual similarity between odorant and tastant. This last result indicates rather that the similarity or confusability between odorant and tastant might not be a necessary condition for enhancement to occur. This last point will be discussed later in the chapter. 4.3.3 Subject-driven factors Additional constraints for the observation of an effect of odour on sweet taste seem to be subject-driven. We already saw that participants' degree of expertise might play an important role in the ability to separate or integrate olfactory and gustatory components of a mixture. Other factors affecting taste odour interactions include participants' culture and individual differences.

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Culture effect A first demonstration of the effect of culture on odour-induced sweetness enhancement resulted from an attempt at replicating Frank and Byram (1988) with French participants (Nguyen, 2000). In both Frank and Byram and Nguyen studies, participants had to rate the sweetness intensity of four whip cream samples containing respectively 0, 0.25, 0.60 and 1.20 M sucrose with and without the presence of a strawberry aroma. Whereas Frank and Byram observed a positive effect of strawberry on sweetness for all sucrose concentrations, Nguyen observed a positive effect only for the second concentration. Moreover, this effect was much smaller than that observed by Frank and Byram (2 vs. 6 points on a 20 points scale). This result can be explained in terms of food consumption habits and cognitive association between odorants and tastants. Whereas the association between strawberry and sweet is very frequent in American food (milk shake, ice cream, yogurts), it is less frequent in French food. This was confirmed in the free association task carried out by Sauvageot et al. (2000). For French subjects, strawberry is more often associated with a sour taste or a red colour than with a sweet taste as it is for American subjects. A link between food consumption habits and taste odour interaction was also reported in Nguyen et al. (2001). French and Vietnamese participants were presented with two sets of samples: a sweet set made of a sucrose solution, a sucrose-vanilla mixture and a sucrose-lemon mixture and a sour set made of an acid solution, a acid-vanilla mixture and a acid-lemon mixture. They were instructed to swallow the samples and to rate their sweet or sour intensity on a 10 cm unstructured line scale. An analysis of variance revealed a one-way interaction between type of mixtures and cultures. Pairwise comparisons showed that this interaction was mainly due to the sweet mixtures. Whereas for French subjects sweetness enhancement was higher in the presence of vanilla than of lemon, the opposite was observed for Vietnamese subjects. As for strawberry aroma, this cultural effect can be put in perspective with culinary association. Vanilla is very often used in France to flavour sweet dishes but not in Vietnam. In contrast, the association between lemon and sweet is less frequent in France than in Vietnam where lemon soft drinks are very popular. A study by van der Klaauw and Frank (1994) showed that in addition to cultural differences, individual differences might play a role in odour±taste interactions. Participants were given three solutions of sucrose (0.18, 0.30, 0.435 M), a mixture of 0.30 M sucrose and strawberry aroma, and a mixture of 0.30 M sucrose and coffee aroma. Their task was either to rate the sweet intensity of the five samples or to match each sample with a sucrose solution from a range of nine concentrations going from 0.13 to 0.55 M. While on the whole strawberry aroma and to a lesser degree coffee aroma enhanced the sweetness of sucrose solutions, participants clearly differed in their probability to show an enhancement. Interestingly, these individual differences were consistent across the two tasks. An individual showing an enhancement effect with the scaling method was likely to show the same effect with the matching task. The authors interpreted these individual differences as an indication that

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some participants are better at separating their perception into olfactory and gustatory components.

4.4

Mechanisms of taste±odour interactions

Despite the different factors that might intervene the odour-induced taste enhancement or suppression effect seems to be a robust effect. The question that naturally arises, however, is at which level does this effect occur? It is unlikely to occur at the receptor level. Since as already mentioned sweet taste enhancements have been observed even in situations in which the odorant and the tastant were presented separately (Sakai et al., 2001; Djordjevic et al., 2004), it is more probable that it occurs at the central processing level. However, the central mechanisms involved in this effect are still controversial and three main views seem to emerge from the literature. 4.4.1 Response bias For some authors such as Clark and Lawless (1994), odour-induced taste enhancement could be explained in terms of response biases. These authors base their argumentation on the fact that a taste enhancement is observed only when subjects are provided with a single scale (cf. sections 4.3.1 and 4.3.2). Clark and Lawless (1994) interpreted this phenomenon as a `dumping' effect. According to their interpretation, when a participant is not given an appropriate rating scale to express a sensation, he/she `dumps' this sensation onto the only available scale(s). This behaviour would `correspond to a general tendency to integrate all information to increase subjective confidence in the judgments' (see Clark and Lawless, 1994, p. 591). For example, when subjects are asked to evaluate the sweetness intensity of an aspartame solution to which a strawberry aroma was added, they `dump' the strawberry sensation onto the sweetness scale and thus a sweetness overestimation is observed. An opposite effect can also be observed when too many scales are used. For example Clark and Lawless (1994) observed that an aspartame solution without any added aroma was rated as less sweet when participants were asked to rate both the sweetness and the flavour intensity than when they were asked to rate only the sweetness intensity. This suppression effect could be due `to a tendency to use response category with equal frequency' (p. 592). Within this framework, it is unclear whether taste±odour interactions are a reliable perceptual effect or simply a measurement artefact. Indeed, intensity rating methods indicate that odours might increase sweetness ratings but does that imply that it increases sweetness perception? To answer this question a few authors examined taste±odour interactions to see if they can be shown with paradigms different from intensity ratings. Djordjevic et al. (2004) for example used a sweet taste detection task. Participants were given pairs of samples made of a weak sucrose solution and a blank solution. They were asked to sip each solution immediately after having

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sniffed or imagined a strawberry or a ham odour and to indicate which of the two solutions tasted stronger. In both perceived and imagined conditions, detection of sucrose was poorer when combined with a ham odour than with no odour. No difference was observed with strawberry odour. This result clearly shows that taste±odour interaction can be obtained with a paradigm free of the response biases described by Clark and Lawless (1994). Nguyen et al. (2000) proposed another bias-free method to measure the effect of vanillin on sucrose. This method, based on Garner (1974) filtering paradigm, evaluates if a categorization task performed on sweetness perception is affected by changes in vanilla aroma. Participants had to make categorization judgements according to the level of the perceived sweetness intensity of samples made of two concentrations of sucrose and two concentrations of vanillin. Results show that categorization performance was systematically lower when the concentration in vanillin changed in addition to the concentration of sucrose. This indicates that participants were unable to ignore the olfactory component when categorizing the solutions according to the gustatory component. Taken together the Djordjevic et al. (2004) and Nguyen et al. (2000) results suggest that taste±odour interactions cannot be explained solely in terms of response biases. 4.4.2 Learned synesthesia For other authors such as Stevenson et al. (1998), the effect of odour on sweet taste perception could be explained in terms of learned synesthesia. Synesthesia is defined as a systematic association between two sensations corresponding to two different sensory modalities. A classical example of synesthesia can be found in Cytowic (1993). For the patient described in this book, each different taste had a different feel. Mint for instance felt like `smooth, cool columns of glass'. Stevenson et al. (1998, see also Stevenson et al., 1995) showed that synesthesia can be learned through a combined exposure to two sensations. Especially, they showed that repeated pairing of a previously neutral odorant with sucrose results in an increased perceived sweetness of the odorant. Participants attended five sessions, one session of pre-test, three sessions of conditioning and a session of post-test. During pre-test, participants had to rate among other ratings (liking, overall intensity, sourness, familiarity) the sweetness of lychee and water chestnut odours. During each conditioning session participants performed six triangle tests among which one consisted of water chestnut (or lychee) paired with sucrose (conditioning trial) and one of lychee (or water chestnut) and water (control condition). The other triangle tests served as fillers. During the post-test, participants had to rate again the sweetness of lychee and water chestnut odours and to answer an awareness test on the same odours. Results indicated that sweetness perceived intensity increased between pre-and post-tests for the odour paired with sucrose (water chestnut or lychee) but not for that paired with water. This increase in perceived sweetness occurred independently of participants' awareness that the

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odour was paired with sucrose during the conditioning test and did not disappear after 24 h of delay. More recently, Prescott et al. (2004), using a similar paradigm, found that even a single co-exposure with sucrose was sufficient to enhance the perceived sweetness of a prune and a water chestnut odour. However they also found that a synthetic perceptual strategy during the co-exposure was necessary to observe a taste enhancement in the presence of the same odours. Indeed, participants instructed to evaluate the overall flavour of sweet-prune (or water chestnut) flavoured solutions during the conditioning phase showed an enhancement whereas participants instructed to evaluate separately the sweetness and the aroma of the same solutions did not. Prescott et al. (2004) concluded that `exposure alone is a necessary, but not sufficient, cause of sweetness enhancement effects' (see Prescott et al., 2004, p. 335). Both Stevenson et al. (1998) and Prescott et al. (2004) results emphasize the role of associative learning in odour-induced sweetness enhancement. However, can we really interpret this effect in terms of learned synesthesia as proposed by Stevenson et al. (1998)? Synesthesia is the involuntary physical experience of a cross-modal association. It is characterized by idiosyncratic, vivid and irrepressible perceptions and does not seem to rely on any form of learning (Cytowic and Wood, 1982). In contrast, taste±odour interactions seem to result from perceptual learning and to be culture-dependent rather than idiosyncratic. An interpretation in terms of central integration as proposed by Prescott et al. (2004) seems more appropriate. Consistent with this interpretation, White and Prescott (2002) showed that an odour can prime the cognitive system to expect a particular type of taste based on past flavour experiences. More specifically, reaction time to identify a taste in the presence of an orthonasally delivered odour decreases if the taste and the odour have been encountered conjointly in a previous conditioning phase. Likewise, White and Prescott (2001) found that participants identified faster a sweet taste in the presence of a congruent odour such as strawberry than in the presence of an incongruent odour such as grapefruit. 4.4.3 Central integration Integration between odour and taste could occur in a brain region called the orbitofrontal cortex (Rolls, 2004). This brain structure receives projections from the olfactory cortex and the gustatory cortex. As already mentioned, Rolls and Bayliss (1994) found in the orbitofrontal cortex of monkeys some neurons that respond to both olfactory and gustatory inputs. Among these bimodal neurons, some responded to odours and tastes that occur together in food. For example, a given neuron will respond preferentially to a sweet taste in a taste discrimination task and to a fruit odour in an odour discrimination task. Moreover, Rolls et al. (1996) showed that the response of some olfactory neurons in the monkey orbitofrontal cortex depends upon the taste with which the odour is associated. Rolls (1997) suggested that bimodal neurons would develop from unimodal

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neurons through associative learning of tastes and odours occurring naturally together in food. Imaging techniques have been used recently to assess the role of central brain structures in flavour perception by humans. Small et al. (1997) used positron emission tomography (PET) to compare cerebral blood flow (CBF) changes due to the processing of olfactory (e.g. strawberry), gustatory (e.g sucrose), and combined olfactory gustatory stimuli (e.g. strawberry, sucrose). They found that bimodal processing of taste and odours produces significant CBF decreases relative to unimodal processing of identical tastes and odours. This decrease in CBF can be related to suppression effects observed in psychophysical experiments. More recently de Araujo et al. (2003), using an event-related functional magnetic resonance imaging (fMRI) study, reported an activity in the orbitofrontal cortex when a tastant (sucrose or monosodium glutamate) and a retronasally delivered odorant (strawberry or chicken-like) were presented simultaneously. This activity was greater than the sum of the activities obtained when tastant or odorant were presented separately, thus indicating that an interaction occurred. A similar finding was also reported by Small et al. (2004) thus indicating that orthonasal and. retronasal olfaction might interact differently with taste. Indeed, Small et al. (1997) found a taste suppression effect with orthonasally delivered odours. This differential effect of orthonasal and retronasal olfaction on taste was not, however, always found in psychophysical studies (Sakai et al., 2001). Small et al. (2004) also showed that superadditive responses were observed only when the taste±odour pair was congruent (sucrose-vanillin as opposed to NaCl-vanillin). Such patterns of superadditivity seem to be the physiological correlate of psychophysical data by Dalton et al. (2000). These authors found that participants could detect the presence of benzaldhyde at 30% below its detection threshold when the odorant was combined with sodium saccharin at 30% below its detection threshold. In a second study, Breslin et al. (2001) failed to find a superadditivity effect when benzaldehyde was presented in combination with monosodium glutamate. 4.4.4 Towards a dual model The literature reviewed so far indicates that two different cognitive mechanisms might be involved in taste±odour interactions. The first one would not require any previous association between odorants and tastants and would give rise to small amplitude effects. It would correspond to a general tendency to integrate all information into a total intensity impression (dumping effect) and thus would be sensitive to the number of rating scales. In contrast, because it acts more like a response bias than a perceptual enhancement, it would not be sensitive to subjects' history and thus would not be mixture dependent. The second one would correspond to a real perceptual enhancement and give rise to high amplitude effects. It would result from the co-encoding of an odorant and a tastant and thus would be mixture and culture dependent (Nguyen, 2000).

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Fig. 4.2 Changes in sour perception (sourness rating for the mixture minus sourness rating for citric acid alone) for vanilla citric acid and lemon citric acid mixtures under two rating conditions (adapted from Valentin and Nguyen, 2001).

To test this dual mechanism hypothesis, Valentin and Nguyen (2001) evaluated the effect of lemon and vanilla aroma on sourness ratings. The experiment consisted of two intensity tasks. In the first intensity task (1 question condition) subjects had to indicate the sourness of an acid solution, a vanillaacid solution and a lemon-acid solution. In the second intensity task (3 questions condition) subjects were given the same solutions but were instructed to rate the sour, vanilla and lemon intensity of the mixtures on three different scales. As illustrated in Fig. 4.2, student t-tests revealed a significant odour-induced taste enhancement for both vanilla and lemon in the 1 question condition with a higher amplitude effect for lemon than for vanilla. In the 3 questions condition, the effect of vanilla on sourness was not significant whereas the effect of lemon is significant. This result indicates that providing several scales to subjects does not have the same effect on odour-induced taste enhancement depending on the association between tastants and odorants. Subjects seem to be able to separate taste and odour sensations only for taste±odour mixtures in which the components are not cognitively associated, similar, harmonious, congruent, and familiar (e.g. acid-vanilla). For other mixtures (e.g. acid-lemon), subjects seem to be able to separate only partially their sensations: providing several scales decreases the odour-induced taste enhancement effect but does not suppress it.

4.5

Implications for food product development

Odour-induced sweet taste enhancement or suppression appear to be a robust effect albeit not a strong one. Indeed, while odour and taste sensation in the mouth can, to some extent, be analysed, sensory integration might be more frequent in natural eating conditions. This is reflected by the spontaneous use of the term `sweet' to describe food related odours (Dravnieks, 1985). The question thus is can we use this natural tendency to integrate sensations to enhance the sweetness of a food product via its aromas? The answer is yes, but the result will depend on several factors. First, all odours will not be equally useful to enhance

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sweet taste. Odours confusable with sweet taste because they have been experienced often in conjunction with a sweet taste are the best candidates to enhance sweet taste. Second, the ability of an odour to enhance sweet taste does not seem to be universal: an efficient sweet taste enhancer in a given culture might not be as useful in another one. Finally interactions between sweet taste and odours might vary as a function of individual sensitivity and strategy in food tasting. Individuals trained to analyse their sensations might be less prone to taste odour interactions than other individuals. Taste odour interactions also have broad implications for descriptive analyses in which multiple attributes of complex foods are rated. For some time scientists in sensory evaluation thought that panellists could be trained to separate their sensations. The data presented here show that this is not always the case. For example, it is quite possible that even for a trained panellist the sweetness produced by a fruity aroma cannot be separated from the sweetness produced by sucrose. This raises the question of what do we want to measure when we ask panellists to rate the sweetness of a food product? The overall sweetness of the product or the sweetness yielded by the taste component? According to Prescott (1999), `this distinction might be artificial [. . .] since the sweetness of the flavour components may be represented cognitively, and even perhaps neurally, as equivalent. They may be functionally equivalent as well' (Prescott, 1999, p. 355). In other words, in some cases, such as understanding consumer preferences, it might be preferable to measure the overall perceived sweetness of a food product. In other cases, where it is necessary to estimate only the sweetness produced by the taste component of a food, the rating scales provided to the panellists should be chosen with care to help them separate their sensations. Indeed instructing a panellist to focus only on taste perception will not be enough to avoid odour-induced taste enhancement.

4.6

Future trends

One potentially interesting line of research will be to further examine the effect of the degree of cultural experience with particular combinations of tastants and odorants in odour-induced sweet taste enhancement. Cultural factors are one of the most powerful determinants of which food we consume. Nevertheless, there has been surprisingly little research on how perception of, and preference for food might vary across cultures. To understand odour-induced sweet enhancement as a function of cultural experience may provide some new insights in the understanding of cultural differences in preferences for sweet foods. A second line of research will be to extend the study of odour±taste interactions to multiple interactions in sweet taste perception. It is well known that when eating food, sensations such as somatosensory, visual or auditory sensations influence food perception and preference (Delwiche, 2004). This may be particularly relevant also from a neuroanatomical point of view to evaluate how the different sensory inputs interact with each other and how they are

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integrated in the brain. In this perspective, imaging techniques will be very useful. Finally, focus should be given to a more general consequence of sweet taste enhancement: the improvement of palatability and the compensation for chemosensory losses in populations such as the elderly. Recently, it has been demonstrated that the addition of a flavour enhancer can improve a number of nutritional and immune parameters such as increasing the number of lymphocyte cells or increasing secretion rate of specific antibodies (Schiffman, 2002). As flavour enhancement can be achieved in several ways, in addition to further investigations into the contribution of neural integration of sensory inputs, it is crucial to examine also other sources of sweet taste enhancement such as modification of receptor mechanisms.

4.7

Sources of further information and advice

4.7.1 Reviews A few reviews are of interest to acquire further information and advice on taste±odour interactions. Prescott (1999) provides a broad body of literature on taste±odour interactions, and also raises the question of the different origins of odour±taste interactions and provides evidence that taste enhancement is a psychological construct. Frank (2002), based on a large number of studies from his laboratory, proposes a model that attempts to characterize taste±odour interactions when making judgements about the sensory magnitude of complex chemosensory stimuli. Recommendations are furthermore made based on studies of response context and flavour research. Delwiche (2004) provides a large scope of investigations on flavour perception. This paper reviews studies that look at the impact of different sensory cues on the perception of taste, odour and taste±odour mixtures, as well as the impact of certain physical interactions on these perceptions such as influence of irritation, temperature, colour, texture and sound. Readers interested in neuroanatomical issues of odour±taste interactions will find some thorough information in Rolls (1999). 4.7.2 Current research and interest groups The Monell Chemical Senses Center in Philadelphia (USA) and the Sensory Science Research Centre at the University of Otago (New Zealand) are the main references in terms of research groups investigating taste±odour interactions.

4.8

References

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and PERRING K D (1990), `Sensory studies with sucrose-maltol mixtures', Chem Senses, 15, 447±456. BRESLIN, P A, DOOLITTLE H and DALTON P (2001), `Subthreshold integration of taste and smell: The role of experience in flavour integration', Chem Senses, 26, 1035. CLARK C C and LAWLESS H T (1994), `Limiting response alternative in time-intensity scaling: an examination of the halo-dumping effect', Chem Senses, 19, 538±594. CLIFF M and NOBLE A C (1990), `Time-intensity evaluation of sweetness and fruitiness and their interaction in a model solution', J Food Sci, 55, 450±454. CYTOWIC R E (1993) The Man Who Tasted Shapes: A Bizarre Medical Mystery Offers Revolutionary Insights into Reasoning, Emotion, and Consciousness. New York: Putnam. CYTOWIC R E and WOOD F B (1982) `Synesthesia I: A review of major theories and their brain basis'. Brain and Cognition, 1, 23±35. DALTON P, DOOLITTLE N, NAGATA H and BRESLIN P A (2000), `The merging of the senses: integration of subthreshold taste and smell', Nature Neurosci, 3, 431±432. DE ARAUJO I E, ROLLS E T, KRINGELBACH M L, MCGLONE F and PHILLIPS N (2003), `Tasteolfactory convergence, and the representation of the pleasantness of flavour, in the human brain', Eur J Neurosci, 18, 2059±2068. DELWICHE J (2004), `The impact of perceptual interactions on perceived flavour', Food Qual Pref, 15, 137±146. DJORDJEVIC J, ZATORRE R J and JONES-GOTMAN M (2004), `Effects of perceived and imagined odors on taste detection', Chem Senses, 29, 199±208. DRAVNIEKS A (1985), Atlas of Odor Character Profiles, Philadelphia: ASTM. FRANK R A (2002), `Response context affects judgments of flavour components in foods and beverages', Food Qual Pref, 14, 139±145. FRANK R A and BYRAM J (1988), `Taste-smell interactions are tastant and odorant dependent', Chem Senses, 13, 445±455. FRANK R A, DUCHENY K and MIZE S J S (1989), `Strawberry odor, but not red color, enhances the sweetness of sucrose solutions', Chem Senses, 14, 371±377. FRANK R A, WESSEL N and SHAFFER G (1990), `The enhancement of sweetness by strawberry odor is instruction-dependent', Chem Senses, 15, 576±577. FRANK R A, SHAFFER G and SMITH D V (1991), `Taste±odor similarities predict taste enhancement and suppression in taste±odor mixture', Chem Senses, 16, 523. FRANK R A, VAN DER KLAAUW N J and SCHIFFERSTEIN H N J (1993), `Both perceptual and conceptual factors influence taste±odor and taste±taste interactions', Percept Psychophys, 54, 343±354. GARNER W R (1974) The Processing of Information and Structure. Potomac, MD: Lawrence Erlbaum Associates. LAVIN J G and LAWLESS H T (1998) `Effect of color and odor on judgments of sweetness among children and adults', Food Qual Pref, 9, 283±289. LAWLESS H and SCHLEGEL M P (1984), `Direct and indirect scaling of sensory differences in simple taste and odor mixtures', J Food Sci, 49, 44±51. MURPHY C and CAIN W C (1980), `Taste and olfaction: independence vs. interaction', Physiol Behav, 24, 601±605. MURPHY C, CAIN W S and BARTOSHUK L M (1977), `Mutual action of taste and olfaction', Sensory Processes, 1, 204±211. Á l'eÂtude des interactions entre les entreÂes sensorielles: NGUYEN H D (2000), Contribution a Effets d'une odeur sur la perception d'une saveur, TheÁse de Doctorat de l'Universite de Bourgogne, Ensbana, Dijon. BINGHAM A F, BIRCH G G, DE GRAAF C, BEHAN J M

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la psychologie cognitive', Proc Colloque Alliance 7, Cedus, Paris 2002. and FRANK R A (1993), `Taste±smell interactions with multiple sweeteners', Chem Senses, 18, 580. VAN DER KLAAUW N J and FRANK R A (1994), `Matching and scaling of taste±smell mixtures: Individual differences in sweetness enhancement by strawberry odor', Chem Senses, 19, 567±568. VAN DER KLAAUW N J and FRANK R A (1996), `Scaling component intensity of complex stimuli: The influence of response alternatives', Environment International, 22, 21±31. WHITE T and PRESCOTT J (2001), `Odors influence speed of taste naming', Poster presented at the XXIII AChems meeting, Sarasota FL, 23±25 April. WHITE T and PRESCOTT J (2002), `Learned associations with smells influence taste naming speed', Poster presented at the European Chemoreception Research Organisation conference. Erlangen, Germany, 23±27 July. VAN DER KLAAUW N J

5 Taste±ingredient interactions modulating sweetness M. Lindley, Lindley Consulting, UK

5.1

Introduction

In mixtures of substances with different taste qualities, the individual components can still be recognised but are usually perceived as less potent than when unmixed. This phenomenon is known as `mixture suppression' (Bartoshuk, 1975) and is normally observed when compounds of differing taste qualities are mixed together. Sensory interactions between sweet tasting compounds, and between sweeteners and compounds of different taste qualities, influence the overall taste quality delivered in many food and beverage products, as well as potentially having important commercial implications. From practical standpoints, interactions between sweet tasting compounds are particularly relevant due to the sometime occurrence of sweetness synergy. Understanding the interactions between sweet tasting compounds and acidulants is also important because of the use of sweeteners and acids in combination in many food and beverage products. Sweet and bitter compounds are found together in some foods and beverages, so it is also appropriate to develop an understanding of these sensory interactions. Additionally, many high potency sweeteners deliver bitterness in addition to sweetness, thus confirming the need to understand how these sensations interact. In contrast, the use of sweet and salty tasting compounds together is not a widespread practice in foods and beverages, although inorganic salts can exert significant influences over the sensory delivery of many sweet compounds. Interactions between ingredients that elicit non-sweet taste sensations with sweet tasting compounds are important influences on overall taste and product

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quality. In addition, however, as has been mentioned, the taste profiles delivered by sweeteners, particularly high potency sweeteners, are often complex mixtures of tastes. High potency sweeteners frequently elicit taste sensations additional to sweetness, with associated bitter tastes being particularly prevalent. Additionally, cooling aftertaste sensations, coupled with liquorice notes, are frequent complications of high potency sweeteners. Ingredient interactions that result in these taste sensations being reduced in their intensity have been widely studied, not least because of the important commercial implications of success.

5.2

Interactions between sweeteners and bitter compounds

Many food and beverage products are both sweet and bitter. Important examples include chocolate confectionery and coffee products, both of which contain intrinsic bitterness that is balanced by added sweetness. The ability of sucrose to balance the bitterness of cocoa and of the bitterness of cocoa to reduce the perceived sweetness of sucrose, both reductions being a consequence of mixture suppression, is arguably the most important market example of this sensory phenomenon. 5.2.1 Mixture suppression of sweet and bitter compounds Mixture suppression effects between sweet and bitter tasting compounds are reliable; mutual suppression of sweetness and bitterness always occurs. There is clear evidence that these mixture suppression effects are not due to any chemical interactions in solution or competition of molecules for common receptor sites, but that the effects are peripherally mediated (Lawless, 1979, 1982). In the first of these studies (Lawless, 1979), removal of sweetness from bitter-sweet mixtures, either by adapting to sucrose or pre-treatment of the tongue with an extract solution of Gymnema sylvestra, caused the perceived intensity of bitterness to increase. Since neither adaptation nor sweetness inhibition by Gymnema affected the concentration of sucrose on the tongue, it was concluded that mixture suppression must be due to neural inhibition. As part of the same study, Lawless also showed that similar dependence of suppression was observed with mixtures of phenylthiocarbamide (PTC) and sucrose in which those assessors that perceived PTC as bitter exhibited stronger suppression of sweetness than assessors that were insensitive to the bitterness of PTC. It was concluded that these observations were also inconsistent with molecular interactions mediating suppression, further supporting the neural inhibition conclusion. The second study by Lawless (1982) used a dorsal flow technique to control the exposure of the tongue to quinine, sucrose and a quinine-sucrose mixture. Previously documented effects of adaptation, mixture suppression and release from suppression were observed, as anticipated. In addition, reliable residual bitter or sweet taste was measured following adaptation to quinine-sucrose and presentation of equi-molar unmixed quinine or equi-molar unmixed sucrose.

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This residual taste was significantly greater than following self-adaptation of the unmixed solutions. It was concluded that these observations suggest a peripheral mechanism is responsible for the suppression between sweet and bitter tastes in a mixture. In contrast, a subsequent study (Kroeze and Bartoshuk, 1985), that also confirmed bitterness suppression in mixtures of quinine hydrochloride and sucrose, concluded that bitterness suppression of quinine hydrochloride in mixtures with sucrose occurs centrally. However, irrespective of the mechanism of mutual mixture suppression of bitterness and sweetness, it is a reliable phenomenon. There are individual differences in sensitivity to these mixture suppression sensory effects that are largely related to the ability of each individual to taste 6-n-propylthiouracil (PROP) or PTC as bitter. It has been demonstrated unequivocally that individuals vary in the extent to which they taste both phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP) as bitter (Fox, 1932; Bartoshuk, et al., 1994). For many years, it was assumed that there are two population sub-groups; those that taste PTC/PROP as intensely bitter at a given concentration and those that taste the same concentration as very slightly bitter or tasteless. Now, however, it is acknowledged that there are in fact three subgroups, non-tasters, medium tasters and super-tasters (Bartoshuk et al., 1992). Notwithstanding this evolution in the classification of PTC/PROP tasters, some studies have shown that there are clear differences in the suppressive effect of quinine hydrochloride on sucrose that appear to be dependent on an individual assessor's classification as super-taster, medium-taster or non-taster of PTC/ PROP. In one comprehensive study, Prescott et al. (2001) demonstrated that quinine suppresses the sweetness of sucrose, but primarily only for super-tasters and medium-tasters. These observations are consistent with the fact that supertasters and medium-tasters rated quinine hydrochloride as more intensely bitter than did non-tasters and so an important conclusion from these studies is that genetic differences in taste sensitivity may influence the perception of taste qualities differently in complex, formulated foods and beverages. If so, a PTC/ PROP super-taster might be more likely than a non-taster to perceive the bitterness of cocoa in chocolate or bitterness in fruit juices, for example, and these groups might therefore be likely to exhibit marked differences in their ratings of product acceptability. In food and beverage companies where product quality and acceptability are not judged in formal sensory assessment by a taste panel, but by key technical or marketing personnel, the potential dangers are obvious. However, other studies have failed to confirm these effects with quinine. For example, Mela (1989) and Schifferstein and Frijters (1991) both failed to demonstrate a relationship between sensitivity to PROP and the perception of quinine hydrochloride taste. Therefore, as has been noted by Mattes (1994), the PROP sensitivity/food dislikes relationships reported for particular foods have tended to be inconclusive, possibly symptomatic of the complexity of assessing food acceptability.

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5.2.2 Bitterness reduction in sweet/bitter high potency sweeteners It has been established that there are clear qualitative differences among sweeteners, principally among the high potency sweeteners, and that these are frequently expressed as being due to bitter or metallic tastes (Schiffman et al., 1979). The high potency sweeteners saccharin, acesulfame-K, stevioside and rebaudioside A are specific examples of sweeteners that also elicit slight bitterness. From a practical standpoint, routes to the amelioration of the bitter aftertaste of saccharin have been the focus of much attention, mainly because during the whole of the 1970s, saccharin was the only permitted high potency sweetener in the USA. A number of ingredient additives have been evaluated for their bitterness reducing characteristics when formulated with saccharin. Specifically, glucono-lactone, sodium gluconate and cream of tartar (potassium bitartrate) have been proposed by Eisenstadt (1972) as a route to bitterness elimination from saccharin. This formulation must be considered to have been a success since it has formed the basis of the successful `Sweet 'n' Low' table-top sweetener product that is based on the sweetener saccharin. Another interesting approach to the elimination of the bitter aftertaste of saccharin was described by Pampiano (1980). It was claimed that when the aqueous extract of Gentiana lutea is added at low levels to saccharin, the bitter aftertaste conventionally associated with saccharin is substantially reduced or eliminated and that higher levels of saccharin may be employed to produce higher sweetness levels than are normally used. This approach is particularly interesting because G. lutea has been utilised in extract form as a tonic and is itself bitter. Gentiopicrin, the active component responsible for the bitter taste of gentian, is present in G. lutea and yet the extract does act to prevent the bitter aftertaste conventionally associated with saccharin itself. In other words, it may be functioning through some form of competitive binding at the receptor for saccharin bitter taste on the tongue. Similar observations were made by Wong et al. (1992) who disclosed that the sweet taste quality of acesulfame-K in an acidic environment may be improved and enhanced by the addition of potassium chloride. They propose that potassium chloride should be added in an amount approximately equal to that of the acesulfame-K. Again, given the intrinsic bitter taste of potassium chloride, the mechanism whereby the bitterness of acesulfame-K is reduced may be due to competitive binding at a bitter receptor site on the tongue. Other ingredients that have been reported (Lindley, 1999) to improve that sensory delivery of saccharin through amelioration of its bitter aftertaste include calcium chloride, arabinogalactan, D-tryptophan and neodeosmin.

5.3

Interactions between sweeteners and acids

Foods and beverages are formulated across a broad range of pH levels and intensities of perceived acidity. The pH range extends from approximately pH

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2.5 to around neutrality and perceived acidities vary from the mild acid taste intensity of fermented milk products such as yoghurt through to the intense acidity of some fruit based hard-boiled confectionery products and some soft drinks. In many food products, sucrose and other carbohydrate sweeteners are used for a variety of reasons in addition to sweetness delivery, including the provision of bulk and the development of texture. Therefore, carbohydrate sweeteners tend to be used at concentrations sufficient to deliver the necessary bulk and texture and not for reasons of sweetness delivery having achieved a particular level. As a result, any sweetness modifying impact of acids on carbohydrate sweeteners is of practical relevance only in that the amount of acid added should be sufficient to achieve the desired sweetness-acidity balance. In contrast, high potency sweeteners are mono-functional ingredients, contributing sweetness alone to the foods and beverages in which they are formulated. In these circumstances, potentiation or inhibition of high potency sweetener derived sweetness by acids can have important commercial implications, particularly since most applications for high potency sweeteners are in media that are acidified and their main use is in beverages that have pronounced acid tastes. 5.3.1 Carbohydrate sweetener±acid interactions Conventional concepts of mixture suppression suggest that an acid taste should suppress the sweetness delivery of a sweet substance. This certainly seems to be the case when sucrose is mixed with acids, as was first described by Pangborn (1961). McBride and Finlay (1990) examined the effects of citric acid on sucrose and also found a suppression of sweetness. Similarly, Schifferstein and Frijters (1990), using the `equi-ratio' method in which the mixing ratio is held constant across a series of solutions, reported that citric acid has a slight suppressive effect on the sweetness of sucrose, and that the suppressive effect of sucrose on the acid taste intensity of citric acid is more marked. Prescott et al. (2001) also showed that the sweetness of sucrose was suppressed by increasing concentrations of citric acid and that increasing concentrations of sucrose suppressed the sourness of citric acid in a progressive fashion. Thus, these and other publications all agree that mixtures of sucrose and citric acid mutually suppress the taste intensities delivered by each component. Of relevance from a practical standpoint, it has been reported that the carbonation of sweetener solutions also affects the perceived intensity of sweetness. Carbonation, which of course releases carbonic acid into beverages, apparently suppresses sucrose sweetness at practical use levels, but, paradoxically, enhances sweetness at concentrations approaching threshold (Odake, 1996). The influence of acidity on the sweetness of fructose is a special case complicated by the fact that fructose in solution is present as a mixture of anomeric forms; -pyranose, -furanose and its keto-hexose form. Of these anomeric conformations, only the -pyranose anomer is believed to be sweet. As the pH of a solution of fructose is reduced, the equilibrium of fructose

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anomers is shifted in favour of the sweet -pyranose form, thus resulting in sweetness being enhanced (Osberger and Linn, 1978). It is for this reason that the sweetness of a fructose solution is at a maximum at cold temperatures and low pH. 5.3.2 High potency sweetener±acid interactions There is very little published literature on the influence of food acids on the sweet taste delivered by high potency sweeteners and much of what has been published is difficult to interpret due to the use of different sensory methods and the failure to quantify sweetness in terms of sucrose equivalence. This is illustrated by early work with aspartame that indicates equivalent potencies at pH 3.2 and in water (Cloninger and Baldwin, 1974). However, there is more reliable evidence that aspartame tastes sweeter relative to sucrose in an acid solution compared to a water solution (Fry, 1993) and there are other indications from the literature that acids may enhance sweetness (Lotz and Meyer, 1994). While it is not clear whether it is the sweetness of aspartame that is actually enhanced or the sweetness of the sucrose reference that is reduced, the commercial consequences are that less sweetener is required than would be expected by examination of sucrose equivalence values in water. Most of the few studies that have been conducted have examined sweetener performance in citric acid buffers. However, since the food acids exhibit different sensory profiles, and indeed are often marketed on that basis, it would be surprising if all food acids interacted in the same way with all high potency sweeteners. Unfortunately, there have been no systematic studies to examine possible differences in sensory effects, although Pettigrew and Silka (1988) did conclude that the sweetness of aspartame is enhanced more by phosphoric acid than it is by citric acid. What is perhaps surprising, given the commercial importance of this observation, is that it is not more widely appreciated within the industry and that the findings have not been judged sufficiently important to stimulate further study in to the phenomenon. As has been mentioned, different food acids exhibit different sensory properties, particularly with respect to their temporal properties. For example, malic acid is marketed as delivering a longer-lasting acid taste relative to the acid taste of citric acid. This difference is claimed to make malic acid more ideally suited for use with high potency sweeteners, particularly with those sweeteners that deliver longer-lasting sweet aftertastes. However, no systematic study appears to have been conducted to develop objective sensory data to underpin these claims and there are also no data examining the impact of acids on blended sweetener performance, but since blends tend to be used in the majority of cases today, this apparent oversight seems particularly surprising. The ability of different acids to modify the temporal properties of specific high potency sweeteners has been examined in individual cases with some potentially important conclusions. For example, Shamil (1998) identified tannic acid compounds as additives that have the capability to reduce the lingering

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sweet aftertaste of sucralose. Data were presented in which a cola beverage sweetened with high fructose corn syrup was compared with one sweetened with sucralose and another sweetened with sucralose plus a small concentration of tannic acid. Sensory assessors were required to rate the intensity of sweetness, sourness, bitterness and sweet aftertaste duration, concluding that only sweet aftertaste duration was affected by the tannic acid. A statistically significant reduction in sweet aftertaste duration was observed. However, although there were no statistically significant differences in the measured intensities of the other attributes, it is interesting that the scores of the tannic acid containing samples for both sourness and bitterness were higher than those for sucralose alone, thus indicating that although tannic acid may indeed reduce the lingering sweetness of sucralose, it may introduce further sensory complications. In fact, it seems likely that tannic acid is reducing the sweet aftertaste of sucralose by taking advantage of the `mixture suppression' sensory phenomenon already discussed. The other high potency sweetener whose temporal profile has been a subject of some scientific study is neotame. Prakash et al. (2001) reported a number of formulation approaches to reducing the lingering sweet aftertaste of neotame, including the use of hydrophobic organic acids (such as cinnamate) or hydroxyamino acids (such as serine and tyrosine). In this study, samples were evaluated at room temperature and panellists trained on sucrose solutions such that a rating of 10 for maximum sweetness intensity equates to a 10% sucrose solution. Starting as soon as each solution was taken into the mouth, perceived sweetness was recorded by moving a cursor along a 15 cm line scale. A number of timeintensity parameters were extracted from the recordings, including maximum sweetness intensity, area under the curve, rising area under the curve, falling area under the curve, time to maximum sweetness and duration of sweetness. Cinnamic acid was found to reduce the duration of sweetness of neotame, as measured by the `duration' and `falling area under the curve' parameters (Table 5.1) and was shown to produce a statistically significant 17% reduction in sweetness duration. The authors conclude it is unlikely that these effects were as a consequence of acid Table 5.1 Temporal profile data for colas sweetened with neotame and various levels of cinnamic acid (Prakash et al., 2001) Parameter

Maximum intensity Area under curve Rising area under curve Falling area under curve Time to maximum intensity (sec) Duration (sec)

Control (Neotame)

Neotame + 10 ppm cinnamic acid

8.56 236 56 180 10.8 59.3

* Statistically significant difference from control (p < 0:05)

8.55 209 55 154 10.8 49.2*

Neotame + 25 ppm cinnamic acid 8.78 221 55 165 10.6 50.3*

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taste delivery from cinnamic acid since further work demonstrated that it is more likely the effects are due to cinnamic acid acting competitively at the receptor than by direct interaction with the sweetener in solution. Since cinnamic acid has also been shown to exhibit other taste modifying characteristics as a bitter taste inhibitor, it seems likely that the effects are due to general taste modification rather than sweetener±acid interactions.

5.4

Interactions of sweet and salty compounds

As has been noted, there are few important examples of foods and beverages that deliver both sweet and salt taste sensations; some savoury soups and sauces, notably those based on tomato, constitute some of the products that are both sweet and salty. Other examples can be found in populations that occasionally sprinkle salt on fruit. Thus, the interactions between these two taste sensations have not been widely examined, although the aforementioned mixture suppression effects have also been reported in mixtures of sucrose and sodium chloride (Bartoshuk, 1975). Other reports suggest that some salts at specific uselevels can cause enhancement of sweet taste (van der Heijden et al., 1983), with potassium acetate, potassium chloride and sodium chloride all claimed to enhance sweetness at specific concentrations (Table 5.2; Birch, 1999). Table 5.2

Sweetness enhancing salts (Birch, 1999)

Salt Potassium acetate Potassium chloride Sodium chloride Potassium carbonate Magnesium acetate

Dosage required for significant enhancement (mg/l) 700 450 300 450 700

It has been reported that the flavour quality of tomato products can be influenced by sweetness. Rosett et al. (1997), working in formulated tomato soups, demonstrated that salt taste intensity correlated positively with tomato flavour and that salt taste was suppressed in versions formulated using milk, concluding that salt taste was probably masked by the lactose-derived sweetness from the milk.

5.5 Interactions of sweet compounds with other sweet compounds Sweetness synergy is said to occur when a mixture of two sweeteners is perceived as being sweeter than would be expected based on the sweetness of the

Taste±ingredient interactions modulating sweetness Table 5.3 1996)

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Synergistic and non-synergistic pairs of high potency sweeteners (Birch,

Sweetener pairs exhibiting strong synergy Sweetener pairs exhibiting little/no synergy Aspartame ± Acesulfame-K Aspartame ± Saccharin Cyclamate ± Aspartame Saccharin ± Cyclamate Acesulfame-K ± Cyclamate

Saccharin ± Acesulfame-K Sucralose ± Saccharin Sucralose ± Acesulfame-K Sucralose ± Aspartame Sucralose ± Cyclamate

individual sweeteners. The sweetness synergy sensory phenomenon is discussed elsewhere in this volume. However, some sweetener±sweetener interactions are also reviewed in this chapter; partly for completeness, but also because of the taste quality benefits that can also be a consequence of blending sweeteners. This issue, in particular, has important implications for food product development. The mechanism whereby some pairs of sweeteners deliver higher sweetness intensities than predicted based on their individual perceived intensities is not known with certainty (Birch, 1996). However, the mechanism of synergy is not particularly important in practical terms; what is important are the commercial benefits of lower sweetener concentrations and hence lower costs. The most obvious practical benefit of sweetness synergy today is that between aspartame and acesulfame-K resulting in substantially lower costs of sweetness than would be the case if using aspartame alone. Some synergistic pairs of sweeteners and non-synergistic pairs of sweeteners are listed in Table 5.3. Examination of the sweetener pairs in this table suggests that sweetness synergy may be little more than a consequence of release from the effects of mixture suppression. A sweetener such as aspartame that is effective at reducing the perception of bitterness synergises with those sweeteners that also elicit a clear bitter taste, such as saccharin and acesulfame-K. Aspartame may simply suppress the bitterness of saccharin or acesulfame-K, thus allowing intrinsic saccharin/ acesulfame-K sweetness previously masked by their inherent bitterness to be released and perceived. While this is a plausible explanation for synergy, what is of practical consequence is the potential that must therefore exist for improvements in taste quality to be a result of such interactions between sweeteners. These improvements may be further enhanced because of the inevitable consequence of using lower concentrations of individual sweeteners in blends than when each might be used as the sole sweetener. Thus, there is a reduced potential for the side-tastes or after-tastes that are an intrinsic part of the taste delivered by high potency sweeteners to be perceived. There has been little academic study of the impact of sweetener blending on taste quality, but examination of market practices demonstrates clearly that industry has adopted the principle with enthusiasm. Products containing three

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and four high potency sweeteners blended together are commonly seen on supermarket shelves. As noted by Shamil (1997), `if you blend many different sweeteners (sometimes up to six or seven), you really do create a very sugar-like taste'. In effect, the greater the number of sweeteners used in a blend, the closer will be the approach to the taste of sucrose.

5.6

Future trends

There is a dearth of quantitative information on the impact of different acids on the performance, both quantitative and qualitative, of different high potency sweeteners and their blends. It would be very helpful to the users of high potency sweeteners to have such information, particularly if the information was relevant to their products. There may be potential for ingredient cost saving through the identification of specific enhancements, and also for improving overall taste quality, possibly by optimising the temporal delivery of sweetness and acidity. Thus, it would be a valuable addition to the literature to complete fully comprehensive quantitative and qualitative studies of sweetener±acid interactions. The experience that has been gained in recent years on blending high potency sweeteners in foods and beverages indicates that this trend will almost certainly continue. All current mainstream high potency sweeteners are synthetic chemicals and so do not offer the marketing benefit of `natural' label declarations. Thus, there is no significant marketing disadvantage of blending sweeteners in products. With the development of a quality natural sweetener, that situation might change, but the immediate approval of such a sweetener is not anticipated. The practical observations are that blending leads to taste quality benefits, and although it would be interesting to see published confirmation of this, even without that endorsement, the practice will continue.

5.7

References

(1975) `Taste mixtures: is mixture suppression related to compression?', Physiol. Behav., 14, 643±649. BARTOSHUK, L.M., FAST, K., KARRER, T.A., MARINO, S, PRICE, R.A. and REED, D.A. (1992) `PROP supertasters and the perception of sweetness and bitterness', Chem. Senses, 17, 594±601. BARTOSHUK, L.M., DUFFY, V.B. and MILLER, I.J. (1994) `PTC/PROP tasting: anatomy, psychophysics and sex effects', Physiol. Behav., 56, 1165±1171. BIRCH, G.G. (1996) `Towards an improved understanding of sweetener synergy', Trends Fd Sci. Technol, 7, 403±407. BIRCH, G.G. (1999) `Modulation of sweet taste', BioFactors, 9, 73±80. CLONINGER, M.R. and BALDWIN, R.E. (1974) `L-Aspartyl-L-phenylalanine methyl ester (aspartame) as a sweetener', J. Fd Sci., 39, 347±351. EISENSTADT, M.E. (1972) `Cyclamate-free calorie-free sweetener', US Patent 3,647,483. BARTOSHUK, L.M.

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(1932) `The relation between chemical constitution and taste', Proc. Nat. Acad. Sci. USA, 18, 115±120. FRY, J.C. (1993) `Relative sweetness of intense sweeteners ± guidelines for the technologist', Food Technol. Europe, Sept./Nov., 114±116. KROEZE, J.H.A. and BARTOSHUK, L.M. (1985) `Bitterness suppression as revealed by splittongue taste stimulation in humans', Physiol. Behav., 35, 779±783. LAWLESS, H.T. (1979) `Evidence for neural inhibition in bittersweet taste mixtures', J. Comp. Physiol. Psychol., 93, 538±547. LAWLESS, H.T. (1982) `Paradoxical adaptation to taste mixtures', Physiol. Behav., 29, 149± 152. LINDLEY, M.G. (1999) `New developments in low-calorie sweeteners', in Corti, A., Low calorie sweeteners: Present and future, Basel: Karger, 44±51. LOTZ, A. and MEYER, E. (1994) `Sweeteners in beverages ± new developments', Internat. Fd Marketing and Technol., 8, 4±6, 8±9. MATTES, R. (1994) `Influences on acceptance of bitter foods and beverages