Novel Enzyme Technology for Food Applications

Novel enzyme technology for food applications

Related titles: Modifying lipids for use in food (ISBN 978-1-85573-971-0) Any oil or fat should have the optimum physical, chemical, and nutritional properties dictated by its end use. Modification of natural fats and oils is therefore important to improve the quality of lipids for use in foods. When lipids are modified, though, compromises have to be made as the physical, chemical and nutritional properties of lipids are not always mutually compatible and this provides an important challenge for food technologists. Edited by an eminent specialist, this collection shows how these challenges have been met in the past, how they are being met today, and how they may be met in the future. Starch in food – Structure, function and applications (ISBN 978-1-85573-731-0) Starch is both a major component of plant foods and an important ingredient for the food industry. Starch in food reviews starch structure and functionality and the growing range of starch ingredients used to improve the nutritional and sensory quality of food. Part I illustrates how plant starch can be analysed and modified, with chapters on plant starch synthesis, starch bioengineering and starch-acting enzymes. Part II examines the sources of starch, from wheat and potato to rice, corn and tropical sources. The third part of the book looks at starch as an ingredient and how it is used in the food industry. There are chapters on modified starches and the stability of frozen foods, starch–lipid interactions and starch-based microencapsulation. Part IV covers starch as a functional food, including the impact of starch on physical and mental performance, detecting nutritional starch fractions and analysing starch digestion. Proteins in food processing (ISBN 978-1-85573-723-5) Proteins are essential dietary components and have a significant effect on food quality. Edited by a leading expert in the field and with a distinguished international team of contributors, Proteins in food processing reviews how proteins may be used to enhance the nutritional, textural and other qualities of food products. After two introductory chapters, the book first discusses sources of proteins, examining the caseins, whey, muscle and soy proteins and proteins from oil-producing plants, cereals and seaweed. Part II illustrates the analysis and modification of proteins, with chapters on testing protein functionality, modelling protein behaviour, extracting and purifying proteins and reducing their allergenicity. A final group of chapters is devoted to the functional value of proteins and how they are used as additives in foods. Details of these books and a complete list of Woodhead’s titles can be obtained by:

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Novel enzyme technology for food applications Edited by Robert Rastall

CRC Press Boca Raton Boston New York Washington, DC

Cambridge England

Published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB21 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 2007, Woodhead Publishing Limited and CRC Press LLC © 2007, Woodhead Publishing Limited. Chapters 12 and 14 were prepared by US government employees; those chapters are therefore in the public domain and cannot be copyrighted. The authors have asserted their moral rights. Every effort has been made to trace and acknowledge ownership of copyright. The publishers will be glad to hear from any copyright holders whom it has not been possible to contact. 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. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing ISBN 978-1-84569-132-5 (book) Woodhead Publishing ISBN 978-1-84569-371-8 (e-book) CRC Press ISBN 978-1-4200-4396-9 CRC Press order number WP4396 The publishers’ policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elementary chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Ann Buchan (Typesetters), Middlesex, England Printed by TJ International Limited, Padstow, Cornwall, England

Contents

Contributor contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv Part I Principles of industrial enzyme technology 1

Discovering new industrial enzymes for food applications . . . . . . . . . 3 Thomas Schäfer, Novozymes A/S, Denmark 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2 Where to screen for new enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.3 How to screen for new enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.4 Summary: which option to choose? . . . . . . . . . . . . . . . . . . . . . . . 13 1.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2

Improving enzyme performance in food applications . . . . . . . . . . . . Ronnie Machielsen, Sjoerd Dijkhuizen and John van der Oost, Wageningen University, The Netherlands; Thijs Kaper and Loren Looger, Carnegie Institution of Washington, USA 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Laboratory evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Examples of improving enzyme stability and functionality by laboratory evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Rational and computational protein engineering . . . . . . . . . . . . . 2.5 Examples of improving enzyme stability and ability by rational protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Examples of combined laboratory evolution and computational design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

16 17 24 28 30 34

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Contents 2.7 2.8 2.9

3

4

5

Summary and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Sources of further information and advice . . . . . . . . . . . . . . . . . . 35 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Industrial enzyme production for food applications . . . . . . . . . . . . . . Carsten Hjort, Novozymes A/S, Denmark 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Traditional sources and processes for industrial enzyme production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Design of expression systems for industrial enzyme production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Development of an enzyme production process . . . . . . . . . . . . . . 3.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . 3.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immobilized enzyme technology for food applications . . . . . . . . . . . . Marie K. Walsh, Utah State University, USA 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Immobilized enzyme technology for modification of acylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Immobilized enzyme technology for modification of carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Immobilized enzyme technology protein modification . . . . . . . . 4.5 Immobilized enzyme technology for production of flavor compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consumer attitudes towards novel enzyme technologies in food processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Helle Søndergaard, Klaus G. Grunert and Joachim Scholderer, MAPP, University of Aarhus, Denmark 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Theoretical approaches to how consumers form attitudes to new food production technologies . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Studies of consumer attitudes to enzyme technologies . . . . . . . . . 5.4 Implications of consumer attitudes to enzyme technologies . . . . . 5.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . 5.7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43 43 44 46 54 56 56 57 60 60 62 68 73 75 77 78

85

85 86 88 94 95 95 96 96

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Part II Novel enzyme technology for food applications 6

7

8

Using crosslinking enzymes to improve textural and other properties of food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Johanna Buchert, Emilia Selinheimo, Kristiina Kruus, Maija-Liisa Mattinen, Raija Lantto and Karin Autio, VTT, Finland 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Types of crosslinking enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Application of crosslinking enzymes in baking and pasta manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Application of crosslinking enzymes in meat and fish processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Application of crosslinking enzymes in dairy applications . . . . 6.6 Other applications of crosslinking enzymes in food manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 Analysing the chemistry of crosslinks formed by enzymes . . . . 6.8 Effect of biopolymer crosslinking on nutritional properties of food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatically modified whey protein and other protein-based fat replacers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jacek Leman, University of Warmia and Mazury in Olsztyn, Poland 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Enhancing the fat mimicking properties of proteins . . . . . . . . . . 7.3 Applications in low-fat foods . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic production of bioactive peptides from milk and whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paola A. Ortiz-Chao and Paula Jauregi, University of Reading, UK 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Angiotensin I-converting enzyme inhibitory peptides . . . . . . . . 8.3 Other bioactive peptides and their health benefits . . . . . . . . . . . 8.4 Production of bioactive peptides from milk and whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6 Sources of further information and advice . . . . . . . . . . . . . . . . . 8.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

101

101 103 109 114 118 122 122 124 126 126

140 140 142 149 152 153

160

160 161 165 170 177 177 177

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Contents Production of flavours, flavour enhancers and other protein-based speciality products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stuart West, Biocatalysts Ltd, UK 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Production and usage of monosodium glutamate (MSG) . . . . . . 9.3 Chondroitin sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Production of aspartame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Enzymes for vanilla extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Enzyme modified cheese as a flavour ingredient . . . . . . . . . . . . 9.7 Enzymes used in savoury flavouring . . . . . . . . . . . . . . . . . . . . . 9.8 Enzymes used in yeast extract manufacture . . . . . . . . . . . . . . . . 9.9 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.10 Sources of further information and advice . . . . . . . . . . . . . . . . . 9.11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 Applications of cold-adapted proteases in the food industry . . . . . . A. Guðmundsdóttir and J. Bjarnason, University of Iceland, Iceland 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Use of proteolytic enzymes in food processing . . . . . . . . . . . . . 10.3 Application of cold-adapted serine proteases in food processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Modifying marine proteases for industrial use . . . . . . . . . . . . . . 10.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Health-functional carbohydrates: properties and enzymatic manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simon Hughes and Robert A. Rastall, University of Reading, UK 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Dietary fibre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Transgalacto-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Gluco-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.7 Alternansucrase–maltose acceptor oligosaccharides . . . . . . . . . 11.8 Resistant starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.9 Arabinoxylan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.10 Oligosaccharides from non-starch polysaccharides . . . . . . . . . . 11.11 Pectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.12 Oligodextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.13 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.14 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183 183 186 188 190 191 193 198 199 200 202 203 205 205 208 209 211 212 212

215 215 215 217 219 222 223 224 226 228 230 232 234 237 237

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12 Flavorings and other value-added products from sucrose . . . . . . . . Gregory L. Côté, United States Department of Agriculture, USA 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Di- and oligosaccharides from sucrose . . . . . . . . . . . . . . . . . . . . 12.3 Polysaccharides from sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Other products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Sources of further information and advice . . . . . . . . . . . . . . . . . 12.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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13 Production of structured lipids with functional health benefits . . . . Xuebing Xu, Janni B. Kristensen and Hong Zhang, BioCentrumDTU, Technical University of Denmark, Denmark 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Production of diglyceride oils . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Production of healthy oils containing medium chain fatty acids 13.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Lipase-catalyzed harvesting and/or enrichment of industrially and nutritionally important fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . George J. Piazza and Thomas A. Foglia, US Department of Agriculture, USA; and Xuebing Xu, BioCentrum-DTU, Technical University of Denmark, Denmark 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Lipase selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Fatty acid harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.4 Structured triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 Single reaction step process for the production of STAG . . . . . 14.6 Multiple reaction step processes for the production of STAG . . 14.7 Nutritional and other uses of structured lipids . . . . . . . . . . . . . . 14.8 Summary and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243 244 257 260 261 262 262 270

270 271 278 282 282 282

285

285 286 294 295 301 307 307 308 309

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

Contributor contact details (* = main contact)

Editor

Chapter 3

R. A. Rastall School of Food Biosciences PO Box 226 Whiteknights Reading RG6 6AP UK

C. Hjort Novozymes A/S Krogshoejvej 36 DK-2880 Bagsvaerd Denmark

email: [email protected]

Chapter 4

Chapter 1 T. Schäfer Novozymes A/S Krogshoejvej 36 DK-2880 Bagsvaerd Denmark email: [email protected]

email: [email protected]

M. K. Walsh Utah State University 8700 Old Main Hill NFS 318 Logan UT, 84322-870 USA email: [email protected]

Chapter 5

R. Machielsen* and J. van der Oost Hesselink van Suchtelenweg 4 6703CT, Wageningen The Netherlands

H. Søndergaard*, K. G. Grunert and J. Scholderer MAPP Aarhus School of Business Halslegaardsvej 10 DK-8210 Aarhus V Denmark

email: [email protected]

email: [email protected]

Chapter 2

xii

Contributor contact details

Chapter 6 J. Buchert*, E. Selinheimo, K. Kruus, M. L. Mattinen, R. Lantto and K. Autio VTT PO Box 1000 FI-02044 VTT Finland email: [email protected]

Chapter 7 J. Leman Faculty of Food Sciences University of Warmia and Mazury in Olsztyn Heweliusza 1 10-718 Olsztyn Poland

email: [email protected] secretarytheresac@ biocats.com

Chapter 10 A. Guðmundsdóttir* Science Institute University of Iceland Læknagardi Vatnsmýrarvegi 16 101 Reykjavík Iceland email: [email protected] J. B. Bjarnason Dunhaga 3 107 Reykjavík Iceland

email: [email protected]

Chapter 8 P. A. Ortiz-Chao and P. Jauregi* School of Food Biosciences University of Reading PO Box 226 Whiteknights Reading RG6 6AP UK

Chapter 11 S. Hughes and R. A. Rastall* School of Food Biosciences PO Box 226 Whiteknights Reading RG6 6AP UK email: [email protected]

email: [email protected]

Chapter 9 S. West Biocatalysts Limited Cefn Coed Nantgarw Cardiff CF15 7QQ UK

Chapter 12 G. Côté NCAUR/ARS/USDA 1815 N. University St Peoria IL 61604 USA email: [email protected]

Contributor contact details

Chapter 13 X. Xu*, J. B. Kristensen and H. Zhang BioCentrum-DTU Technical University of Denmark Building 227 DK-2800 Kgs. Lyngby Denmark email: [email protected]

Chapter 14 G. J. Piazza* and T. A. Foglia US Department of Agriculture Agricultural Research Service

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Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor PA 19038 USA email: [email protected] X. Xu BioCentrum-DTU Technical University of Denmark Building 227 DK-2800 Kgs. Lyngby Denmark email: [email protected]

Preface

Enzymes have been used in the food industry for many years. They have largely been used as processing aids and they have many attributes that make them fit for this purpose. They are generally non-toxic and speed up chemical reactions with great specificity at low temperatures and pressures and at near-neutral pH. A large industry exists to serve this need across the world. One of the limitations of enzyme application in the food industry is the lack of availability of enzymes with the required properties at an acceptable price. Whilst desired enzyme activities are frequently known somewhere in the biological world, they are often unsuitable for commercial application. In recent years, however, there has been increasing sophistication in our ability to isolate novel enzymes from biological sources and an expansion of the range of sources of enzymes to include, for example, extremophiles. Such organisms frequently have enzymes with higher pH and temperature optima and can extend the range of processes in which enzymes can be used. We now have the ability to rationally engineer or artificially evolve desired catalytic properties into enzyme molecules. These new technologies will ultimately remove many of the limitations currently restricting the application of enzymes in the food industry and will open up many more possibilities. Technological aspects are dealt with in Part I – Principles of industrial enzyme technology. Chapters 1 and 2 deal with the discovery of novel enzymes for food applications and the improvement of enzymes for food applications. Chapters 3 and 4 then examine the production of industrial enzymes and their immobilisation in the context of food applications. Part I is concluded by Chapter 5 on consumer attitudes to novel enzyme technologies. Concurrent with these technological developments has been the advance in our knowledge of the role of specific food components in health and disease. This has led to a significant increase in interest in functional food ingredients – compounds that are specifically added (or whose levels are deliberately increased) in foods to

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Preface

provide a specific health attribute beyond nutrition. Examples include prebiotic oligosaccharides to improve gut health, bioactive peptides to help reduce blood pressure, and nutritionally enhanced fats. Governments around the world are also taking heed of modern nutritional knowledge and are increasingly looking to the food industry to manufacture foods with a healthier profile. These nutritional developments are starting to provide a new range of application areas for novel enzymes and enzyme technologies and it is these applications that are discussed in Part II – Novel enzyme technology for food applications. Chapters 6, 7 and 8 deal with enzymatic modification of proteins to achieve cross-linking, to generate fat replacers and to manufacture bioactive peptides respectively. Protein modification also features in Chapter 9 on production of flavours and flavour enhancers and in Chapter 10 on the application of novel cold-adapted proteases. The focus then moves to carbohydrates, in Chapter 11 on health-functional carbohydrates and Chapter 12 on value-added products from sucrose. Finally, the manufacture of lipids with health and other functional attributes is discussed in Chapters 13 and 14. This volume aims to give the reader an overview of recent developments in enzyme technology in the food industry rather than an exhaustive account of traditional applications. The aim is to increase awareness of and stimulate interest in developing novel enzyme technologies to meet the new and changing needs of the food industry. Professor Robert Rastall University of Reading

Part I Principles of industrial enzyme technology

1 Discovering new industrial enzymes for food applications Thomas Schäfer, Novozymes A/S, Denmark

1.1

Introduction

Enzymes have been exploited by humans for thousands of years. Traditional foods and beverages like cheese, yoghurt and kefir, bread, beer, vinegar, wine and other fermented drinks, as well as paper and textiles, were produced with the help of enzymes which were present in starting materials as early as 6000 BC in China, Sumer and Egypt. The epoch of classical biotechnology was marked by the landmark discoveries of microbes by Leeuwenhook, of fermentations as biological processes by Pasteur, of enzymes as proteins by Buchner and of the first enzyme crystal structures by Sumner. The modern era of industrial enzymology began in 1913 when Otto Röhm was granted a patent for the use of a crude protease mixture isolated from pancreases in laundry detergents. In the following years an increasing number of enzymes were found in microorganisms and these microbes were cultured in large scale fermentations to produce enzymes. However, the number of enzymes that could be produced in this fashion was limited, because not all microbes are amenable to large scale fermentation. The pioneering work of Avery and MacLeod, Hershey and Chase, Watson and Crick, Cohen and Boyer and many others who introduced the era of recombinant biotechnology revolutionized industrial enzyme screening and production. With the advent of genetic engineering, genes encoding interesting enzymes could be transferred to and expressed in selected host microbes for production on an industrial scale. Today, gene technology plays a major role in both the discovery

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Novel enzyme technology for food applications

of novel enzymes and the optimization of existing proteins, and is basis for the production of the majority of industrial enzymes. Food applications of enzymes represent a wide and highly diverse field including baking, dairy, juice, vegetable processing and meat. The enzymes are used to obtain a number of benefits, like more efficient processes, leading to reduced use of raw materials, improved or consistent quality, replacement of chemical food additives and avoidance of potential harmful by-products in the food.

1.1.1 Technologies for discovery of industrial enzymes Nature holds a wonderful diversity of organisms and the corresponding wealth of enzymes and has often been the starting point for the identification of novel enzymes. For a variety of applications even Nature’s assortment faces some limitations or it is too time consuming and difficult to look into Nature’s diversity. This imposes a challenge for scientists to optimize existing natural enzymes and to generate additional ‘artificial’ diversity to tailor-make enzymes for a given application. Natural diversity approaches and optimization strategies are complementary routes and both are equally important in developing a high-quality diversity of enzymes (Nedwin et al., 2005). Today, discovery of enzymes for the food industry is not only a multidisciplinary effort involving a wide array of different screening technologies, but is also based on close interaction between food scientists who understand or model the application and biotechnologists who can deliver enzymes for initial trials. Each screening project is new and challenging. Accordingly, each project needs to be uniquely designed to solve the specified application problems in a certain industrial application and for each project the expert team needs to have members with exactly the competencies needed to find a solution. It is obvious that major enzyme companies have to master a variety of technologies which, often in combination with each other, lead to the solution. For all approaches it is important to stress that it is not the broadest possible diversity, but rather the highest possible quality of diversity which will lead to the ultimate goal, namely a novel product that addresses exactly the specific demands of the industrial application. In this respect selection/ deselection via perfectly designed assays is of utmost importance, indicating the significance of linking process understanding to biochemistry.

1.2

Where to screen for new enzymes

One of the main questions which has to be answered in the very beginning of each discovery initiative is ‘where to look for diversity?’ (Bull et al., 2000; Fig. 1.1). There are various potential sources, as input to screening programs is basically divided into (a) natural enzyme diversity and (b) artificial diversity, which are comprehensively reviewed by Schäfer and Borchert (2004) and Aehle (2004). Here the basic principles will be summarized.

Discovering new industrial enzymes for food applications

Fig. 1.1

5

Overview of the main approaches to diversity input in screening programs.

1.2.1 Nature’s diversity: an unlimited source of enzymes The challenge is that Nature’s diversity is virtually infinite and that living microorganisms have inhabited virtually all ecological niches on planet Earth during 3.5 billion years of evolution. The number of described bacterial and fungal species is huge, new isolations are added daily so that the actual number can only be extrapolated roughly. From bioinformatics analysis of the genomes it can be assumed that a bacterial genome on average contains about 4000 enzyme coding genes, while for fungi the number of enzyme encoding genes can be up to 20,000 (Hirose et al., 2000; Dunn-Coleman and Prade, 1998). The art of screening obviously consists of having the right tools to find the ‘needle’ in this ‘haystack’ of biodiversity; no scientist will start looking into the totally available biodiversity, but will look into groups of carefully selected microorganisms. Considering these numbers and using best practice, it is obvious that all screening efforts face a limitation in that we are only scratching the surface. Microorganisms, namely bacteria, fungi and archaea, which are normally stored in culture collections of the groups performing the screening or in public collections, where the strains are accessible for everyone who is interested, often comprise the biological starting material. On top of the cultivated diversity, complex gene libraries compiled from natural material without prior cultivation (Handelsman, 2005) can be generated and used to discover industrial enzymes (Short, 1997) and other natural compounds (Brady et al., 2001). Today, it is generally accepted that only minor numbers among the whole of the microbial diversity have been cultured or might even be amenable to growth in the laboratory (Torsvik et al., 2002) thereby leaving not only a huge set of questions concerning our understanding of the role of microbes in their habitats, but also an enormous potential for yet undiscovered physiological and biochemical

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Novel enzyme technology for food applications

traits including enzyme genes in the so-called metagenome (Lorenz and Schleper, 2002; Rondon et al., 1999). It is estimated that 1 g of soil contains more than 4000 different bacterial genomes, that is about 16 million enzyme encoding genes. By isolating the genetic material, be it DNA or RNA, directly from the soil and cloning this into suitable host complexes, ‘environmental libraries’ can be constructed. These gene libraries need to be screened as described below using either sequencebased techniques or activity assays including some novel constraints caused by the complexity of the library.

1.2.2 Bioinformatics and genomics Input to screening efforts can also come from genes or genomes described by researchers worldwide over time. The updated status of established genomes and those underway can be obtained by visiting the homepage of the TIGR institute (http://www.tigr.org/) or the homepage of the DOE Joint Genome Institute (http: //genome.jgi.org/). The use of existing gene information can potentially shortcut the flow to a new product candidate, although in most instances a gene is described by a sequence only, that is, no biochemical information is available. By using sophisticated software tools within the new discipline of bioinformatics those genes can be aligned to existing ones, grouped into enzyme families in order to predict ideally their putative biochemistry, that is, enzyme activity (Henrissat and Bork, 1996). This is also where one of the major pitfalls lies, namely that the original description of the enzyme can turn out to be incorrect. An additional complexity is the fact that roughly 30% of all gene sequences from genomes are new, that is they do not resemble any biochemical description of the corresponding protein. Interesting hits found in this way can subsequently be analysed in more detail but this requires cloning and expression of the gene (see below) followed by purification and characterization of the corresponding enzyme, which is a tedious and resource-intensive effort when many genes are of potential interest. Accordingly, this comprises one of the major bottlenecks in genomics as the protein can only be characterized very late in the process and the chance of failure is high. Searching of gene databases, both generated in-house and external ones, is a daily complement to the work of a screening scientist. In addition to screening the external world of sequence data for novel enzymes, the discovery scientist must also determine whether any enzymes found are novel and whether their use is protected by patents. Whole genome sequencing combined with bioinformatics, array studies and proteomics are novel key technologies for the targeted improvement of production strains. This has illustratively been described for lysine production in Corynebacterium glutamicum (Ohnishi et al., 2002). Whole genome sequencing which completely maps all genes is, however, not ideal for discovery of selected genes, for example those encoding for enzymes and especially for those enzymes that match defined application criteria. Assuming an average genome size for a bacterium of about 4 Mb, for yeast of 13 Mb (Zagulski, et al., 1998) and for filamentous fungi in the order of 30–40 Mb (Dunn-Coleman and Prade, 1998;

Discovering new industrial enzymes for food applications

7

Radford and Parish, 1997), the costs of sequencing programs of total genomes are unreasonably high for discovery purposes, especially considering the wide diversity of microbes that are interesting for enzyme screening. From the 4100 open reading frames (ORFs) of the Bacillus subtilis genome, only a fraction may be relevant for industrial enzymes. For many industrial applications, extracellular enzymes are of major importance and it is estimated that B. subtilis produces 150– 180 secreted proteins (Hirose et al., 2000), while the number of secreted enzymes for filamentous fungi might be in the order of 200–400 corresponding to their larger genome sizes. This indicates that only 2–5% of the ORFs in a complete genome are of primary interest for enzyme discovery. Accordingly, whole genome sequencing can hardly be justified for enzyme discovery purposes. As a consequence alternative approaches have been developed to mine selectively microbial genomes for secreted enzymes. Those will be described in more detail in Section 1.3.4.

1.2.3 Protein optimization of enzymes In cases where enzymes found from natural sources cannot provide the performance needed for a given application, protein optimization offers an attractive option. In many applications the enzymes are very much stressed by, for example, high temperatures, extreme pH values or the presence of metal ions, which are known to induce unfolding of the protein. Several strategies can be followed in order to optimize the properties of enzymes found in Nature. A simplified way of looking at protein optimization technologies is to divide the field into rational protein engineering and random molecular evolution (Fig. 1.1). This is discussed briefly below and in more detail in Chapter 3. Rational protein engineering is based on the knowledge of a given enzyme structure and the corresponding biochemistry, for example the substrate specificity, the temperature tolerance, inhibition by metal ions, and so on, the combination thereof comprising the structure–function relationship. This is the parameter that will be modified as changes in structure will lead to changes in functionality. The challenge is to introduce changes that lead to improved functionality rather than inferior variants of an enzyme. A key is the ability to create protein variants with designed and deliberate amino acid alterations at any desired position that provides the capability for precise probing of structure–function relationships in proteins (Bott, 2005). The gene is mutated selectively at specified sites and the corresponding enzyme is expressed and subsequently tested to verify the hypothesis behind the mutation. Positive mutations are collected and analysed in more detail, for example which amino acids in the enzymes were changed, at the position where the mutations are located. These mutations are eventually combined to find the ultimate combination of positive mutation events. As easy as this might sound, this approach represents a considerable challenge for researchers and in many instances the experiments have failed. Several years of hands-on experience of biochemistry, bioinformatics and structure–function analysis are a prerequisite for success. Importantly, it must

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Novel enzyme technology for food applications

be acknowledged that we still only have limited understanding of protein function and that only a small part of the huge natural enzyme diversity has been analysed on a structural level, resulting in severe limitations for rational protein engineering. We are still far from predictability, that is, knowing which amino acid change will result in which consequence. Years of trial and error have, however, increased our knowledge, especially of selected enzyme classes which are of major importance for industrial enzymes. Accordingly, engineered variants of a number of hydrolytic enzymes such as proteases, amylases, lipases and cellulases are commercially available today. In contrast, no prior knowledge of structure–function relationships is needed to carry out molecular evolution experiments. Here the basic principle is to carry out random introduction of mutations, thereby generating DNA libraries consisting of up to millions of variant genes. The DNA variation is expressed into protein diversity in a variant library, for example in Escherichia coli or Saccharomyces cerevisiae, the library is subjected to a screening procedure using a functional assay (see below) and the best performing mutants are collected. There is a tight connection between the selected variant protein and its encoding gene, which makes it easy to sequence the enzyme coding gene and detect the mutation, in this case after the modified phenotype was detected. Random mutation leads to millions of mutants of a given gene and smart screening systems are needed to identify the best performing variants. Robotics equipment for colony picking, colony transfer into screen-able formats, often microtitre plates, and addition of assay components are a prerequisite for this approach (Eijsink et al., 2005).

1.3

How to screen for new enzymes

After the question ‘where to screen?’ has been answered the next question is ‘how to screen?’ (Bull et al., 2000). There are several possibilities for this and variations of these themes (Fig. 1.2). The following paragraphs will describe some of the most prominent screening approaches.

1.3.1 Functional biochemical assays The most preferred screening route for novel enzymes is via functional screening assays, where the biochemical activity can be detected. Ideally, this will also show how well the positive hits meet application requirements to be tested; a detected amylase can, for example, also be tested for activity at elevated temperature, high or low pH or under other hostile conditions by using the same assay principle. Biochemical assays allow the screening of living microorganisms, of gene libraries constructed from cultivated microbes and from the environment (metagenomes), of artificial evolution libraries, as well as of rationally designed protein variants for a wanted enzyme activity. Many of the assays can be implemented on agar plates, where growing colonies can be tested for activity, or on a smaller scale using

Discovering new industrial enzymes for food applications

9

Screening output or ‘how to screen’ Functional screening

Biochemical screening assays Molecular screening

Sequence based screening Secretomics

Major screening approaches

Genomics/ Bioinformatics Transcriptomics

Proteomics Expression

Fig. 1.2

Gene trapping technologies Genome mapping and analysis

Gene regulation/expression

Protein analytics

Protein production

Overview of the main technologies for screening for industrial enzymes.

microtitre plates. The latter is often a challenge but is a prerequisite for highthroughput screening which is needed especially for screening metagenome and artificial evolution libraries. Accordingly, a lot of effort is invested in developing screening assays and novel screening technologies including high-throughput technologies that give enhanced freedom in assay design. Accordingly a variety of publications and patent applications cover this field (Joo et al., 1999; Ruijssenaars and Hartmans, 2001; Meeuwsen et al., 2000; Preisig and Byng, 2001; Kongsbak et al., 1999; Short and Keller, 2001; Schellenberger, 1997). To include application-relevant parameters is very difficult, as the biophysical matrix in the target application is often very complex and it can be difficult to identify relevant screening criteria, for example for enzymes in bread making. Potential enzyme candidates can be identified (for example an amylase), but it is difficult to qualify these in small scale assays which mimic the application. Accordingly, full scale baking trials are the only way of evaluating the enzyme candidates, which is both a time and resource requiring approach. To overcome this, hypotheses concerning the most relevant application parameters are made and assays are developed which are as close to real conditions as possible. Ideally, functional screening procedures help to select the best performers in a given library. The best candidates might be chosen as product candidates directly, or alternatively they can form the starting point for a repeated round of the directed evolution cycle if a performance gap still exists. In the second round, enzymes from natural diversity screens or protein engineered variants might be included if they show beneficial characteristics for a given application, again pointing at the fact that screening approaches are complementary and the combination of several of them often leads to success.

10

Novel enzyme technology for food applications

1.3.2 Primary and secondary screening The above illustrates one of the main approaches to screening, that is, sequential primary and secondary screening. This is the general pattern for all new screening programmes. First, screening criteria are defined which include the substrate to be converted and conditions such as temperature, pH, presence or absence of ions. These are used to design functional screening assays that are able to (a) detect the desired enzyme activity in the primary round and (b) select the best performing enzymes in the secondary round. The primary screening is often very broad and all microbes, clones or variants which are positive in the chosen assay are selected. Accordingly, the primary screening assay has low selectivity in order to capture a wide variety of positive hits. In the next round these hits are subjected to a secondary screening where a highly selective enzyme assay is applied that allows ranking and selection of the best-performing enzyme candidates. It must be noted that screening is seldom a simple linear process but usually involves learning loops running in several cycles.

1.3.3

Expression cloning for further characterization and application testing Ideally, the enzymes that are tested in industrial applications are monocomponent, that is, free of other potentially misleading enzyme activities. To deliver these pure enzymes, cloning of genes and expression of proteins is an important step. In most cases a technique called expression cloning is used which is an effective means of isolating a gene from a gene library based on its encoded activity (Dalbøge and Heldt-Hansen, 1994). In brief, the genetic material, either DNA or mRNA, is isolated and purified, cut into small pieces (in case of DNA) to separate all genes physically and randomly from each other, set into suitable vector systems, and transferred into a screening host to form a gene library. Host strains ideally do not produce the targeted enzyme activity and often E. coli or S. cerevisiae are used for this purpose. In this step a 10 000–50 000 colony gene library is tested with the biochemical assay, and single colonies expressing the cloned enzyme can be isolated. Ideally the selected clones only produce the enzyme of interest. Subsequently the corresponding gene can be isolated, sequenced and used for further analysis and optimization. Monocomponent enzymes produced by recombinant DNA technology are preferred in small-scale applications to correlate measured effects clearly to a given protein rather than a complex mixture of enzymes. This is important when comparing the protein with existing enzymes (Can the enzyme be patented? Is it better than the benchmark?), for further improvement by protein optimization and also to obtain an initial idea of whether the enzyme can be produced under economically promising conditions (Chapter 3). Only at the point of working with a pure enzyme can the hypothesis underlying the assay be tested, that is, do the selected candidates fail or pass the real application test? Both failure and success can be used to optimize the assay and thereby generate even more and better diversity. Accordingly the quality and nature of the screening assay has a central

Discovering new industrial enzymes for food applications

11

role during the whole selection/deselection programme as the quality of the assay determines the quality of the resulting candidate. The key to successful screening of industrial enzymes is not to detect a large diversity of proteins, but rather those few that can perfectly match the application conditions. Again, the importance of cooperation between food scientists and the biochemists performing the screening cannot be ranked highly enough.

1.3.4

Molecular screening approaches for identification of genes and proteins Sequence-based approaches, also described as molecular screening, complement functional screens. They are based on similarities between enzyme-encoding gene sequences (Dalbøge and Lange, 1998, Precigou et al., 2001). Sequence information from a set of related enzyme genes is used to identify evolutionarily conserved regions and to design polymerase chain reaction (PCR) primers to amplify genes from other organisms. Using this method, a number of genes which are homologous to the initial gene sequences can quickly be identified. The limitation of the method is that enzyme variants rather than totally novel enzymes are detected. The advantages, on the other hand, are that this method is not dependent on growing the strains in the laboratory or on the active expression of a protein, which makes this an interesting option for screening metagenomic libraries. Secretome studies, transcriptomics and proteomics are gaining more importance as screening tools (Nedwin et al., 2005). A fast and efficient approach for trapping of genes which encode secreted enzymes from a genome (hence secretomics) is transposon-assisted signal trapping (TAST) of gene libraries (Duffner et al., 2001). A genomic or cDNA library is treated with a transposon carrying a reporter gene which codes for a secreted protein with its own secretion signal sequence removed. A signalless beta-lactamase has been used as reporter which can, upon insertion in a gene with an active secretion signal, be transported out of the cell as a fusion protein. This results in ampicillin-resistant phenotypes that can be selected on agar plates. Genes encoding secreted proteins are subsequently sequenced and identified by comparison to databases using bioinformatic tools. In contrast to traditional screening of gene libraries with functional assays for selected enzyme activities, the entire genome, as represented in the library, is trapped for known and novel enzymes simultaneously on a gene level. The disadvantage of the method is that genes are identified but are not directly available for testing which means that all relevant ORFs have to be expressed individually. Alternative approaches have been developed to mine microbial genomes selectively for secreted enzymes. One alternative is to clone and sequence expressed sequenced tags (ESTs) randomly. This has been used to identify novel enzymes in Fusarium (Berka et al., 2003). The principle is based on large-scale isolation and (partial) sequencing of randomly selected, anonymous cDNA clones which express the enzyme linked to the gene derived from the cDNA. By

12

Novel enzyme technology for food applications

sequencing the gene and comparing it with other genes in the databases, previously unknown genes have been identified in a variety of organisms. When gene sequences are analysed from cells grown on different nutrient sources, it is possible to discover and catalogue novel enzymes that are produced specifically on those nutrients. For this kind of comparative analysis signal trapping, EST analysis, transcriptomics using DNA microarrays or proteomics might be used. DNA microarray technology is a popular tool for studying gene expression and gene regulation by monitoring mRNA formation on a genomic scale (DeRisi et al., 1997; Berka et al., 2003). This technology can also be applied in enzyme screening as it is possible to detect genes induced under specific physiological conditions (Yaver et al., 2003; Diehn et al., 2000; Bashkirova and Berka, 2003). In principle the genome of the microorganism to be investigated needs to be known, all ORFs, that is enzyme-encoding genes, need to be amplified by PCR and plotted on a carrier material, for example a glass slide. When a strain is grown on different specific substrates, genes involved in utilization of that specific substrate are induced and RNA is collected. The mRNA can be isolated and labelled with fluorescent dyes of different colours, corresponding to, for example, the different growth substrates. This differential, medium dependent expression allows detection of genes that are important for degradation of the particular substrate by comparing hybridization of differently labelled mRNAs to the DNA on the glass slide. Using this technology, global gene expression profiling of Ceriporiopsis subvermispora was performed to discover novel peroxidases enzymes whose expression is induced during growth on thermomechanical pulp (Yaver et al., 2003). Thus, shotgun genomic DNA microarrays appear to be a viable approach for identifying novel enzymes involved in the degradation of complex substrates. A method combining suppression subtractive hybridization (SSH) and DNA microarray techniques was used to identify biomass-induced genes in the cellulolytic fungus Trichoderma reesei (Bashkiro and Berka, 2003). The degradation of cellulosic biomass is the result of the concerted effort of many fungal enzymes, though only a few enzymes have been identified and characterized. The cDNA libraries generated by SSH allowed for the selection of differentially expressed mRNAs, as well as enrichment of rare mRNAs and equalization of the cDNA pool. DNA sequence analysis and bioinformatics were used to assemble the clones into approximately 90 previously unrecognized genes/proteins. Thus, the combination of SSH and cDNA microarray technologies has proved to be a useful tool for discovering new differentially expressed enzyme genes involved in biomass utilization. All these different genetic approaches have drastically increased the amount of data available in both industrial and public databases. Additionally, hundreds of genome projects have led to an explosion of data, which again underlines the need for new tools within the discipline of bioinformatics to compare, sort and finally select the most relevant data.

Discovering new industrial enzymes for food applications

1.4

13

Summary: which option to choose?

The discussion above has outlined the complexity, not only of the individual methods which have to be mastered, but also of the interplay between these techniques. As each screening program is new and possesses unique challenges, a variety of questions have to be answered: Which technology or which combination of technologies will most probably result in a new product candidate? Which route gives highest chance of success? Which route is fastest to pursue? And which will deliver the optimal result in terms of quality and patentability? Accordingly, no clear, straightforward answer can be given, but often accumulated knowledge is crucial in deciding which route to follow. Technological improvements have contributed to shorten the time from idea to product significantly. In the mid-1990s, it took approximately five years from the creation of a gene bank to selling the product in the market place. In 2000, this was reduced to approximately 26 months. Today, in many cases, it is possible to go from enzyme identification to shipment of large (tonnes) quantities of safe and approved product in the technical field in approximately 12 to 24 months. Within the food and animal feed areas the approval process extends the launch time in the order of an additional one to two years. Once enzyme discovery is made, enzyme scale-up progression begins. This includes strain evaluation, selection, fermentation development and optimization. These technologies are described in Chapter 3.

1.5

References

Aehle W. (2004), Enzymes in Industry, Wiley-VCH, Weinheim. Bashkirova E. and Berka R. M. (2003). ‘Towards the discovery of new enzymes involved in biomass degradation: combination of SSH and microarray technologies to identify Trichoderma reesei biomass-induced genes’, 25th Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, Colorado, USA, May 4–7. Berka R. M., Nelson B. A., Zaretsky E. J., Yoder W. T. and Rey M. W. (2003). ‘Genomics of Fusarium venenatum: An alternative fungal host for making enzymes’, Applied Mycology and Biotechnology, 4, 5682–5687. Bott R. (2005). ‘Analyzing the three-dimensional structures of variant enzymes’. In Svendsen A., Enzyme Functionality, Marcel Dekker, New York, p 35 Brady S. F., Chao C. J., Handelsman J. and Clardy J. (2001). ‘Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA’, Organic Letters, 3(13), 1981–1984. Bull A. T., Ward A. C. and Goodfellow M. (2000). ‘Search and discovery strategies for biotechnology: the paradigm shift’, Microbiology and Molecular Biology Reviews, 64(3), 573–606,CP3. Dalbøge H. and Heldt-Hansen H. P. (1994). ‘A novel method for efficient expression cloning of fungal enzyme genes’, Molecular and General Genetics, 243(3), 253–260. Dalbøge H. and Lange L. (1998). ‘Using molecular techniques to identify new microbial biocatalysts’, Trends in Biotechnology, 16(6), 265–272. DeRisi J. L., Iyer V. R. and Brown P. O. (1997). ‘Exploring the metabolic and genetic control of gene expression on a genomic scale’, Science, 278(5338), 680–686. Diehn M., Eisen M. B., Botstein D. and Brown P. O. (2000). ‘Large-scale identification of

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secreted and membrane-associated gene products using DNA microarrays’, Nature Genetics, 25(1), 58–62. Duffner, F., Wilting R. and Schnorr K. (2001), Signal Sequence Trapping, Novozymes A/S, Patent: WO 20017–1315-A. Dunn-Coleman N. and Prade R. (1998). ‘Toward a global filamentous fungus genome sequencing effort’, Nature Biotechnology, 16(1), 5. Eijsink V. G. H., Gåseidnes S., Borchert T. V. and van den Burg B. (2005), “Directed evolution of enzyme stability’, Biomolecular Engineering, 22(1–3), 21–30, Handelsman J. (2005). ‘Metagenomics or megagenomics?’, Nature Reviews Microbiology, 3(6), 457–458. Henrissat B. and Bork P. (1996). ‘On the classification of modular proteins’, Protein Engineering, 9(9), 725–726. Hirose I., Sano K., Shioda I., Kumano M., Nakamura K. and Yamane K. (2000). ‘Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study’, Microbiology-UK, 146, 65–75. Joo H., Arisawa A., Lin Z. L. and Arnold F. H. (1999). ‘A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases’, Chemistry and Biology, 6(10), 699–706. Kongsbak L., Jørgensen K. S., Jørgensen C. T., Husum T. L., Ernst S. and Møller S. (1999), A Fluorescence Polarisation Screening Method, Novozymes A/S, Patent WO 9945–143A. Lorenz P. and Schleper C. (2002). ‘Metagenome – a challenging source of enzyme discovery’, Journal of Molecular Catalysis B: Enzymatic, 19, 13–19, Meeuwsen P. J. A., Vincken J. P., Beldman G. and Voragen A. G. J. (2000). ‘A universal assay for screening expression libraries for carbohydrases’, Journal of Bioscience and Bioengineering, 89(1), 107–109. Nedwin G. E., Schäfer T. and Falholt P. (2005). ‘Enzyme discovery – Screening, cloning, evolving’, Chemical Engineering Progress, 101(10), 48–55. Ohnishi J., Mitsuhashi S., Hayashi M., Ando S., Yokoi H., Ochiai K. and Ikeda M. (2002). ‘A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant’, Applied Microbiology and Biotechnology, 58(2), 217–223. Precigou S., Goulas P. and Duran R. (2001). ‘Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction’, FEMS Microbiology Letters, 204(1), 155–161. Preisig C. and Byng G. (2001). ‘Applications of mass spectrometry in screening for new biocatalysts’, Journal of Molecular Catalysis B: Enzymatic, 11(4–6), 733–741. Radford A. and Parish J. H. (1997). ‘The genome and genes of Neurospora crassa’, Fungal Genetics and Biology, 21(3), 258–266. Rondon M. R., Goodman R. M. and Handelsman J. (1999). ‘The Earth’s bounty: Assessing and accessing soil microbial diversity’, Trends in Biotechnology, 17(10), 403–409. Ruijssenaars H. J. and Hartmans S. (2001). ‘Plate screening methods for the detection of polysaccharase-producing microorganisms’, Applied Microbiology and Biotechnology, 55(2), 143–149. Schellenberger V. (1997), Compartmentalization Method for Screening Microorganisms, Genencore International, Patent WO 9737–036A1. Schäfer T. and Borchert T. V. (2004). ‘Bioprospecting for industrial enzymes: importance of integrated technology platforms for successful biocatalyst development’, in Bull A. T., Microbial Diversity and Bioprospecting, ASM Press, Washington, 375–390. Short J. M. (1997). ‘Recombinant approaches for accessing biodiversity’, Nature Biotechnology, 15(13), 1322–1323. Short J. M. and Keller M. (2001), High-Throughput Screening for Novel Enzymes, Diversa Corporation, Patent US 6,174, 673 B1. Torsvik V., Ovreas L. and Thingstad T. F. (2002). ‘Prokaryotic diversity – Magnitude, dynamics, and controlling factors’, Science, 296(5570), 1064–1066.

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Yaver D. S., Weber B. and Murrell J. (2003). ‘Global expression profiling of the lignin degrading fungus Ceriporiopsis subvermispora for the discovery of novel enzymes’, Applied Mycology and Biotechnology, 3, 261–269. Zagulski M., Herbert C. J. and Rytka J. (1998). ‘Sequencing and functional analysis of the yeast genome’, Acta Biochimica Polonica, 45(3), 627–643.

2 Improving enzyme performance in food applications Ronnie Machielsen, Sjoerd Dijkhuizen and John van der Oost, Wageningen University, The Netherlands; Thijs Kaper and Loren Looger, Carnegie Institution of Washington, USA

2.1

Introduction

Biocatalysis is gradually taking over from chemical catalysis in many industrial applications. Enzymes are environmentally friendly, biodegradable, efficient, and low cost in terms of resource requirements; as such they provide benefits compared with traditional chemical approaches in various industrial processes. In many instances, however, natural enzymes do not perform optimally in a particular unnatural process and, as such, can be unsuitable for large-scale industrial applications (Schoemaker et al., 2003). Reflecting their participation in complex metabolic networks inside living cells, enzymes are often inhibited by their own substrates or products, either of which may severely limit the productivity of an industrial biocatalytic process. During natural evolution, enzymes are optimized and often highly specialized for certain biological functions within the context of a living organism. In contrast, biotechnology needs enzymes that have (i) a high activity over longer periods of time (a feature that might clash with the need for rapid protein turnover inside a cell), (ii) a high stability under harsh physical (high temperature) and chemical (non-aqueous solvents) conditions and (iii) a high specificity and selectivity that does allow the enzyme to generate efficiently specific products that are not necessarily present in nature. There are three major and principally different routes to obtain enzyme variants with improved features: (i) isolating enzyme variants from organisms living in appropriate environments, (ii) rationale-based mutagenesis and (iii) laboratory

Improving enzyme performance in food applications

17

evolution. The first option assumes that the desired enzyme is around and has been generated by natural evolution. To allow rationale-based engineering, a highresolution three-dimensional model and insight into the structure–function relations of the biocatalyst of interest are required. Laboratory evolution offers a way to optimize enzymes randomly in the absence of structural or mechanistic information (Bornscheuer and Pohl, 2001). In this chapter, the available techniques for engineering enzymes are described. The engineering approaches are subdivided into directed (rational protein engineering) and random (laboratory evolution) techniques. In addition, different selection and (high-throughput) screening methods are described, a crucial development that allows screening of large mutant libraries.

2.2

Laboratory evolution

Laboratory evolution has emerged as a powerful tool for improving biocatalysts as well as for broadening our understanding of the underlying principles of substrate specificity, stereoselectivity and the responsible catalytic mechanism. In contrast to rational protein design (discussed below), laboratory evolution does not require knowledge of the three-dimensional structure of a given enzyme or about the relationship between structure, sequence and mechanism. Laboratory evolution experiments implement a simple, iterative Darwinian optimization algorithm. Molecular diversity is typically created by random mutagenesis [for example error-prone polymerase chain reaction (PCR)] and/or recombination of a target gene or a family of related target genes. Improved variants are identified in a screen (or selection) that accurately reflects the properties of interest. The gene(s) encoding those improved enzymes are, if necessary, used as parents for the next round of evolution. The basis of laboratory evolution, also referred to as directed evolution, was laid by Pim Stemmer in 1994. He proposed a recombination system for genes (‘gene shuffling’, or ‘molecular breeding’) by using random fragmentation of two or more genes and their subsequent reassembly (Stemmer, 1994 a,b). Compared with the error-prone PCR method in which few point mutations are introduced, gene shuffling results in blocks of mutations. Since then, numerous variations on this theme have been developed, each specific to certain types of proteins or desired outcomes (Yuan et al., 2005). Currently, laboratory evolution principles have been used to improve a variety of enzyme properties: enantioselectivity (Reetz et al., 2004), catalytic efficiency or rate (Van der Veen et al., 2004, 2006), enzyme stability (Kim et al., 2003; Eijsink et al., 2005), pH activity profile (Bessler et al., 2003), enzyme functionality in organic solvents (Castro and Knubovets, 2003), product inhibition (Rothman et al., 2004) and substrate specificity (Zhang et al., 1997; Yano et al., 1998; Oue et al., 1999). Further developments in the field of laboratory evolution take the procedures to a different level. Enzymatic pathways (Masip et al., 2004; Umeno et al., 2005) and genomes (Patnaik et al., 2002; Zhang et al., 2002; Dai and Copley, 2004) are now subjected to various shuffling

18

Novel enzyme technology for food applications

Fig. 2.1

Key steps in a typical laboratory evolution experiment.

protocols, with various outcomes. Unlike natural recombination, the genetic material (gene, operon, genome) of more than two parents may be shuffled in a single laboratory evolution experiment.

2.2.1 Techniques used in laboratory evolution The major steps in a typical laboratory evolution experiment are outlined in Fig. 2.1. The genetic diversity for evolution is created by mutagenesis and/or recombination of one or more parent sequences. These altered genes are cloned back into a plasmid for expression in a suitable host organism. Clones expressing improved enzymes are identified in a high-throughput screen (see Section 2.2.2) or in some cases by selection, and the gene(s) encoding those improved enzymes are isolated and if necessary recycled to the next round of laboratory evolution. The most widely used approaches for generating diversity are random point mutagenesis and in vitro recombination. The commonly used technique to create random point mutants is error-prone PCR. The fidelity of the DNA polymerase is decreased (i) by adding divalent cations (for example Mn2+) to substitute for the optimal Mg2+ in the PCR mixture or (ii) by employing high error rate DNA polymerases (for example Taq polymerase lacking proofreading activity, Mutazyme which is designed to make mistakes) that will incorporate mismatching bases at a

Improving enzyme performance in food applications

19

A

Fragmentation

B Reassembly of fragments (recombination)

C

Selection or screening

D Fig. 2.2 Basic DNA shuffling scheme. (A) The starting pool of homologous genes can be either a library of random mutants of a parental gene (e.g. by error-prone PCR) or a family of related genes. The pool of genes is fragmented with DNaseI. (B) Pool of random DNA fragments. (C) The fragments are reassembled into full length genes by repeated cycles of PCR without added primers. During this step the fragments prime each other in the homologous regions, resulting in recombination when fragments derived from one parental gene prime another one, causing a template switch (crossovers shown by the black line). Reassembly of the random fragments into full length genes results in frequent template switching and recombination. (D) A selected pool of improved recombinants provides the starting point for another round of mutation and/or recombination.

controllable rate during gene amplification. A low error rate (2–3 base substitutions or ~1 amino acid changes) accumulates mostly adaptive mutations, whereas higher error rates merely generate neutral and deleterious mutations (Arnold et al., 2001). DNA shuffling methods enable the in vitro recombination of DNA sequences that are more or less related, either orthologous genes or error-prone PCR variants (Fig. 2.2). DNA shuffling requires a degree of identity >55–65%. Techniques such as incremental truncation for the creation of hybrid enzymes (ITCHY) are variant methods that allow for recombination of sequences with a lower degree of homology. In the last decade a multitude of methods have been developed to enable shuffling of genes with lower homology, to improve mutant libraries by negative selection for wild-type sequences or to obtain a less biased library (Kurtzman et al., 2001). Most new methods have been invented to solve the drawbacks of the original protocol or to circumvent patent limitations. Although these alternative procedures aim to solve one problem, they usually appear to create another one. For example, it is difficult to start with highly different parent genes and still reach a high recombination frequency. Methods aiming at higher

20

Novel enzyme technology for food applications

recombination frequencies start with more homologous parents, thereby introducing fewer mutations. On the other hand, methods have been developed to recombine genes without any homology. These processes result mainly in only one crossover, although some methods do generate multiple crossovers at fixed places. Another way of obtaining a high recombination frequency is by starting from synthetic oligonucleotides. The advantage of this method is that any mutant can be constructed and thereby the largest possible sequence space can be explored. An additional advantage is the possibility of using codons other than the original ones in order to obtain more homology. Furthermore, the preferred codon usage for the expression host can be applied in the synthetic oligonucleotides. The largest disadvantages of synthetic methods are the high costs and the large size of the library, which quickly exceeds the most elaborate screening and selection methods (Ostermeier, 2003). Other conflicting parameters seem to be speed and bias. Recombination methods that aim to generate unbiased libraries all consist of numerous steps in order to achieve this, resulting in more elaborate procedures, while quick protocols normally result in more wild-type backgrounds and a biased library. Most of the methods mentioned can result in good mutant libraries. Therefore, the choice for one or another strategy is usually led by the size of the protein, the goal of the research, the existence of homologous proteins, the selection and/or screening capacity and practical issues like the equipment and expertise in the research group. Table 2.1 summarizes some of the methods that have been successfully utilized for laboratory evolution of a variety of proteins. This is not a complete list, as new techniques and strategies for laboratory evolution are constantly arising.

2.2.2 Selection and screening The success of a laboratory evolution experiment depends greatly on the method that is used to select the best mutant enzyme. Since most laboratory evolution experiments generate a huge mutant library, it is very important to develop an efficient method of screening this library for the desired property. Both selection and screening strategies have been developed for all kinds of enzyme functions (Boersma et al., 2007). The big challenge in these strategies is generally to make the improved function quantifiable, that is to differentiate signal from noice. Enzymatic assays have to be sufficiently sensitive and specific to identify positive mutants (Zhao and Arnold, 1997). Selection is based on the fact that mutants with the desired enzyme function have an advantage over wild-type enzymes; variants are selected because, under certain conditions, they enable the host to grow. Selection in the laboratory mimics the natural survival-of-the-fittest strategy and is the most efficient method to find the best mutant, since only mutants of interest will appear. Unfortunately, this approach is not possible for all enzymatic activities. For in vivo selection this means that only enzyme activities with a growth or survival advantage can be used. Only a few industrially interesting enzymes are essential for the bacterial cell

Improving enzyme performance in food applications

21

themselves, so most selection methods are based on enzymatic activities that lead to the generation of a product that is essential for growth of the expression host. The coupling of the desired enzymatic reaction to survival in the selection step often requires the development of complex, non-trivial and intelligent assays (Taylor et al., 2001). Sometimes, this means that the substrates in these selection systems are not the desired substrates, but analogues thereof. This may result in the selection of undesired mutants with activity towards the analogue and not towards the wanted substrate. It is, therefore, very important to choose the selection substrate carefully, since the first law of laboratory evolution is: ‘you get what you select/screen for’ (Schmidt-Dannert and Arnold, 1999). In vitro selection is usually based on binding the enzyme to the desired substrate or a transition state analogue, although strategies in which catalytic properties are used for selection are also described (Boersma et al., 2007). These methods are mostly based on a physical linkage between phenotype and genotype. The first established and most used technique is phage display, which has been successfully used to find improved enzymes. In this system, the enzyme of interest is fused to a coat protein of a filamentous phage and as such displayed on the outside of the phage, where in principle it is able to retain enzymatic activity. Since the gene encoding the displayed protein is present in the phage particle, the gene of the mutant enzyme with the desired property is linked to its phenotype (FernandezGacio et al., 2003). When the displayed proteins may be toxic to filamentous phage assembly or incompatible with the bacterial secretion pathway, lytic phages can be used that allow displayed sequences to minimize negative selection. Other in vitro selection methods with a physical phenotype–genotype linkage are cell-surface display, ribosome display, plasmid display and mRNA-protein fusion (Lin and Cornish, 2002; Becker et al., 2004). Recently, a different approach was described to maintain a linkage between genotype and phenotype. In vitro compartmentalization is a method in which compartments are formed as aqueous droplets in water-in-oil emulsions which contain only one gene and a complete transcription/translation machinery (Tawfik and Griffiths, 1998). These droplets mimic a bacterial cell by keeping the gene and its product together. The droplets containing an enzyme with the desired activity can be selected by fluorescence activated cell sorting (FACS) or, when the gene is physically bound to the substrate, by breaking the droplets and fishing out the desired product (Griffiths and Tawfik, 2000). The advantages of in vitro over in vivo selection are the larger sample size of a mutant library that can be handled and as such the larger amount of possible enzyme variants to be tested. Drawbacks are that making the right water-in-oil emulsions with only one gene per droplet is tricky, and that the efficiency of the in vitro transcription and translation can be a bottleneck. Another way of finding the desired mutant enzyme is by screening. In screening methods all mutants have to be tested for the desired enzymatic reaction, even those that might not be active or accurately folded. The advantage is, however, that almost every enzymatic reaction can be tested, since the activity does not have to be dependent on growth rate or the formation of essential products. This can be done in a qualitative way by relatively simple visual screens such as the formation

22

Table 2.1

Laboratory evolution techniques Description

Literature

Error-prone PCR

Introduces random point mutations by imposing imperfect, and thus mutagenic, reaction conditions. Mutagenesis in vivo is performed by transforming a plasmid containing the gene to be mutagenized to a mutator E. coli strain. These strains lack DNA repair mechanisms or contain a modified polymerase with lower fidelity. With this approach, it is possible to create a library of mutants containing all possible mutations at one or more pre-determined target positions in a gene sequence.

Leung et al., 1989 Cadwell and Joyce, 1992 Greener et al., 1997 Camps et al., 2003

In vivo random mutagenesis

Saturation mutagenesis

DNA shuffling Synthetic shuffling

Staggered extension process (StEP) in vitro recombination

Random chimeragenesis on transient templates (RACHITT)

DNA is randomly digested and allowed to recombine to form novel sequences. Using degenerate oligonucleotides, every amino acid from a set of parents is allowed to recombine independently of every other amino acid. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage can also be incorporated. StEP consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerasecatalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. DNA shuffling method for generating highly recombined genes. The approach relies on the ordering, trimming and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold.

Miyazaki and Arnold, 1999 Zheng et al., 2004 Wong et al., 2004 Wong et al., 2005 Stemmer, 1994 a,b Minshull and Stemmer, 1999 Ness et al., 2002 Ostermeier, 2003

Zhao et al., 1998 Aguinaldo and Arnold, 2003 Zhao, 2004

Coco et al., 2001 Coco, 2003

Novel enzyme technology for food applications

Technique

Sequence independent site-directed chimeragenesis (SISDC) Structure based combinatorial protein engineering (SCOPE) Incremental truncation for creation of hybrid enzymes (ITCHY)

Degenerate homoduplex gene family recombination (DHR)

This procedure maintains alignment between two parental genes, and produces cross-overs mainly at structurally related sites along the sequences. A combination of PCR reassembly and in vivo recombination in yeast produces highly shuffled libraries. Procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This approach seeks to randomly swap polymorphisms between a collection of polymorphic genes. This technique is different from shuffle techniques in that random segments of genes are not recombined, but homologous segments containing point mutations are.

Hiraga and Arnold, 2003 O’Maille et al., 2002 O’Maille et al., 2004 Ostermeier et al., 1999 Horswill et al., 2004

Lutz et al., 2001 Kawarasaki et al., 2003 Sieber et al., 2001 Udit et al., 2003 Abecassis et al., 2000 Gibbs et al., 2001 Coco et al., 2002

Improving enzyme performance in food applications

Creating multiple-crossover DNA libraries independent of sequence identity (SCRATCHY) Sequence homology-independent protein recombination (SHIPREC) Combinatorial libraries enhanced by recombination in yeast (CLERY) Degenerate oligonucleotide gene shuffling (DOGS)

By using inserted marker tags this technique allows at discrete sites for the recombination of sequences that are not related at all. A semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. Incremental truncation, a method for creating a library of every one base truncation of dsDNA, creates diversity by changing the length of a gene. The combination of two incremental truncation libraries creates diversity by fusing two gene fragments. Performing ITCHY between two different genes generates libraries of fusion proteins in a DNA-homology independent fashion. This technique is based on the ITCHY technique but includes an extra round of gene shuffling.

23

24

Novel enzyme technology for food applications

of coloured or fluorescent products or halos around a colony on a plate. For protein functions such as catalysis of a specific reaction or substrate specificity this is very difficult or even impossible. Quantitative methods are better suited to screening for these enzymatic activities, but are usually more labor-intensive. This means that on a normal time scale only small libraries can be tested, or that high-throughput screening (HTS) has to be employed. HTS demands miniaturization and automation of enzymatic assays. In the past decade, a lot of research has been focused on finding better, cheaper, quicker and more accurate HTS assays (Cohen et al., 2001; Aharoni et al., 2005). This has made HTS feasible for many laboratories all over the world, resulting in many smart enzymatic screening methods. A screening method is used to find the best enzyme from a large pool of mutants. The number of clones that results depends on the accuracy of the assay and should be optimized for every laboratory evolution experiment. For this optimization one should consider (i) the size of the library, (ii) the number of (false) positive and false negative colonies that can be allowed, (iii) the costs of the assay and (iv) the possibility of performing a more accurate assay with the best mutants. Most screening assays are based on spectrophotometric methods in 96 or 384 wells plates (most often colorimetric detection) or fluorescent methods. Usually raw cell extracts are incubated with the substrate or an analogue thereof; this is followed by measuring either substrate consumption, cofactor conversion or product generation. Another possibility is the use of a discontinuous assay in which the product is used by a second enzyme, which allows for the indirect analysis of product formation of the enzyme of interest. More HTS methods are developed every day, both for specific enzymatic reactions and general applications, making screening the method of choice for many researchers.

2.3

Examples of improving enzyme stability and functionality by laboratory evolution

2.3.1 Enzyme stability Naturally available enzymes are often not optimally suited for industrial applications. This incompatibility generally relates to the stability of the enzymes under process conditions. Although it sometimes is beneficial to adapt industrial processes to the mild and environmentally friendly conditions favoured by the enzyme, the use of more extreme conditions is often desirable. Despite many successful efforts to understand the structural basis of protein stability, there is still no universal strategy for stabilizing any protein by a limited number of rationally designed mutations (Eijsink et al., 2004). It is concluded that protein stability appears to be the result of many small stabilizing features. Although the rational design (see Section 2.4) of enzyme stabilization has been successful in several instances (e.g. Nielsen and Borchert, 2000 for α−amylase; Van den Burg et al., 1998 for protease), it relies heavily on the availability of a high-resolution 3D

Improving enzyme performance in food applications

25

structure. Therefore, the random techniques used in laboratory evolution can be a powerful alternative for improving enzyme stability. The method used for screening the libraries obtained during laboratory evolution is extremely important. For instance, the distinction between thermal tolerance and real thermal stability is important when considering thermal stability. Thermal tolerance refers to the ability to withstand incubation at elevated temperatures, without necessarily being active at those temperatures. Real thermal stability refers to enzymes that not only withstand elevated temperatures, but that also retain activity at these temperatures. Clearly, these two types of properties need different screening regimes. It is important to ensure that the screening procedure accounts for all the properties that one wishes to improve, while ensuring that other qualities that are important for a certain process (for instance activity) are preserved. Screening directly for activity under denaturing conditions (for example high temperature, extreme pH, organic solvents) is a simple and good method, but this may pose some practical problems. For example, in the case of thermal stability one faces the limitation that most high-throughput microplate readers can only reach temperatures of about 50 °C. Most strategies for screening for enzyme stability rely on measuring residual enzyme activity after exposure to a denaturing challenge on a filter or in microtitre plate wells (Eijsink et al., 2005). There are alternative methods for stability selection based on phage display (e.g. the Proside method, a phage-based method for selecting thermostable proteins; Sieber et al., 1998; Martin et al., 2003) or the use of extremophilic microorganisms as expression host, for example Thermus thermophilus has been used for the selection of thermostable selection markers (e.g. Hoseki et al., 1999 for kanamycin; Brouns et al., 2005 for bleomycin resistance) and enzymes (e.g. Tamakoshi et al., 2001 for 3-isopropylmalate dehydrogenase). The value of laboratory evolution in stabilization of enzymes was illustrated by Richardson et al. (2002), who used it for the development of a stable amylase for starch liquefaction in corn wet milling. Corn wet milling is an example of a multistep industrial process in which improvement of enzyme performance is desired. The process would benefit from an α-amylase capable of liquefying starch at pH 4.5 and 105 °C without the addition of calcium. High-throughput screening of microbial DNA libraries from various environments was used to identify αamylases with activity at low pH and high temperature in the absence of calcium. This large screening effort yielded 15 primary hits and three variants that have good properties with respect to at least one of the desired phenotypes were selected for optimization by DNA shuffling. This next step was performed by gene reassembly, random ligation of a pool of restriction site-determined fragments of about 150 bp, from all three selected genes. The resulting library of 21,000 clones was first screened for activity at pH 4.5 and high temperature. Subsequently, improved variants resulting from this screening (<100) were tested for thermal stability, liquefaction ability, expression levels and dependence on calcium. After this biochemical and process-specific characterization of the best variants, one αamylase with exceptional process compatibility was identified (Richardson et al., 2002).

26

Novel enzyme technology for food applications

Xylanases catalyse the hydrolysis of xylan, a major constituent of hemicellulose. The enzyme is the focus of much attention because of its potential for use in industrial processes, including the paper and pulp industries and food and feed industries. Many efforts have been made to improve the properties of xylanase in handling industrial tasks. In particular, thermostability is a major target of such modifications. Recently, Miyazaki et al. used laboratory evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. At first, random mutagenesis (error-prone PCR) was carried out for the entire sequence of the xylanase gene and the mutant library was screened for thermostability. Next, site-saturation mutagenesis was employed to optimize the sequence at three positions, which were identified as positions that are involved in thermostability by the random mutagenesis approach. Improved variants were then recombined by DNA shuffling. This combination of random mutagenesis, saturation mutagenesis, and DNA shuffling yielded a thermostable variant, which contained three amino acid substitutions. The half-inactivation temperature (the midpoint of the melting curves) for the xylanase variant compared with the wildtype enzyme after incubation for 10 min was elevated from 58 to 68 °C and at 60 °C the thermostable variant retained full activity for more than 2 h, whereas the wild-type enzyme inactivated within 5 min (Miyazaki et al., 2006).

2.3.2 Enantioselectivity By inverting the enantioselectivity of a key enzyme in a multi-enzyme pathway, a process for the production of L-methionine in E. coli has been improved. Through random mutagenesis, saturation mutagenesis and screening the enantioselectivity of a D-hydantoinase was inverted (from enantiomeric excess eeD = 40% to eeL = 20%) and its total activity with D,L-5-(2-methylthioethyl)hydantoin (D,L-MTEH) as substrate was increased fivefold. Introduction of the evolved L-hydantoinase into a recombinant whole-cell catalyst increased productivity for L-methionine and decreased accumulation of an undesired intermediate, compared with cells with the wild-type pathway. It is worth noting that a single amino acid substitution was sufficient to invert the hydantoinase enantioselectivity. Highly D-selective variants (eeD = 90%) containing a single amino acid substitution were also found (May et al., 2000). Until recently the number of studies on the laboratory evolution of enantioselective biocatalysts has been limited, one reason being the lack of generally applicable high-throughput screening methods. However, several efficient high-throughput ee assays have been developed lately, so that today the analytical bottleneck for improving the enantioselectivity of many enzymes can be solved. The most efficient ee assays are based on mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR) which allow 1000–10,000 samples to be evaluated per day (Reetz, 2004).

2.3.3 Genome shuffling Current methods for the improvement of industrial microorganisms range from the

Improving enzyme performance in food applications

27

random approach of classical strain improvement (CSI; sequential random mutagenesis and screening) to the highly rational methods of metabolic engineering. The first strategy is resource intensive and time-consuming, requiring many generations of mutation and selection to allow accumulation of multiple beneficial mutations in a single strain. The efficacy of the second strategy is limited by the ability to predict which mutations will improve a particular phenotype. Thus, it is not possible to take advantage of mutations in genes that are not obviously related to the phenotype of interest but may nevertheless improve microbial fitness or performance under a particular set of conditions. Genome shuffling is a random method that is much more efficient for evolution of strains of microbes with desirable phenotypes. Genome shuffling is a process that combines the advantage of multi-parental crossing allowed by family DNA shuffling with the combination of entire genomes normally associated with conventional breeding. The driving force for the accelerated evolution is the recombination of multiple parents in a recursive manner. The advantage of genome shuffling over CSI has been demonstrated for Streptomyces fradiae, a commonly used strain for commercial production of the complex polyketide antibiotic tylosin (Zhang et al., 2002). To generate a diverse population for genome shuffling, a low-production parental strain was subjected to one round of CSI using nitrosoguanidine as mutagen. Two rounds of genome shuffling based on recursive protoplast fusion of mixed populations and screening for tylosin production resulted in mutant strains with productivities similar to that of the commercial strain SF21. However, while it took 20 years and about 1 000 000 assays for the 20 rounds of CSI required to obtain SF21, similar results were produced with 24 000 assays in 1 year of genome shuffling. Patniak et al. (2002) demonstrated the use of genome shuffling for improved acid tolerance in production of lactic acid by lactobacilli. Lactobacilli are of interest in the commercial production of lactic acid, a compound that has long been used as a food additive and as an important feedstock for the production of other chemicals such as 2,3-pentadione. Improving the growth and lactic acid production of lactobacilli at low pH could decrease waste and the cost of product purification. Growth at low pH involves multiple widely distributed loci and without an understanding of the mechanism of pH tolerance in bacteria, a rational approach to engineering pH tolerance was impractical. In addition, the Lactobacillus strain used in their study is of commercial interest, but has not been genetically characterized. Lactobacillus strains with improved low-pH tolerance were first obtained by CSI in order to generate the initial biodiversity pool and then shuffled for five rounds by recursive protoplast fusion. The shuffled population contained new strains that grew at substantially lower pH (pH 3.8) than the wild-type strain does and improved strains that produced threefold more lactic acid than the wildtype strain at pH 4.0 (Patnaik et al., 2002). Genome shuffling was also used to improve the degradation of pentachlorophenol (PCP) by the Gram-negative bacterium Sphingobium chlorophenolicum (Dai and Copley, 2004). Degradation of pentachlorophenol, a highly toxic anthropogenic pesticide, is slow and S. chlorophenolicum cannot tolerate high levels of

28

Novel enzyme technology for food applications

PCP. To generate a diverse population for genome shuffling, S. chlorophenolicum was subjected to one round of CSI using nitrosoguanidine as mutagen. Three successive rounds of protoplast fusion were carried out and after each round, the concentration of PCP in the plates used for selection was increased. Several strains obtained after the third round of shuffling grew on plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of concentrations higher than 0.6 mM. Some mutant strains were able to degrade 3 mM PCP completely in liquid media, whereas no degradation could be achieved by the wild-type strain. The examples described all use recursive protoplast fusion as a useful method for genome shuffling, but other mechanisms of genetic exchange (such as conjugation and phage-mediated transduction) are also useful for the genomic shuffling of bacteria and lower and higher eukaryotes. As most of these approaches rely on natural homologous recombination, organisms modified by these means are not considered to be ‘genetically modified’. Genome shuffling thus represents a practical method for the rapid manipulation of the complex phenotypes of whole cells and organisms (Stephanopoulos, 2002; Zhang et al., 2002).

2.4

Rational and computational protein engineering

The ‘holy grail’ of rational protein engineering and computational design is the accurate de novo design of proteins with specified characteristics. However, the astronomical number of possible interactions in an average protein presents computational problems and currently limits de novo prediction of protein structure to small proteins (Baker, 2006). As most proteins of interest are fairly large, this implies that proteins of known structure (or which are highly homologous to a protein of known structure) will be the most amenable to rational and computational engineering. This is in contrast to the aforementioned laboratory evolution methods, which require no knowledge of protein structure. A high-resolution structure is most preferable, but a reliable model (supplemented by sequences of homologues, mutant characterization, reaction mechanism and the like) may also facilitate design. Rational molecular engineering requires the establishment of a set of desired design goals and a paradigm with which to rank proposed sequences and structures. For computational design, this takes the form of a quantitative molecular fitness function for the system. This typically involves ‘first principles’ biophysical components (Kraemer-Pecore et al., 2001), for example ‘van der Waals’ steric contacts, hydrogen bonds, electrostatics and cavity formation, but may also include indirect terms such as statistical preponderance of side chains in given combinations or protein microenvironments. Depending on the level of sophistication of the engineering experiment, terms describing bond length stretching, bond angle bending and quantum mechanical terms may be added to the fitness function. Recent developments in computational protein engineering include the repeated cycling between different optimization techniques (for example Monte Carlo and gradient minimization; Baker, 2006), dead-end elimination and hydrogen bond

Improving enzyme performance in food applications

Fig. 2.3

29

Fitness of an enzyme for functions 1 and 2 versus sequence space.

inventorying (Looger et al., 2003)), design of sequence libraries instead of individual sequences (Saraf et al., 2006) and the simultaneous design of multiple molecular components. Recent design goals of rational and computational engineering have been thermostabilization (Korkegian et al., 2005), altering of the specificity of a superfamily of bacterial binding proteins (Looger et al., 2003), re-engineering protein–protein interactions (Kortemme et al., 2004), alteration of enzyme substrate specificity (Ashworth et al., 2006; Park et al., 2006) and de novo design of activity (Dwyer et al., 2004). It is likely that the greatest advances will be made by a judicious combination of rational and evolutionary techniques. Both methodologies are employed to select particular regions of sequence space for experimental analysis. The metaphor of climbing a mountain range has been proposed for sequence space optimization; laboratory evolution may be thought of as ascending to the highest peak within a given mountain range (path A in Fig. 2.3). It seems likely that rational design will facilitate more dramatic sequence space movements, as >20 simultaneous mutations may be required for a novel function within a given scaffold. It thus seems appropriate to describe rational design as being capable of discovering a new island in sequence space and landing on the beach (path B in Fig. 2.3), after which laboratory evolution may discover the peak of the island (path C in Fig. 2.3).

2.4.1 Laboratory techniques for the construction of designed sequences Once individual sequences or sequence libraries have been designed, they must be fabricated in the laboratory and tested. Quickchange mutagenesis (Stratagene) is a reliable method for introducing mutations, but is limited to a single mutagenic oligonucleotide per round, whereas Kunkel mutagenesis (Kunkel, 1985) efficiently introduces multiple mutagenic primers simultaneously. ‘Inside-out’ PCR techniques have also been employed for the assembly of target sequences. Finally, rational design may be employed to design sequence libraries, which can be input to laboratory evolution methods.

30

2.5

Novel enzyme technology for food applications

Examples of improving enzyme stability and ability by rational protein engineering

2.5.1 Enzyme stability Proteins exhibit marginal stabilities equivalent to only a small number of weak intermolecular interactions (Jaenicke, 2000). Therefore, a single successfully engineered stabilizing interaction can lead to a significant increase in overall protein stability. Factors that contribute to protein stability are packing of the hydrophobic core, disulfide and salt bridges, hydrogen bonding and secondary structure propensities (Strickler et al., 2006). The mobility of a single loop can determine the overall stability of a protein. Eight designed mutations mainly in a 14-residue surface loop increased the kinetic stability of the moderately stable metalloprotease from Bacillus stearothermophilus CU21 more than 350-fold at 100 ºC while retaining wild-type catalytic properties (Van den Burg et al., 1998). Since inactivation of the enzyme was thought principally to occur by autodegradation, the mutations were designed to reduce the mobility of the surface loop. The stabilized protein contained an engineered disulfide bridge, a rigidifying Xaa→Pro mutation and five residues present in thermolysine, a close thermostable homologue of the protease. The study showed that protein stability is acquired by a combination of multiple stabilizing interactions and that low-temperature activity and enzyme stability can be uncoupled. The importance of efficient packing of the hydrophobic core in protein stability was shown by computational redesign of the labile, 153 amino acid, dimeric yeast cytosine deaminase (Korkegian et al., 2005). Using the RosettaDesign scoring function and a Metropolis Monte Carlo search algorithm, target sequences were threaded onto the backbone of the available 3D-structure of the enzyme and mutated towards a lower energy. Residues in and directly surrounding the active site, as well as those involved in oligomerization were excluded, subjecting about 40% of the residues to the design. Half of those emerged as wild-type after the design. Of the 33 predicted substitutions, those at the protein surface were ignored. Finally, three mutations, all resulting in a more efficient packing of the hydrophobic core, were found to be stabilizing (Fig 2.4a). The triple mutant resulted in a 30-fold increase of kinetic stability at 50 ºC. One remaining challenge for computational redesign algorithms is the modelling of the interactions of buried polar and charged side chains (Korkegian et al., 2005). Interactions at the protein surface also contribute to thermostability, as protein structures of proteins from hyperthermophilic origin have indicated (Vieille and Zeikus, 2001), and provide another possibility for improving the stability of enzymes. The contribution of surface salt bridges to stability depends on the spatial orientation of the involved residues, their desolvation upon salt bridge formation and the local context of the salt bridge (Makhatadze et al., 2003). By combining a genetic algorithm for evolution of protein sequences with a computational evaluation of surface charge–charge interactions, five proteins ranging from 72–100 amino acids in size were stabilized up to 18 kJ mol–1 resulting from the introduction of only a few salt bridges (Fig 2.4b, Strickler et al., 2006).

Improving enzyme performance in food applications Wild-type protein

31

Designed protein

(a)

(b)

(c)

(d)

(e)

Fig. 2.4 Examples of designed proteins discussed in the text. I are wild-type proteins and II are designed proteins. (a) Improved packing of hydrophobic core in yeast cytosine deaminase (Korkegian et al., 2005). (b) Optimized surface charge distribution of ubiquitin (Strickler, 2006). (c) Designed trinitrotoluene-binding protein (Looger et al., 2003). (d) Designed affinity of homing endonuclease I-MsoI for a novel recognition sequence (Ashworth et al., 2006). (e) Designed cefotaxime hydrolase (β-lactamase) (Park et al., 2006).

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Another computational technique, which has been experimentally validated, is the design of particular stabilizing interactions such as disulfide bonds. Recently the computational design of a disulfide-stabilized double mutant of the penicillin acylase from Alcaligenes faecali has been reported (Wang et al., 2006). The mutant exhibits a 50% increased half-life at 55 °C and will be useful for antibiotic biosynthesis.

2.5.2 Reaction mechanism The glycoside hydrolases present a diverse group of enzymes that are of great significance for the food industry. Consequently, many studies have been undertaken to engineer their activity. Glycoside hydrolases that use the retaining mechanism for cleavage of glycosidic bonds have been engineered into ‘glycosynthases’ and ‘thioglycoligases’ for the exclusive synthesis of oligosaccharides and thioglycosides by engineering of the catalytic residues in the active site. To obtain glycosynthases, the catalytic nucleophile of retaining glycosidases was removed (Mackenzie et al., 1998). These inactivated enzymes are able to synthesize oligosaccharides from glycosyl fluorides with inverted anomeric stereochemistry and suitable acceptors in high yields. This approach has been extended to several other glycoside hydrolases like exo-β-glycosidases (Trincone et al., 2000), endo-β-glycosidases (Fairweather et al., 2002, 2003; Van Lieshout et al., 2004), endo-β-xylanases (Sugimura et al., 2006), exo-α-glucosidases (Okuyama et al., 2002) and exo-α-xylosidases (Moracci et al., 2001). Thioglycoligases lack the catalytic general acid/base residue of retaining glycosidases and use dinitrophenyl glycosides as donors and deoxythiosugars as acceptors (Mullegger et al., 2005). Cyclodextrin glucano transferases (CGTases) of the α-amylase family convert starch into a mixture of α, β and γ-cyclodextrins, circular donut-shaped dextrin molecules of 6, 7 or 8 glucose molecules with a hydrophilic exterior and a hydrophobic core that can be used for micro-encapsulation of hydrophobic molecules, thus protecting it from an aqueous environment. One of the applications of cyclodextrins in food is as a flavour preservative. Based on comparison of crystal structures substrate binding sites have been probed by site-directed mutagenesis and resulted in optimized CGTase variants with tailored α, β and γ cyclodextrin yields (Wind et al., 1998). In addition, CGTase could be converted from a glucano transferase into a starch hydrolase by a single site-directed mutation in acceptor substrate binding site +2 (Leemhuis et al., 2002). An additional substitution in acceptor subsite +1 resulting in the same reversal of reaction specificity was identified by a laboratory evolution approach (Leemhuis et al., 2003).

2.5.3 Ligand and substrate specificity There have been several successful alterations in the specificity of receptors and enzymes. A number of members of the bacterial periplasmic binding protein (PBP)

Improving enzyme performance in food applications

33

superfamily have been structurally solved in the ligand-free open and/or ligandbound closed form (Dwyer et al., 2004), allowing careful study of this general hinge-bending mechanism of ligand binding. The modularity of this conformational change has allowed the computational diversification of these binding proteins for a variety of ligands. Metal-binding sites (Marvin and Hellinga, 2001) and nascent metalloenzyme active sites (Benson et al., 2002) have been designed into family members. Subsequently, binding sites for trinitrotoluene, serotonin, lactate and a soman nerve gas analogue were designed into proteins with a cognate specificity for sugars and amino acids (Fig 2.4c; Looger et al., 2003; Allert et al., 2004). In several cases, the proteins were produced in bacteria as part of a synthetic signal transduction cascade, harnessing the designed binding event to initiate expression of a reporter gene. The interaction of proteins with other proteins (Kortemme et al., 2004) and with nucleic acids (Ashworth et al., 2006) has also been systematically altered by computational design. A recent experiment of particular note is the in silico affinity maturation of a therapeutic antibody by computational protein design (Clark et al., 2006), in which the already-tight binding affinity of a clinically relevant antibody was computationally improved by an order of magnitude. The DNA-binding and DNA-cleaving specificity of the homing endonuclease IMsoI has been altered by computational design (Ashworth et al., 2006). A single base-pair substitution known not to be well recognized by the wild-type enzyme was modelled in complex with the enzyme in silico, and interfering side chains were identified. Computational design was used to repack the protein–nucleic acid interface, with particular emphasis on the formation of precise hydrogen bonds with the novel DNA bases introduced. A double mutant was predicted and shown to cleave the target DNA with 10 000-fold increased efficiency relative to the wild-type enzyme; then it was crystallized and shown to adopt the target interface structure (Fig 2.4d). In a demonstration of complementary methods for modelling and design, the known crystal structure of human butyrylcholinesterase was used to create a model of cocaine docked into the active site (Pan et al., 2005). Molecular dynamics was then employed to model the ensemble of conformations, which the substrate can assume in the binding pocket and in silico mutagenesis was used to select a quadruple mutant predicted to stabilize the hydrolytic transition state of cocaine better than the wild-type protein. The mutant was experimentally characterized and shown to catalyse the breakdown of cocaine roughly 500-fold faster than the wild-type enzyme. An enzyme useful for ‘green synthesis’ of the flavour vanille, the vanillylalcohol oxidase (VAO), has been improved by alteration of its stereospecificity (van den Heuvel et al., 2000). The architecture of the active site of VAO has been perturbed by its rational engineering; transfer of the reactive side chains to the opposite face of the active site cavity leads to a significant increase in specificity (>80% enantiomeric excess) for the desired substrate enantiomer. Crystal structure determination of the designed double mutant indeed has verified the proposed active site structure.

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2.6

Novel enzyme technology for food applications

Examples of combined laboratory evolution and computational design

As stated above, the most successful designs are likely to result from the combined efforts of rational/computational design and laboratory evolution (Chica et al., 2005). Computational methods may be used directly to design sequence libraries (Voigt et al., 2001); alternatively, designed sequences may be optimized by evolutionary methods. Feedback of experimental results into modelling steps may increase the accuracy of fitness functions used in the initial design steps. The following examples demonstrate the power of combining rational protein engineering and laboratory evolution. The E. coli ribose-binding protein, a member of the penicillin binding protein (PBP) superfamily and catalytically inert, has been converted into a triose phosphate isomerase (TIM) enzyme via computational design followed by laboratory evolution (Dwyer et al., 2004). A model of the target active site geometry was constructed by analysis of the crystal structure complex of a transition state analogue in wild-type TIM. Binding pocket side chains and a transition state model were simultaneously optimized in silico to produce a set of putative active sites, which were then repacked to produce a set of final designs. The resulting designs contained 18 to 22 mutations, exhibited a rate enhancement of more than 105 over that of the uncatalysed reaction and are biologically active. The best design exhibited a Kcat/Km ratio for the conversion of dihydroxyacetophenone phosphate to glyceraldehyde-3-phosphate, which was still about three orders of magnitude less than the ratio for wild-type triose phosphate isomerase. Subsequently, a laboratory evolution approach has been used to improve the Kcat/Km ratio of the designed enzyme. Km is a constant that is equal to the substrate concentration at which an enzyme reaction proceeds at half the maximum velocity. This represents (for enzyme reactions exhibiting Michaelis–Menten kinetics) the affinity for the substrate. The larger the Km, the weaker the binding affinity of enzyme for substrate. The catalytic constant, Kcat, is also called the turnover number of the enzyme, that is, the number of reaction processes (turnovers) that each active site catalyzes per unit time. The Kcat/Km ratio is a measure of the enzyme’s catalytic efficiency. Final protein variants (~3–5 additional mutations) were selected with sufficient activity to complement a TIM-deficient strain. As is often the case, many of the accumulated changes identified by laboratory evolution were localized at the protein surface, in regions distant from the active site and their effect on activity is therefore difficult to rationalize and, with present knowledge, impossible to predict (Dwyer et al., 2004). A similar approach was used to alter the substrate specificity of the (α/β)8 hydrolytic enzyme glyoxalase II from S-D-lactoylglutathione to the antibiotic cefotaxime (Park et al., 2006). Rational design was used to graft the active site loops from β-lactamase into glyoxalase, with insertions and deletions both used to select optimal grafts. Initial designs were subjected to numerous rounds of PCR shuffling followed by increasingly stringent antibiotic selection. Final designs were 59% identical to the parent glyoxalase scaffold and produced a 100-fold increased cefotaxime resistance in transformed bacteria (Fig 2.4e).

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Combined rational and evolutionary protein engineering has also been applied to enzymes of industrial relevance. The resistance of α-amylase to both thermal (Nielsen and Borchert, 2000) and pH-induced (Nielsen et al., 2001) unfolding has been increased by both techniques. Rational methods, such as the introduction of prolines, optimization of a metal-binding site and improvement of electrostatic interactions, were used to render the α-amylase scaffold more robust. Subsequent random mutagenesis identified further beneficial mutations not predicted by any of the rational improvements.

2.7

Summary and future trends

The literature contains a large number of studies in which enzymes have been improved by rational or random approaches. It has been shown that both approaches have strengths, but also weaknesses. Rationale-based engineering on the one hand requires for instance a high-resolution three-dimensional model and insight into the structure–function relationships of the biocatalyst of interest. Laboratory evolution on the other hand requires the ability to screen large libraries; when major adjustments are desired, the leap in sequence space may be too big to bridge. In other words, the chance is extremely small of successfully obtaining a protein variant with more than five specific amino acid substitutions – this will only work when extremely large libraries can be screened (205 = 3.2 × 106 combinations). For this reason, the random approach heavily depends on the starting material (the gap in sequence space should be relatively limited) and the efficiency of screening and selection. Progress in the random approach of laboratory evolution will be focused on improved techniques to generate diversity, but more importantly on the development of efficient (simple and cheap) methods for the screening of large mutant libraries for a large number of enzyme features. At the same time rational protein engineering will take advantage of (i) the increased computer power and optimized combinatorial algorithms, and (ii) the increasing knowledge of protein structures that is due to the fast growing PDB (not in the least because of structural genomics initiatives) and to the insight from previous design and laboratory evolution studies. Clearly, these developments will have a decisive influence on the future use of the independent rational and random approaches in enzyme engineering. Moreover, the complementary use of both rational design and laboratory evolution appears to be a very promising path towards the production of proteins with new and improved properties.

2.8

Sources of further information and advice

The following websites may be useful to those interested in laboratory evolution: 1. Arnold Laboratory, Caltech: http://www.che.caltech.edu/groups/fha/ 2. Bornscheuer Laboratory, University of Greifswald: http://www.chemie.unigreifswald.de/~biotech/

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3. Georgiou Laboratory, University of Texas: http://www.che.utexas.edu/ georgiou 4. Hilvert Laboratory, Swiss Federal Institute of Technology: http://www.protein. ethz.ch/ 5. Reetz laboratory, Max-Planck-Institut für kohlenforschung: http://www.mpimuelheim.mpg.de/kofo/institut/arbeitsbereiche/reetz//reetz_e.html 6. Zhao Laboratory, University of Illinois: http://www.chemeng.uiuc.edu/~zhao grp/ 7. Maranas Laboratory, Pennsylvania State University: http://fenske.che.psu.edu/ faculty/cmaranas/ 8. Benkovic Laboratory, Pennsylvania State University: http://research.chem. psu.edu/sjbgroup/ 9. Cornish Laboratory, Columbia University: http://www.columbia.edu/cu/chemistry/fac-bios/cornish/group/index.html 10. Schwaneberg Laboratory, International University Bremen: http://www. faculty.iu-bremen.de/zakhartsev/BCE2/ 11. Maxygen Company, Redwood City, CA, USA: http://www.maxygen.com/ index.php 12. Codexis Company, Redwood City, CA, USA: http://www.codexis.com/wt/ home/index 13. Diversa Company, San Diego, CA, USA: http://www.diversa.com The following websites may be useful to those interested in rational and computational protein engineering: 1. Richardson Laboratory, Duke University: http://kinemage.biochem.duke.edu 2. Baker Laboratory, University of Washington: http://depts.washington.edu/ bakerpg 3. Hellinga Laboratory, Duke University: http://www.biochem.duke.edu/ Hellinga/hellinga.html 4. Mayo Laboratory, Caltech: http://www.mayo.caltech.edu/ 5. Arnold Laboratory, Caltech: http://www.che.caltech.edu/groups/fha/ 6. Maxygen Company, Redwood City, CA, USA: http://www.maxygen.com/ index.php

2.9

References

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Ostermeier M. (2003). ‘Synthetic gene libraries: in search of the optimal diversity’. Trends Biotechnol, 21(6), 244–247. Ostermeier M., Shim J. H. and Benkovic S. J. (1999). ‘A combinatorial approach to hybrid enzymes independent of DNA homology’. Nat Biotechnol, 17(12), 1205–1209. Oue S., Okamoto A., Yano T. and Kagamiyama H. (1999). ‘Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues’. J Biol Chem, 274(4), 2344–2349. Pan Y., Gao D., Yang W., Cho H., Yang G., Tai H. H. and Zhan C. G. (2005). ‘Computational redesign of human butyrylcholinesterase for anticocaine medication’. Proc Natl Acad Sci USA, 102(46), 16656–16661. Park H. S., Nam S. H., Lee J. K., Yoon C. N., Mannervik B., Benkovic S. J. and Kim H. S. (2006). ‘Design and evolution of new catalytic activity with an existing protein scaffold’. Science, 311(5760), 535–538. Patnaik R., Louie S., Gavrilovic V., Perry K., Stemmer W. P. C., Ryan C. M. and Del Cardayre S. (2002). ‘Genome shuffling of Lactobacillus for improved acid tolerance’. Nat Biotechnol, 20(7), 707–712. Pelletier J. and Sidhu S. (2001). ‘Mapping protein–protein interactions with combinatorial biology methods’. Curr Opin Biotechnol, 12(4), 340–347. Reetz M. T. (2004). ‘Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications’. Proc Natl Acad Sci USA, 101(16), 5716– 5722. Reetz M. T., Torre C., Eipper A., Lohmer R., Hermes M., Brunner B., Maichele A., Bocola M., Arand M., Cronin A., Genzel Y., Archelas A. and Furstoss R. (2004). ‘Enhancing the enantioselectivity of an epoxide hydrolase by directed evolution’. Org Lett, 6(2), 177– 180. Richardson T. H., Tan X., Frey G., Callen W., Cabell M., Lam D., Macomber J., Short J. M., Robertson D. E. and Miller C. (2002). ‘A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable α-amylase’. J Biol Chem, 277(29), 26501–26507. Rothman S. C., Voorhies M. and Kirsch J. F. (2004). ‘Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase’. Protein Sci, 13(3), 763–772. Saraf M. C., Moore G. L., Goodey N. M., Cao V. Y., Benkovic S. J. and Maranas C. D. (2006). ‘IPRO: an iterative computational protein library redesign and optimization procedure’. Biophys J, 90(11), 4167–4180. Schmidt-Dannert C. and Arnold F. H. (1999). ‘Directed evolution of industrial enzymes’. Trends Biotechnol, 17(4), 135–136. Schoemaker H. E., Mink D. and Wubbolts M. G. (2003). ‘Dispelling the myths – Biocatalysis in industrial synthesis’. Science, 299(5613), 1694–1697. Sieber V., Pluckthun A. and Schmid F. X. (1998). ‘Selecting proteins with improved stability by a phage-based method’. Nat Biotechnol, 16(10), 955–960. Sieber V., Martinez C. A. and Arnold F. H. (2001). ‘Libraries of hybrid proteins from distantly related sequences’. Nat Biotechnol, 19(5), 456–460. Stemmer W. P. C. (1994a). ‘Rapid evolution of a protein in vitro by DNA shuffling’. Nature, 370(6488), 389–391. Stemmer, W. P. C. (1994b). ‘DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution’. Proc Natl Acad Sci USA, 91(22), 10747– 10751. Stephanopoulos G. (2002). ‘Metabolic engineering by genome shuffling’. Nat Biotechnol, 20(7), 666–668. Strickler S. S., Gribenko A. V., Keiffer T. R., Tomlinson J., Reihle T., Loladze V. V. and Makhatadze G. I. (2006). ‘Protein stability and surface electrostatics: a charged relationship’. Biochemistry, 45(9), 2761–2766. Sugimura M., Nishimoto M. and Kitaoka M. (2006). ‘Characterization of glycosynthase

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mutants derived from glycoside hydrolase family 10 xylanases’. Biosci Biotechnol Biochem, 70(5), 1210–1217. Tamakoshi M., Nakano Y., Kakizawa S., Yamagishi A. and Oshima T. (2001). ‘Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus’. Extremophiles, 5(1), 17–22. Tawfik D. S. and Griffiths A. D. (1998). ‘Man-made cell-like compartments for molecular evolution’. Nat Biotechnol, 16(7), 652–656. Taylor S. V., Kast P. and Hilvert D. (2001). ‘Investigating and engineering enzymes by genetic selection’. Angew Chem Int Ed Engl, 40(18), 3310–3335. Trincone A., Perugino G., Rossi M. and Moracci M. (2000). ‘A novel thermophilic glycosynthase that effects branching glycosylation’. Bioorg Med Chem Lett, 10, 365– 368. Udit, A. K., Silberg, J. J. and Sieber, V. (2003), Sequence homology-independent protein recombination (SHIPREC). Methods Mol Biol, 231, 153–163. Umeno D., Tobias A. V. and Arnold F. H. (2005). ‘Diversifying carotenoid biosynthetic pathways by directed evolution’. Microbiol Mol Biol Rev, 69(1), 51–78. Van den Burg B., Vriend G., Veltman O. R., Venema G. and Eijsink V. G. (1998). ‘Engineering an enzyme to resist boiling’. Proc Natl Acad Sci USA, 95(5), 2056–2060. Van den Heuvel R. H. H., Fraaije M. W., Ferrer M., Mattevi A. and Van Berkel W. J. H. (2000). ‘Inversion of stereospecificity of vanillyl-alcohol oxidase’. Proc Nat Acad Sci, 97(17), 9455–9460. Van der Veen B. A., Potocki-Veronese G., Albenne C., Joucla G., Monsan P. and RemaudSimeon M. (2004). ‘Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity’. FEBS Lett, 560(1–3), 91–97. Van der Veen B. A., Skov L. K., Potocki-Veronese G., Gajhede M., Monsan P. and RemaudSimeon M. (2006). ‘Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution’. FEBS Journal, 273(4), 673–681. Van Lieshout J., Faijes M., Nieto J., Van der Oost J. and Planas A. (2004). ‘Hydrolase and glycosynthase activity of endo-1,3-β-glucanase from the thermophile Pyrococcus furiosus’. Archaea, 1, 285–292. Vieille C. and Zeikus G. J. (2001). ‘Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability’. Microbiol Mol Biol Rev, 65(1), 1–43. Voigt C. A., Mayo S. L., Arnold F. H. and Wang Z. G. (2001). ‘Computational method to reduce the search space for directed protein evolution’. Proc Natl Acad Sci USA, 98(7), 3778–3783. Wang T., Zhu H., Ma X., Ma Y. and Wei D. (2006). ‘Structure-based stabilization of an enzyme: the case of penicillin acylase from Alcaligenes faecalis’. Protein Pept Lett, 13(2), 177–183. Wind R. D., Uitdehaag J. C. M., Buitelaar R. M., Dijkstra B. W. and Dijkhuizen L. (1998). ‘Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1’. J Biol Chem, 273(10), 5771–5779. Wong T. S., Tee K. L., Hauer B. and Schwaneberg U. (2004). ‘Sequence saturation mutagenesis (SeSaM), a novel method for directed evolution’. Nucleic Acids Res, 32(3), e26. Wong T. S., Tee K. L., Hauer B. and Schwaneberg U. (2005). ‘Sequence saturation mutagenesis with tunable mutation frequencies’. Anal Biochem, 341(1), 187–189. Yano T., Oue S. and Kagamiyama H. (1998). ‘Directed evolution of an aspartate aminotransferase with new substrate specificities’. Proc Natl Acad Sci USA, 95(10), 5511–5515. Yuan L., Kurek I., English J. and Keenan R. (2005). ‘Laboratory-directed protein evolution’. Microbiol Mol Biol Rev, 69(3), 373–392.

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3 Industrial enzyme production for food applications Carsten Hjort, Novozymes A/S, Denmark

3.1

Introduction

Enzymes have been used for food processing for as long as man has processed foods. However, deliberate use of enzymes is relatively new. As we have learned how to use enzymes for food more deliberately, the need for controlled methods of production of these enzymes has become more and more necessary. Traditionally food enzymes have been obtained from a number of different sources, for example by extraction from plants or animals. However, enzyme production based on extraction from plant or animal sources suffers from a number of drawbacks. The raw materials are often limited in supply and often the enzyme content in the raw material is very low. So substantial processing of huge amounts of raw materials is needed to obtain an acceptable purity of the enzyme product. As the raw materials are very different for different enzymes and the extraction and purification process is dependent on the enzyme properties, a specialized process for each enzyme product is required. So the enzyme producer cannot use generic production processes and multipurpose process equipment. In many cases the raw material itself raises serious concerns about the safety of the enzyme product. Also the quality and composition of the raw materials for enzyme extraction may be highly variable adding complexity to the extraction process. A different way of producing food enzymes is by microbial fermentation. Microbial fermentation processes and the raw materials used can be much better controlled than the production of raw materials for extraction from plants and

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animals. Also generic processes and multipurpose production facilities can be used for an array of different enzyme products. The use of microbial enzymes for food processing is not a new invention. Traditional processes, like the miso process, were the starting point for developing industrial enzyme processes based on submerged fermentation in stainless-steel tanks in large volumes. The first industrial production of enzymes using microorganisms was launched in the late part of the 19th century, but it was not until after World War II that the industry became significant. The early processes were based on microorganisms such as Aspergillus niger, Aspergillus oryzae and Bacillus licheniformis. These organisms produced significant amounts of enzymes that were found to be applicable in various food processes and they could be demonstrated to be safe. Significant development of the organisms was achieved using classical mutagenesis. Even though major screening programs were conducted by enzyme companies it proved to be difficult to isolate microorganisms that could both produce large amounts of enzyme in an industrial setup and at the same time were risk class 1 organisms. This was a severe limitation for development of enzyme products before the advent of recombinant DNA technology. With the introduction of genetically modified production strains in the late 1980s, the industry was transformed once again. It was now possible to transfer genes from organisms that were not suitable for industrial production into industrial host organisms and to produce large amounts of enzymes from any enzyme class in a safe way. In parallel, protein engineering technologies were developed enabling the properties of the enzymes to be altered to suit the application better. Recombinant DNA technology has also opened up new avenues for food enzyme production. Production of enzymes in recombinant plants like soy, corn, tobacco and rice has been exploited for years but not generally commercialized yet. This is mainly due to resistance from customers, consumers and legislators.

3.2

Traditional sources and processes for industrial enzyme production

Traditionally food enzymes have been obtained from a number of different sources. Enzymes have been extracted from plant materials in more or less crude form. A good example of this is the protease papain used for example in meat processing (Tainter and Buchanan, 1951). The enzyme has traditionally been extracted from the papaya plant in a very crude form and gradually more and more pure qualities have been made available. Similarly enzymes have been extracted from animal materials. One example of this is bovine chymosin for cheese processing. This has traditionally been extracted from calf stomach in a very crude form. A better understanding of the action of the enzyme paved the way for a more controlled production process and, in 1874, Christian Hansen developed an industrial production process for production of this enzyme (Nielsen et al., 1994). The process has been constantly developed to produce a steadily more pure product, and with the advent of recombinant DNA

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technology, this enzyme and a range of similar proteases have been produced in micro-organisms (Berka et al., 1991). Extraction of enzymes from natural sources such as plants and animals has severe drawbacks that have limited the expansion of this approach. For each enzyme, a dedicated production facility has to be developed and optimized. For example, the papain process has very little in common with the chymosin process. The availability of raw materials may vary from year to year and the quality of the raw materials may also be highly variable. In addition to this, the raw material itself raises serious concerns about the safety of the enzyme product. This is especially true for raw materials of animal origin. For chymosin produced from calf stomach, bovine diseases like bovine spongiform encephalopathy (BSE) are an obvious concern. The limitations of food enzyme processes based on extraction called for an alternative production process for industrial food enzymes. Production based on microbial fermentation soon became the industry standard. Microbial production allowed for multipurpose facilities where different microorganisms producing different enzyme products could be used in the same production line. Relatively cheap raw materials of a uniform quality like starch, glucose syrup, glycerol, soy grits, yeast extract and inorganic salts were required (Aunstrup, 1974). Furthermore the safety issues concerning enzymes from animal origin are not present for microbial enzyme processes unless media components of mammalian origin are used in the process. However, a number of other safety issues have to be addressed for microbial fermentations as will be discussed later in this chapter. The use of microbial enzymes for food processing is not a new invention. In Japan miso, soy and sake have been produced for centuries using A. oryzae and A. sake to degrade rice to the product koji (Kitamoto, 2002). Koji essentially consists of enzymes produced by the two Aspergillus strains. A number of different enzyme activities are important for the performance of the koji including amylases, proteases and phytases (Fujita et al., 2003). The koji process served as the inspiration for the first commercial microbial production process that was started in Chicago in 1894 by the Japanese–American enzyme pioneer Jockichi Takamine (Bennet, 2001). He used A. oryzae to produce the product takadiastase, which was basically a mixture of Aspergillus amylases and proteases produced in a modified koji process. Microbial fermentation processes had already been developed for citric acid production and amino acid production before the market for industrial food enzymes really matured (Röhr et al., 1983). The technology used for these products was readily adapted by the enzyme producers. So the microbial enzyme fermentation processes developed then and still used today were mainly submerged fermentation in stainless steel but also to some extent on-surface fermentation as will be discussed later in this chapter. Before it became possible to genetically modify the production strains used, only microorganisms that could be found in nature could be used for enzyme production. So enzyme producers conducted major screening programs for enzyme producing microorganisms. This task was far from trivial. Not all microorganisms

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are readily cultured in a stainless-steel setup, so even though the production of a wanted enzyme component could be demonstrated on agar plates, the isolated microorganism often failed to grow in submerged fermentation. In an industrial setup it is only feasible and advisable to use safe microorganisms that do not produce toxins and do not have any pathogenic potential against humans, animals or plants. In short, only organisms proven to be risk class 1 organisms can be used for commercial production. In addition to producing the wanted enzyme activity, the selected organism will usually produce a number of other enzyme activities that may either interfere with the application or may reduce the stability of the enzyme product. Lipases are, for example, unwanted in dairy processing enzymes like chymosin as they hydrolyse triglycerides of the lower fatty acids which leads to liberation of fatty acids like butyric acid with dire consequences for taste and smell. Proteases produced by the production strain may degrade the enzyme product and thus lead to dramatically reduced product stability (Mattern et al., 1992). These limitations did not mean that some deficiencies in a potential production strain could not be corrected. Using classical mutagenesis with known mutagens such as UV light, radioactive irradiation, nitrosoguanidine or other chemical mutagens followed by screening or selection when possible, substantial strain improvement could be obtained (Ford, 1999). These methods were the only real strain improvement methods before recombinant DNA technology methods became available, but they are still used extensively today. Using these methods it is possible to isolate mutants with improved yield, mutants deficient in unwanted side activities, mutants that are better adapted to submerged fermentation or mutants deficient in production of unwanted metabolites (Rasuoli and Kulkarni, 1994).

3.3

Design of expression systems for industrial enzyme production

The advent of recombinant DNA technology has opened up a vast array of possibilities for improvements in technologies for food enzyme production. The most obvious advantage of recombinant DNA technology is that it is possible to transfer genes from any microorganism, plant or animal into a host cell organism, so that food enzymes from any source can be produced at least theoretically. However, the technology also has opened up a number of opportunities to improve the production strains with respect to a number of parameters other than just the enzyme yield. In this section, development of an optimal expression system for product yield, product purity, product stability and product safety will be discussed. When the development of a commercial production strain for a new enzyme product is initiated, the properties of the enzyme product have been formulated, the production economics have been calculated and usually a customer is ready and waiting for the product. The only thing missing is a production strain and a

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production process for that strain. Consequently speed is essential in this part of a food enzyme development project. So in addition to delivering the required properties (yield, purity and stability), the timeframe for construction of the production strain must be minimal and predictable and the strain should be constructed in such a way that product and production approval can be obtained as fast as possible without compromising safety. The safety and approval aspects of developing a new production stain are of utmost importance so these aspects are as important as the more technical aspects in choosing how to design an optimal expression system. The main questions to consider for an expression system from a regulatory point of view are (OlempskaBeer et al., 2006):

• Does the host strain have a history of safe use? • Is the system based on secretion of the enzyme product or intracellular accumulation?

• What protein side activities does the host produce and will they end up in the • • • •

product? Is the host strain capable of producing toxic metabolites? Does the host strain sporulate and is that important? How is the vector designed, and what is the fate of the transforming DNA? Does the final production strain harbour antibiotic resistance markers?

3.3.1 Does the host strain have a history of safe use? A history of safe use is a very important part of the safety assessment as it demonstrates that the selected host strain can actually be used for safe production. If a history of safe use can be demonstrated for a product, it can obtain GRAS (generally regarded as safe) status with the US Food and Drug Administration (US FDA) (Gaynor, 2006). GRAS status for a product produced by an expression system is a very important part of establishing a history of safe use of the expression system for new products produced by the same expression system. In continuation of the ‘history of safe use’ concept, a new concept named the ‘safe strain lineage’ has been suggested (Pariza and Johnson, 2001). The idea behind this concept is that if an expression system with a history of safe use has been used for production of several enzyme products and if a strain lineage has been created from that expression system through incremental modifications that can be thoroughly risk assessed, then the entire strain lineage can be considered safe. Filamentous fungi, bacteria and yeast are used for food enzyme production. Table 3.1 lists the most commonly used organisms for food enzyme production. Aspergillus oryzae has traditionally been used for production of koji as mentioned earlier. Aspergillus niger is a member of the black Aspergilli and has been used for enzyme production for the starch industry for almost a century. Other closely related black Aspergilli like A. foetidus, A. awamori, A. aculeatus and A. japonicus are also used to a greater or lesser extent (Bennet and Klich, 1992). Trichoderma reesei and its close relatives are used to some extent, but for food applications suffer from a very high protein background.

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Table 3.1

Most commonly used microorganisms for food enzyme production

Filamentous fungi

Yeast Bacteria

Aspergillus oryzae Aspergillus niger Fusarium venenatum Trichoderma reesei Saccharomyces cerevisiae Klyveromyces lactis Bacillus licheniformis Bacillus subtilis Escherichia coli

Barbesgaard et al., 1992 Schuster, 2002 Royer et al., 1995 Nevalainen et al., 1994 Sleep et al., 2001 van den Berg et al., 1990 de Boer et al., 1994 de Boer and Diderichsen, 1991 Flamm, 1991

Among the yeasts, Klyveromyces lactis has been the most frequently used yeast for food enzyme production. Bakers’ yeast, Saccharomyces cerevisia has not been used for this purpose up to now, mainly owing to low secreted enzyme yields. However, systematic strain development is about to change this, as a yeast system based on a modified 2 µm vector now reaches yields similar to those of filamentous fungi and other yeasts (Sleep et al., 2001). A number of other yeast systems can produce enzymes at very high yields including methylotrophic yeasts Hansenula polymorpha (Ramezani-Rad et al., 2003) and Pichia pastoris (Ciofalo et al., 2006). Most food enzymes produced in bacteria are from Bacillus species, either Bacillus licheniformis or Bacillus subtilis. Strain development in these species has been greatly facilitated by the early genome sequencing projects in bacteria (Ray et al., 2004; Veith et al., 2004). Other organisms like Escherichia coli are used, but not to a very large extent.

3.3.2

Is the system based on secretion of the enzyme product or intracellular accumulation? Nearly all food enzymes are secreted enzymes. It is usually very costly to obtain a sufficiently pure product, not containing an unacceptable level of host cell proteins and host cell DNA, if the enzyme product is intracellular. However a number of intracellular enzymes are high-volume products used in the food industry. One example is glucose isomerase used for production of high fructose corn syrup (HFCS). Glucose isomerase products are formulated as immobilized enzymes so that the glucose substrate for the enzyme can be reacted in a reactor without addition of soluble enzyme product. This is an elegant way of solving the impurity issue without adding cost as the impurities such as host cell DNA and host cell protein are either also immobilized together with the enzyme and thus retained from the HFCS product or the impurity is not immobilized and thus removed during production of the immobilized enzyme product. 3.3.3

What protein side activities does the host produce and will they end up in the product? As most food enzyme products are secreted, the following discussion concentrates

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on these. Removal of unwanted protein activity is important not just from a regulatory point of view but also from a total process view. As will be discussed later, the typical food enzyme production process consists of fermentation, recovery and formulation of the enzyme product. Very little or no purification is usually applied as most purification methods simply carry too much cost. Consequently, in order to obtain a fairly pure product that is acceptable both from an application point of view and from a regulatory point of view, it is necessary to inactivate the genes encoding the major secreted proteins of the host strain. Inactivation of genes is usually done by gene disruption. In filamentous fungi and in yeast, gene disruption is usually done by transforming the host strain with a linear DNA fragment harbouring the disruption cassette (Fincham, 1989). The disruption cassette consists of a DNA fragment identical to the 5' end of the gene which should be disrupted followed by a selection marker and then followed by a DNA fragment identical to the 3' end of the gene which should be disrupted. Gene disruptions and replacements can be done in the same way in Bacillus or it can be done in two steps using a temperature sensitive circular plasmid (Hamilton et al., 1989). The principles of gene replacements and disruptions are shown in Fig. 3.1. A more recent method for preventing formation of a gene product is by silencing the gene rather than disrupting it. This can be done by RNAi first described in higher eukaryotes (Fire et al., 1998) but later also described in filamentous fungi (Maiyuran et al., 2005). Briefly explained, if an inverted repeat of the gene to be downregulated is expressed in the host organism, a chain of events in the host strain leads to specific degradation of the mRNA of the target gene and so no gene product is formed. A particular troublesome side activity is protease activity as this is not just unnecessary contaminating protein but will potentially degrade the enzyme product. As most microorganisms produce several different proteases with different functions, it is usually not sufficient to disrupt a single protease gene. From the genome sequence of A. oryzae, it is predicted that this fungus harbours 135 protease encoding genes (Machida et al., 2005). As some of the proteases are very dominating and since some proteases are maturating other proteases from inactive holoenzymes to active apoenzymes, the protease activity in the supernatant solution can usually be reduced dramatically by simply disrupting a few key enzymes (Lehmbeck, 1996). In A. niger, it has been found that all the extracellular proteases are regulated by a common transcription activator named prtT. This activator is required for expression on the extracellular proteases but not for the intracellular proteases such as the vacuolar proteases. Disruption of this transcription factor results in a very protease-weak host cell with no adverse phenotypes (Hjort et al., 1998).

3.3.4 Is the host strain capable of producing toxic metabolites? The microbial host strain may be capable of producing a number of metabolites. If these metabolites are toxic, for example mycotoxins, the formation of these metabolites is a serious safety concern that needs to be addressed. Other metabolites

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Novel enzyme technology for food applications

(a)

(b)

Fig. 3.1 Gene replacement and disruption. (a) A linear gene disruption or gene replacement fragment has been transformed into a strain and recombines by double overcross with the chromosome or the strain so that the DNA in between the homologous fragments is replaced. This is illustrated here by the pyrG gene which is often used for gene disruptions. (b) A circular plasmid with a temperature sensitive origin (ori) has been transformed into a strain and is forced to recombine by elevating the temperature by single overcross with the chromosome or the strain so that the plasmid is integrated into the chromosome. In a second step at the permitted temperature the second homologous fragments recombine so that a replacement takes place as in Fig. 3.1(a). This is illustrated here by the kanamycin resistance marker gene (kan) which is often used. By switching the order of the kanamycin marker and the 3' homologous fragment a marker free disruption can be made.

may be unwanted because of technical or economical concerns not because of safety concerns.. Most bacteria and fungi used for food enzyme production have the genetic potential for producing toxic secondary metabolites or primary metabolites under exceptional conditions. A. oryzae has the entire aflatoxin cluster. It has however not been possible under any conditions to provoke A. oryzae to produce aflatoxins. A thorough analysis of the aflatoxin gene cluster of the A. oryzae type strain RIB40 demonstrated that a number of mutations have occurred in some of the genes so

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that the cluster of this strain is inactive (Tominaga et al., 2006). Even so, one approach to make sure that aflatoxin production is impossible is to remove some or all of the genes necessary for production. In the commonly used A. oryzae strain IFO4177, the aflatoxin gene cluster has been removed by classical mutagenesis followed by southern blot analysis. At the same time at least one gene from the biosynthesis of another toxic secondary metabolite, cyclopiazonic acid was deleted (Christensen et al., 2000). The relatively new expression system Fusarium venenatum was similarly found to have the genetic potential for production of a class of toxic secondary metabolites known as trichothecenes and the strain MLY3 was indeed demonstrated to produce the compound diacetoxyscirpenol. Again the biosynthetic pathway was known and an early gene, tri5, in the pathway was identified and disrupted (Royer et al., 1999). Not all unwanted metabolites are unwanted for safety reasons. The primary metabolite oxalic acid is produced by A. niger under certain fermentation conditions. It is a moderately toxic compound but can easily be removed by precipitation with calcium. However precipitates of calcium oxalate in the enzyme products are not wanted and they are costly to remove. Furthermore as fairly large concentrations of oxalate can build up, a lot of carbon source is wasted on oxalate production and large amounts of calcium need to be added to the fermentation. So the product quality and the production economy could be greatly improved by reengineering of A. niger metabolism to prevent formation of oxalic acid. To do this, a biosynthesis route was established and a key gene, the oah gene encoding the oxaloacetate hydrolase was identified (Pedersen et al., 2000a). Disruption of this gene resulted in a modified A. niger strain that did not produce oxalic acid even under highly inducing conditions but otherwise had an almost unaltered metabolism (Pedersen et al., 2000b).

3.3.5 Does the host strain sporulate and is this important? Spores are structures for dormant survival which have very different structures and functions in filamentous fungi, yeast and bacteria. The imperfect fungi Aspergillus and Trichoderma produce conidia spores. Sporulation is triggered by starvation of one or more medium components and is for most strains only abundant on surface growth, not in submerged fermentation unless the strain is subjected to starvation of essential nutrients like a carbon source or a nitrogen source (Broderick and Greenshields, 1982). These conidia are easily inactivated by chemicals or elevated temperature and they are thus considered neither a safety nor a process concern. On the contrary, they are necessary for propagation of the production strain and for the host strain prior to transformation. In addition the known mutations that can either reduce or abolish sporulation in these fungi also have other adverse phenotypes like slow growth, altered morphology or reduced protein production (Gems and Clutterbuck, 1994). Yeast strains such as S. cerevisiae and Pichia pastoris form sexual ascospores, but these spores are sexual structures useful for strain development and sporulation

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is not an issue for production. Bacillus strains form heat stable spores as survival structures. These spores are, in addition, resistant to a number of chemical inactivation procedures. Thus, sporulation in Bacillus is an issue from a safety and product quality point of view as it is difficult to ensure absence of spores in the final product. So the Bacillus strains that are used for enzyme production are sporulation deficient. Sporulation-deficient strains are isolated either as classical mutants that have been subjected to a mutagen and then screened or as defined mutants disrupted in one of the genes required for sporulation (Fleming et al., 1995).

3.3.6

How is the vector designed and what is the fate of the transforming DNA? The design of the expression vector is one of the key aspects of the construction of a recombinant production organism. The expression vector in general consists of the product gene to be expressed, a promoter upstream of the product gene that controls the expression, a terminator sequence downstream of the product gene that terminates the transcription, a selectable marker for selection of the transformants and DNA fragments that have been used to construct the expression vector typically in E. coli. The selectable marker and the DNA fragments used for construction may or may not be present in the final production strain as there are ways to remove these after transformation. The expression vector can either be integrated into the chromosome of the host organism or it can be autonomously replicated as an episomal vector. For most expression systems, integration into the chromosome is preferred as it usually gives better genetic stability. For the filamentous fungi Aspergillus, Trichoderma and Fusarium, all expression vectors for commercial production are integrative vectors that are integrated into the chromosome of the host organism, as the only autonomously replicating vectors for filamentous fungi are highly mitotically unstable (Aleksenko et al., 1996). Some vectors are designed for integration into specific loci such as the amyloglycosidase locus of A. niger (van Dijck et al., 2003) or the cellobiohydrolase I locus of Trichoderma reseei (Keranen and Pentilla, 1995), whereas others are designed for ectopic integration into the genome. The promoters and terminators used for Aspergillus strains are mostly based on amylase promoters or amyloglycosidase promoters. The reason for the choice of these promoters is mostly that they are very well expressed and that they are induced by either glucose or maltodextrins but repressed by carbon catabolite repression. This means that these promoters can easily be controlled in a fermentation process using standard carbon sources. A detailed understanding of the regulation of a promoter can provide the basis for designing even stronger promoters with a regulation that better suits the production environment that the strain will be used in. The regulation of the TAKA amylase promoter of A. oryzae is now well understood. The carbon catabolite repression is mediated by the global regulator creA, whereas the induction is mediated by a specific transcription activator, amyR (Petersen et al., 1999).

Industrial enzyme production for food applications

53

Understanding of this promoter has enabled development of promoters with substantially higher strength than the original wild type promoter. In T. reseei the usual choice is the CBH1 promoter as this is the strongest promoter among the cellulase promoters of this strain. Also there are a number of modified versions of this promoter based on a detailed understanding of the regulation (Ilmen et al., 1996). The selection markers used for the filamentous fungi are typically auxotrophic markers or the amdS marker from A. nidulans (Wernars et al., 1985). The amdS gene encodes an acetamidase enabling transformants to hydrolyse acetamide to acetic acid and ammonia. The selection principle is that after transformation, the transformed protoplasts are plated onto a minimal plate with acetamide as sole nitrogen or carbon source. Thus, only transformants that express the amdS gene at a sufficiently high level can grow. As the amdS gene is fairly poorly expressed in most fungi, this secures selection for a fairly high copy number. As the DNA is integrated into the chromosome, it is usually integrated in multiple copies. Transformants with a high copy number typically produce higher amounts of the enzyme product. For the Bacillus systems stable autonomously replicating vectors do exist, but the mitotic stability of these is also limited and so chromosomal integration is also preferred in Bacillus. The homologous recombination frequency in Bacillus is quite high, so all expression systems are based on targeted integration. The typical promoters used in Bacillus are amylase or protease promoters as the natural expression of the corresponding amylases and proteases is very high (Diderichsen and Joergensen, 1997). In addition a number of optimized promoters have been developed for Bacillus including hybrid promoters harbouring mRNA stabilizing elements (Widner et al., 2000).

3.3.7

Does the final production strain harbour antibiotic resistance markers? The use of antibiotic resistance markers for enzyme production strains is highly undesirable and, if such markers are used in the expression vector, they must be removed in the final production strain and the antibiotic marker removal must be confirmed, for example by a Southern blot. Unfortunately, the most efficient selection markers used in Bacillus are antibiotic resistance markers. Using Bacillus promoters isolated from nature, multi copy integration of the expression plasmid is required to obtain optimal expression. Traditional multi copy Bacillus strains have been constructed by integrating a single copy of an expression plasmid harbouring an antibiotic resistance marker in a preselected locus. The copy number is then increased by growing the transformant in the presence of increasingly higher concentrations of the antibiotic corresponding to the antibiotic resistance marker. Hereby cells having gene duplicated the expression plasmid are enriched. The result is multi copy strains with the expression plasmid organized in a head to tail fashion (Albertini and Galazzi, 1985). No markers such as the amdS marker in fungi to secure high copy number using simple enzymatic selection rather than antibiotic resistance have been described.

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Novel enzyme technology for food applications

However, by using very strong promoters and single copy integration, it is possible to construct industry relevant Bacillus production strains that are free of antibiotic resistance markers (Widner et al., 2000).

3.4

Development of an enzyme production process

Food enzymes are produced using the same processes that are used for other enzyme products. The production process consists of fermentation of the production strain, recovery of the enzyme, purification and formulation. These are independent steps, but they are viewed as highly integrated by enzyme producers (Fish, 1984). A fermentation process that can be run in a short time with a high yield is obviously the main objective for fermentation scientists, but if the product that is delivered for recovery is highly viscous, contains a high level of fine particulate matter (‘fines’), is high in protease activity and so on, the product will be difficult to manage in downstream processing and the entire process will be suboptimal. So the needs of downstream processing have to be considered both when designing the production strain as described above and when designing the fermentation process. The fermentation process can be conducted either as a surface fermentation or as submerged fermentation (Rana and Bhat, 2005). Today enzymes are produced almost exclusively by submerged fermentation processes. This is mainly because surface fermentation is labour intensive and the downstream processing is more complicated (Aunstrup, 1979). However automation of the processes may change this situation. Submerged fermentation processes to produce industrial enzymes start with inoculating a vial of the production strain into a flask containing an agar medium or in some cases liquid medium. After outgrowth of that culture and in the case of filamentous fungal production strains, sporulation, the culture is transferred to a seed fermenter, which is a small fermenter in which the biomass for the main fermentation is generated. The seed fermentation allows the cells to adapt to the environment and nutrients used in the rest of the process. The fermentation process is illustrated in Fig. 3.2. Following seed fermentation, the cells are transferred to the main fermenter, where temperature, pH and dissolved oxygen can be controlled to optimize enzyme production. The fermentation process is either executed as a batch, fed batch or continuous process. In the batch process all media components are added from the start of the fermentation. A fed batch fermentation is similar to a batch fermentation, but the production strain is fed with additional medium during the fermentation. In a continuous fermentation a steady state is reached by supplying fresh medium with simultaneous harvest from the tank. When the main fermentation is complete, the mixture of cells, nutrients and enzymes, referred to as the broth, is ready for downstream processing. For intracellular enzymes, the downstream processing is fairly complex. If a soluble enzyme is wanted, the product must first be released from the cells by cell

Industrial enzyme production for food applications

Fig. 3.2

55

Industrial submerged fermentation. In the illustrated setup it is possible to run batch, fed batch and continuous fermentation.

disruption. If an immobilized enzyme product is wanted it is sometimes possible simply to isolate the cells and chemically crosslink them. As intracellular enzymes are not very common and as the processes for intracellular enzymes are not generic, they will not be discussed in more detail here. A more generic process can be designed for extracellular enzymes. The first step for this is recovery. The supernatant solution containing the enzyme is separated from the biomass. This is achieved by various chemical treatments of the fermentation broth to remove particles and unwanted polymers and to ensure efficient separation, followed by removal of the biomass using either centrifugation or filtration. Following the separation, the enzyme is concentrated by means of ultrafiltration, diafiltration or evaporation. Having recovered the enzyme, purification is sometimes necessary. As mentioned before, enzyme production is a very costsensitive industry and many of the purification unit operations that can be used are simply too expensive. This is especially true for capture column chromatography with subsequent elution of the purified protein. So for enzyme production, purification often consist of less expensive unit operations such as selective precipitation, adsorption of impurities to a resin in a column or crystallization by

56

Novel enzyme technology for food applications

which very pure enzyme products can be obtained. Liquid two-phase systems, for example based on polyethylenglycol stabilized systems, have been suggested, but are probably not in commercial use (Linder et al., 2004) The final step in the process is formulation of the enzyme product. The enzymes can be formulated either as liquid products or as granulates dependent on the application of the enzyme. The critical issues of the formulation are to secure stability of the enzyme product, release of the enzyme in the application and to prevent enzyme dust formation that can cause an allergic reaction.

3.5

Future trends

A very significant number of the food enzymes produced today are based on recombinant production strains. Unfortunately acceptance of food products based on recombinant DNA technology is still not absolute. For many food segments, the food producers and their customers still demand products based on non-recombinant raw materials, additives and processing aids. As long as non-recombinant products are needed to serve some segments, it is hard to anticipate any significant development of the product range and of the technology used to produce enzymes for these segments. Enzyme producers address the concerns of the regulatory authorities, the food producers and the end consumers in a number of ways. In relation to construction of production organisms and production processes, a number of changes have already occurred. Antibiotic resistance markers are being phased out and are only used by a few enzyme producers for new production strains. The most critical secondary metabolites of the production strains used have been dealt with and this is now being adapted into a safety and regulatory framework by the safe strain lineage approach. There is no doubt that the production strains used will be further engineered to produce increasingly pure enzyme products.

3.6

Sources of further information and advice

A detailed review of protease production both by extraction from plants and animals as well as microbial production has been prepared by Sumantha and coworkers (Sumantha et al., 2006). The book Genetically Engineered Food: Methods and Detection (Heller, 2006) is a thorough review of regulatory requirements and detection of recombinant food enzymes in food products. The regulatory framework for enzyme production strains and processes can be followed on the web pages of EFSA (www.efsa.europa.eu) for Europe and the FDA (www.fda.gov) for the USA.

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3.7

57

References

Albertini A. M. and Galizzi A. (1985). ‘Amplification of a chromosomal region in Bacillus subtilis’. J. Bacteriol., 162, 1203–1211. Aleksenko A., Nikolaev I., Vinetski Y. and Clutterbuck A. J. (1996). ‘Gene expression from replicating plasmids in Aspergillus nidulans’. Mol. Gen. Genet., 253, 242–246. Aunstrup K. (1974). ‘Industrial production of microbial enzymes’, in: B. Spencer (ed), Industrial Aspects of Biochemistry, Federation of European Biochemical Societies, Amsterdam, The Netherlands, 23–46. Aunstrup K. (1979). ‘Production isolation and economics of extracellular enzymes’. Appl. Biochem Bioeng., 27–70 Barbesgaard P., Heldt-Hansen H. P.and Diderichsen B. (1992). ‘On the safety of Aspergillus oryzae: a review’. Appl. Microbiol. Biotechnol. 36, 569–572. Bennet J. W. and Klich M. A. (1992), Aspergillus, Biology and Industrial Applications, Butterworth-Heinemann, Stoneham, USA. Bennet J. W. (2001), Aspergillus and Koji: History, practice and molecular biology, SIM News, 51, 65–71. van den Berg J. A., van der Laken K. J., van Ooyen A. J. J., Renniers T. C. H. M., Rietveld K., Schaap A., Brake A. J., Bishop R. J., Schultz K., Moyer D., Richman M. and Shuster J. R. (1990). ‘Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin’. Bio/Technology, 8, 135–139. Berka R. M., Kodama K. H, Rey M. W, Wilson L. J, Ward M. (1991). ‘The development of Aspergillus niger var. awamori as a host for the expression and secretion of heterologous gene products’. Biochem. Soc. Trans., 19, 681–685. de Boer A. S. and Diderichsen B. (1991). ‘On the safety of Bacillus subtilis and B. amyloliquefaciens: a review’. Appl. Microbiol. Biotechnol., 36, 1–4. de Boer A. S., Priest F. and Diderichsen B. (1994). ‘On the industrial use of Bacillus licheniformis: a review’. Appl. Microbiol. Biotechnol., 40, 595–598. Broderick A. J. and Greenshields R. N. (1982). ‘Semi continuous and continuous production of Aspergillus niger spores in submerged liquid culture’. J. Gen. Microbiol., 128, 2639– 2646 Christensen B., Moellgaard H., Kaasgaard S. and Lehmbeck J. (2000), Methods for Producing Polypeptides in Aspergillus Mutant Cells. International Patent WO 00/39322. Ciofalo V., Barton N., Kreps J., Coats I. and Shanahan D. (2006). ‘Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil’. Regul. Toxicol. Pharmacol., 45(1), 1–8 (2006). Diderichsen B. K. and Joergensen S. (1997), Bacillus Promoter Derived from a Variant of a Bacillus licheniformis α Amylase Promoter. United States Patent 5,698,415. van Dijck P. W. M., Selten G. C. M. and Hempenius R. A. (2003). ‘On the safety of a new generation of DSM Aspergillus niger enzyme production strains’. Regul. Toxicol. Pharmacol., 38, 27–35. Fincham J. R. (1989). ‘Transformation in fungi’. Microbiol. Rev., 53(1), 148 –170. Fire A., Xu S., Montgomery M. K., Kostas S. A., Driver S. E. and Mello C. C. (1998). ‘Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans’. Nature, 391, 806–811. Fish N. M. and Lilly M. D. (1984). ‘The interactions between fermentation and protein recovery’. Biotechnology, 2, 623–627. Flamm E.L. (1991). ‘How FDA approved chymosin: a case history’. BioTechnology, 9, 349– 351. Fleming A., Tangney M., Jørgensen P. L., Diderichsen B. and Priest F. G. (1995). ‘Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis’. Appl. Environ. Microbiol., 61, 3775–3780. Ford C. (1999). ‘Improving operating performance of glucoamylase by mutagenesis’. Curr. Opin. Biotechnol., 10, 353–357.

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Fujita J., Shigeta S., Yamane Y. I., Fukuda H., Kizaki Y., Wakabayashi S. and Ono K. (2003). ‘Production of two types of phytase from aspergillus oryzae during industrial koji making’. J. Biosci. Bioeng., 95, 460–465. Gaynor P. (2006). ‘How U.S. FDA’s GRAS notification program works’. Food Safety Mag., 11, 16–19. Gems D. H., Clutterbuck A. J. (1994). ‘Enhancers of conidiation mutants in Aspergillus nidulans’, Genetics, 137, 79–85. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P. and Kushner S. R. (1989). ‘New method for generating deletions and gene replacements in Escherichia coli’. J. Bacteriol., 171, 4617–4622. Heller K. J. (2006). ‘Genetically Engineered Food: Methods and Detection, 2nd edition, Wiley-VCH, Weinheim, Germany. Hjort C., van den Hondel A. M. J. J., Punt P. and Schuren F. H. J. (1998), Fungal transcriptional activator useful in methods for producing polypeptides. International Patent Application, PCT WO 0020596. Ilmen M., Onnela M. L., Klemsdal S., Keranen S. and Penttila M. (1996). ‘Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei’. Mol. Gen. Genet., 253, 303–314. Keranen S. and Penttila M. (1995). ‘Production of recombinant proteins in the filamentous fungus Trichoderma reesei’. Curr. Opin. Biotechnol., 6, 534–537. Kitamoto K. (2002). ‘Molecular biology of the koji molds’. Adv. Appl. Microbiol., 51, 129– 153. Lehmbeck J. (1996), Host Cell Producing Reduced Levels of a Metalloprotease and Methods for Using the Host Cell in Protein Production. International Patent Application, PCT WO96/29391. Linder M. B., Qiao M., Laumen F., Selber K., Hyytia T., Nakari-Setalaanddie T. and Penttila M. E., (2004). ‘Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems’. Biochemistry, 43, 11873–11882 Machida M., Asai K., Sano M., Tanaka T., Kumagai T., Terai G., Kusumoto K., Arima T., Akita O.,. Kashiwagi Y., Abe K., Gomi K., Horiuchi H., Kitamoto K., Kobayashi T., Takeuchi M., Denning D. W., Galagan J. E., Nierman W. C., Yu J., Archer D. B., Bennett J. W., Bhatnagar D., Cleveland T. E., Fedorova N. D., Gotoh O., Horikawa H., Hosoyama A., Ichinomiya M., Igarashi R., Iwashita K., Juvvadi P. R., Kato M., Kato Y., Kin T., Kokubun A., Maeda H., Maeyama N., Maruyama J., Nagasaki H., Nakajima T., Oda K., Okada K., Paulsen I., Sakamoto K., Sawano T., Takahashi M., Takase K., Terabayashi Y.,Wortman J. R., Yamada O., Yamagata Y., Anazawa H., Hata Y., Koide Y., Komori T., Koyama Y., Minetoki T., Suharnan S., Tanaka A., Isono K., Kuhara S., Ogasawara N., Kikuchi H. (2005). ‘Genome sequencing and analysis of Aspergillus oryzae’. Nature. 438, 1157–1161. Maiyuran S., Udagawa H. and Brody H. (2005), Methods for Eliminating or Reducing the Expression of a Gene in a Filamentous Fungal System. International PCT Patent Application WO 2005/056772. Mattern I. E., van Noort J. M., van den Berg P., Archer D. B., Roberts I. N. and van den Hondel C. A. (1992). ‘Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases’. Mol. Gen. Genet., 234, 332–336. Nevalainen H., Suominen P. and Taimisto K. (1994). ‘On the safety of Trichoderma reesei’. J. Biotechnol., 37, 193–200. Nielsen P. H., Malmos H., Damhus T., Diderichsen B., Nielsen H. K., Simonsen M., Schieff H. E., Oestergaard A., Olsen H. S., Eigtved P., Nielsen T. K. (1994). ‘Enzyme applications (industrial)’. In Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 9, 4th edition. John Wiley and Sons, New York, 567–620. Olempska-Beer Z. S., Merker R. I., Ditto M. D. and DiNovi M. J. (2006). ‘Food-processing enzymes from recombinant microorganisms – a review’. Regul. Toxicol. Pharmacol., 45, 144–158.

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Pariza M. W. and Johnson E. A. (2001). ‘Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century’. Regul. Toxicol. Pharmacol., 33(2), 173–186 Pedersen H., Hjort C.and Nielsen J. (2000a). ‘Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger. Mol. Gen. Genet., 263(2), 281– 286. Pedersen H., Christensen B., Hjort C. and Nielsen J. (2000b). ‘Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger’. Metab. Eng., 2, 34–41. Petersen K. L., Lehmbeck J. and Christensen T. (1999). ‘A new transcriptional activator for amylase genes in Aspergillus’. Mol. Gen. Genet., 262, 668–676. Ramezani-Rad M., Hollenberg C. P., Lauber J., Wedler H., Griess E., Wagner C., Albermann K., Hani J., Piontek M., Dahlems U.and Gellissen G. (2003). ‘The Hansenula polymorpha (strain CBS4732) genome sequencing and analysis’. FEMS Yeast Res., 4, 207–215. Rana N. K. and Bhat T. K., (2005). ‘Effect of fermentation system on the production and properties of tannase of Aspergillus niger van Tieghem MTCC 2425’. J. Gen. Appl. Microbiol., 51, 203–212. Rasouli I. and Kulkarni P. R. (1994). ‘Enhancement of beta-galactosidase productivity of Aspergillus niger NCIM-616’. J. Appl. Bacteriol., 77, 359–361. Ray M. W., Ramaiya P., Nelson B. A., Brody-Karpin S. D., Zaretsky E. J., Tang M., Lopez de Leon A., Xiang H., Gusti V., Clausen I. G., Olsen P. B., Rasmussen M. D., Andersen J. T., Jørgensen P. L., Larsen T. S., Sorokin A., Bolotin A., Lapidus A., Galleron N., Ehrlich S. D. and Berka R. M.(2004). ‘Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species’. Genome Biol., 5, R77.1–R77.12. Röhr M., Kubicek C. P. and Kominek J. (1983). ‘Citric acid’, in G. Reed and H. J. Rehm (eds), Biotechnology, vol. 3, Verlag Chemie, Weinheim, 420–454. Royer J. C., Moyer D. L., Reiwitch S. G., Madden M. S., Jensen E. B., Brown S. H., Yonker C. C., Johnstone J. A., Golightly E. J., Yoder W. T. and Shuster J. R. (1995). ‘Fusarium graminearum A 3/5 as a novel host for heterologous protein production’. BioTechnology, 13, 1479–1483. Royer J. C., Christianson L. M., Yoder W. T., Gambetta G. A., Klotz A. V., Morris C. L., Brody H. and Otani S. (1999). ‘Deletion of the trichodiene synthase gene of Fusarium venenatum: two systems for repeated gene deletions’. Fungal Genet. Biol., 28, 68–78. Schuster E., Dunn-Coleman N., Frisvad J .C. and van Dijck P. W. M. (2002). ‘On the safety of Aspergillus niger – a review’. Appl. Microbiol. Biotechnol., 59, 426–435. Sleep D., Finnis C., Turner A. and Evans L. (2001). ‘Yeast 2 micron plasmid copy number is elevated by a mutation in the nuclear gene UBC4’. Yeast, 18 (5), 403–421. Sumantha A., Larroche C. and Pandey A. (2006). ‘Microbiology and industrial biotechnology of food-grade proteases: A perspective’. Food Technol. Biotechnol., 44(2), 211–220. Tainter M. L. and Buchanan O. H. (1951). ‘Papain’. Ann. N. Y. Acad. Sci., 54(2), 147–259. Tominaga M., Lee Y.-H., Hayashi R., Suzuki Y., Yamada O., Sakamoto K., Gotoh K. and Akita O. (2006). ‘Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains’. Appl. Environ. Microbiol., 72, 484–490. Veith B., Herzberg C., Steckel S., Feesche J., Maurer K. H., Ehrenreich P., Bäumer S., Henne A., Liesegang H., Merkl R., Ehrenreich A. and Gottschalk G. (2004). ‘The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential’. J. Mol. Microbiol. Biotechnol. 7, 204–211. Wernars K., Goosen T., Wennekes L. M., Visser J., Bos C. J., van den Broek H. W., van Gorcom R. F., van den Hondel C. A. and Pouwels P. H. (1985). ‘Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene. Curr. Genet., 9, 361–368. Widner B., Thomas M., Sternberg D., Lammon D., Behr R. and Sloma A. (2000). ‘Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes’. J. Ind. Microbiol. Biotechnol., 25, 204–212.

4 Immobilized enzyme technology for food applications Marie K. Walsh, Utah State University, USA

4.1

Introduction

There are very few examples of commercial processes that utilize immobilized enzymes for food constituent modifications. In order for the immobilized process to be more economical or more useful than the soluble enzyme, either the cost per unit of product must be less or the product formed can only be produced with an immobilized enzyme. The two most successful examples of the use of immobilized enzymes are the production of high-fructose corn syrup and trans-free oils. Highfructose corn syrup can only be produced using the immobilized form of glucose isomerase (Swaisgood, 2003) and for immobilized lipases, the enzymes are more stable and active in low aqueous systems when immobilized. Therefore the use of the immobilized form of the enzymes for these processes is economical. The use of enzymes for food constituent modification has several advantages over the use of chemicals. The reactions are specific with generally fewer side reactions. For example it is possible to degrade starch into dextrins with acid, but this results in side reactions such as browning and off flavors; therefore enzymes are generally used to produce glucose and fructose syrups from starch. Also it is possible to modify the composition and hence functionality and nutritional impact of triacylglycerols chemically with hydrogen to produce saturated fatty acids, but this also results in trans fatty acids which have been shown recently to have antinutritional properties. The chemical method also requires downstream processing to remove impurities. Therefore the use of lipases is desired to change the fatty acid composition in triacylglycerols, as will be discussed later, to produce higher

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61

melting point fats/oils which do not require post processing to remove contaminants and, more importantly, do not contain trans fatty acids. Immobilized enzymes now have a proven track record of success with over 40 years of research into the development of industrial scale applications (Wiseman and Woods, 2004). Several potential advantages of immobilized enzymes include: (i) greater productivity per unit of enzyme since the enzyme is reused, (ii) precise control over the reaction, which is often automated and continuous, (iii) material handling is minimized, (iv) product does not contain the biocatalyst, (v) enzyme activity may be enhanced and/or stabilized and (vi) a unique product may be produced (Swaisgood, 2003, 2004). Since the biocatalyst is often used in a reactor, it is fairly easy to automate the process and control the extent of the reaction by simply altering the flow rate through the reactor (residence time). Economics play the most critical part in the use of immobilized enzymes over their soluble counterparts in food processing. The end result must be a reduced cost per unit of product produced with the immobilized enzyme. The major factors that determine the cost of an immobilized enzyme process include enzyme purification and immobilization, the matrix cost, regenerative capability of the biocatalyst, upand down-stream processing and sanitation requirements (Swaisgood, 2003). The half-life of the bioreactor factors into the economics of the system, with longer half lives being more economical. Examples of commercial bioreactor half lives are in the ranges of months to years and reactors can be operated through several halflives (Swaisgood, 2003). In addition to the half-life of the reactor, sanitation requirements and optimum temperature influence the economics of the process. It is favorable to operate the reactor at temperatures greater than 60 °C to control microbial growth. With high temperatures the reactors may not need to be cleaned or sanitized as frequently. The type of support and immobilization method is also important. While it would be favorable to immobilize the enzyme covalently onto an inert matrix, this renders the matrix non-reusable. Also, the chemical immobilization of an enzyme may reduce the enzyme activity owing to modifications or steric blocking of the active site. A common method of covalent immobilization involves glutaraldehyde crosslinking. The addition of a spacer can improve the enzyme activity (Li and Walsh, 2000; Nam and Walsh, 2005) by distancing the enzyme from the support to ensure appropriate contact with the substrate. Adsorption (electrostatic, biospecific, hydrophobic) of enzymes to a support is more common than chemical immobilization since the matrix can be stripped of the enzyme, reloaded, and reused, and there are generally fewer steps involved compared with covalent immobilization (Swaisgood, 2003). A drawback to the adsorption method is that the enzyme is in an equilibrium with the environment and desorption will occur over time leading to a decrease in activity. There are over 100 possible matrices with examples including glass, silica, celite, agarose, Sephadex, Sepharose, nylon, polystyrene, polyacrylamide, polyvinyl alcohol, polyethylene and glycol derivatives (White and Kennedy, 1980; Cao, 2005). A matrix with less attrition also contributes to the bioreactor productivity. Matrices for the entrapment, encapsulation, or containment of whole or dead cells are reviewed in

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Bickerstaff (1997) and specifically Kourkoutas et al. (2004) for immobilization technologies and support materials in alcoholic beverage production. There are many factors which contribute to the productivity of the biocatalyst (bioreactor), with the amount of product produced per unit of biocatalyst per half-life being the most critical (Swaisgood, 1991, 2003, 2004).

4.2

Immobilized enzyme technology for modification of acylglycerols

Lipases (EC 3.1.1.3) catalyse the hydrolysis of ester linkages in acylglycerols in aqueous environments at the oil–water interface. Under non-aqueous environments, lipases can catalyse the synthesis or interesterification of triacylglycerols. The optimum water levels for lipase-catalysed reactions vary from 0.042 (%v/v) to as high as 50 (%v/v) depending on whether net esterification or net hydrolysis is sought (Balcao et al., 1996). The interesterification reactions can be divided into three different processes: (i) acidolysis is the exchange of fatty acids between acylglycerols and free fatty acids: (ii) transesterification is the exchange of fatty acids between two acylglycerols: and (iii) glycerolysis is a reaction between free fatty acids or acylglycerols and glycerol. These activities are used to produce transfree and structured triacylglycerols. Recently, the use of the immobilized form of the lipase is preferred since immobilization improves the lipase stability and activity and the enzyme can be reused. The numerous types of supports used for immobilized lipases are reviewed in Balcao et al. (1996) and Malcata et al. (1990), with a majority of the immobilization methods employing non-covalent interactions. Obviously, non-covalent interactions (electrostatic and hydrophobic) predominate and dictate the strength of the interactions which then determines the bioreactor productivity. Balcao et al. (1996) provide an excellent review of 185 immobilized lipase reactors with respect to immobilization type, support, enzyme source, and reactor configurations. Table 4.1 lists the characteristics of some commercially available immobilized lipases. 4.2.1 Production of trans-free oils Regular soy oil contains approximately 54% linoleic acid (18:2), 23% oleic acid (18:1), 11% palmitic acid (16:0), 8% linolenic acid (18:3), and 4% steric acid (18:0). This fatty acid profile is high in unsaturated fatty acids, which is healthy, but shows poor oxidative stability. To improve oxidative stability, partial hydrogenation is often used but this does produce trans fats, which have adverse health effects. Partial hydrogenation is carried out at high temperatures (140–225 °C) and pressures (414 kPa) and the reaction is catalysed by a nickel catalyst in the presence of pure hydrogen. The process typically takes about 2 h after which the addition of citric acid is required to eliminate nickel soaps. The oil is further processed by filtration, bleaching and deodorization (Nawar, 1996). Changing the fatty acid content can be done chemically by interesterification,

Immobilized enzyme technology for food applications Table 4.1

63

Characteristics of some commercially available immobilized enzymes

Name

Supplier

Organism

Specificity

Lipozyme TL IM

Novozyme A/S

Thermomyces lanuginose, TLL-1 Rhizomucor miehei, RML Candida antartica lipase B Pseudomonas cepacia lipase, PCL Burkholderia cepacia lipase (formerly Pseudomonas fluorescens lipase PFL)

sn-1,3 specific Silica granules

Lipozyme RM Novozyme IM Novozyme 435 Novozyme Lipase PS-C

Amano

Lipase AK-C

Amano

Matrix

sn-1,3 specific Macroporous ion exchange resin non-specific Macroporous acrylic resin non-specific Ceramic particles

sn-1,3 specific Ceramic particles

which is simple and inexpensive but random. The chemical process uses a highly reactive catalyst (sodium methylate or sodium ethylate) to shift the fatty acids on a triacylglycerol randomly. No trans fatty acids are produced but the product requires thorough purification to remove by-products after the interesterification reaction. The purification steps include washing with water, bleaching and deodorizing. The fatty acid content of triacylglycerols can also be changed enzymatically with lipases in very low aqueous environments. This reaction is mild and specific and requires little downstream processing. An example reaction utilizing an immobilized lipase, commonly a sn-1,3 specific lipase, in a low aqueous environment with soy oil and free fatty acid (oleic acid) can result in the production of soy oil with a higher oleic acid content with improved oxidative stability. It is very important to control the water level in the reaction. Initially, some water is required for hydrolysis of fatty acids from the triacylglycerol molecules. When the water has been used for hydrolysis to produce the free fatty acids, the direction of lipase action is reversed and they catalyse the synthesis of triacylglycerols with new fatty acid profiles. The final oil does need to be deodorized to remove residual free fatty acids. The commercial production of enzymatically interesterified oils for the production of trans-free fats is being done in the USA as well as several other countries. The process is based on Novozymes immobilized lipase system. To produce the immobilized lipase (Lipozyme TL IM), the lipase and liquid binder are sprayed by atomization onto a silica carrier with a particle size below 100 µm. During the granulation, the silica particles become agglomerated into larger, porous particles (300–1000 µm) with the enzyme distributed evenly over the silica surface area (Berben et al., 2001). The mean diameter of the particles is 600 µm and the surface area is approx 50 m2 gm–1 (Berben et al., 2001). The silica is porous but still is

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Novel enzyme technology for food applications

mechanically stable for both batch and fixed-bed column operations (www.novozymes.com). The materials in this process are all food grade and the resulting catalyst has an activity on average based on the matrix weight of 400 IUN g–1 (inter-esterification unit novo per gram) and on the volume basis (packed bed) of 170 M-IUN m–3 (www.novozymes.com). The Novozymes immobilized lipase, Lipozyme TL IM, is a 1,3 specific lipase from Thermomyces lanuginose that shows a degree of conversion between 30 and 90% (www.novozymes.com). The Lipozyme TL IM can be used in batch format or in a reactor termed a Plug and Play reactor (www.novozymes.com). This reactor is a fixed bed continuous flow reactor secured between the raw material tank and the end product tank. The Plug and Play reactor holds approximately 400 kg of Lipozyme TL IM wetted in approximately 750 kg of oil and is designed for operating pressures of 5–250 kPa. In order to operate, the oil is pumped down through the reactor and distributed evenly over the matrix by a double-cone flow distributor. It is necessary to dry the oil before interesterification to ensure limited formation of free fatty acids. The oil is interesterified as it passes through the bed and the residence time can be adjusted to control the degree of conversion. A 1 m3 column can produce 50 tonnes of interesterified fat/oil per day. At the exit end of the reactor, there is a slit screen to trap broken matrix particles (www.novozymes.com). For the batch process, the Lipozyme TL IM is mixed with the oil and held in a tank for a predetermined length of time to achieve the desired degree of conversion. The matrix can be separated from the oil by sedimentation or filtration (www.novozymes.com). For both processes, deodorization of the oil is necessary to remove free fatty acids, but no washing or bleaching is necessary as in the chemical method. According to Novozymes A/S (www.novozymes.com) the immobilized lipase system is economical compared with both partial hydrogenation and chemical interesterification if operating and investment cost are considered. In December 2001, Karlshamns AB in Sweden was the first company in the world to use Novozymes immobilized lipase system for the production of modified vegetable fats. This system uses a Plug and Play reactor for the interesterification of palm or stearin oil, palm kernel and coconut oil at 70 °C. Ukraine KMT was the second plant to use the immobilized enzyme in the Plug and Play continuous reactor with a combination of palm and sunflower oils in 2003. By January 2004, KMT was using a reactor with a capacity of 400 kg of enzyme and produced 800 –900 kg of interesterified oil per hour (www.novozymes.com). KMT produces a range of products aimed at the confectionery, baking and margarine markets using different mixtures of hydrogenated sunflower oil, unhydrogenated sunflower oil and palm olein as raw ingredients (www.novozymes.com) The first commercial production of trans-free oils using Novozyme’s immobilized lipase system in the USA was in 2002 by Archer Daniels Midland Company (ADM) in Quincy, Il. At this facility more than 6 million kg of interesterified oil has been produced. ADM has opened a new enzymatic interestification facility in Mankato, Min. This is a joint ADM and Novozymes venture for the use of lipases to produce healthier oils and fats for use in margarines, baking and confectionery. ADM’s products in the NovaLipidTM family include shortenings, margarine and

Immobilized enzyme technology for food applications

65

liquid oils with zero/low trans fat that are made from corn, sunflower and/or soy oils. The interesterification process used to create the NovaLipidTM line of fats can result in products with 20–40% stearic acid. For example, combining about 25% fully saturated soybean oil, which is trans fat free but also rich in stearic acid, and 75% liquid soybean oil can result in a final product that is virtually trans-free, yet solid at room temperature with functional properties suited for baking applications.

4.2.2 Production of cocoa butter equivalents Lipases are used to change the melting properties of fats to create a higher value product. Cocoa butter has a melting point of 37 °C which is attributed to its fatty acid content. The major components of cocoa butter are 1(3)-palmitoyl-3(1)stearoyl-2-mono-olein (POS) and 1,3-distearoyl-2-mono-olein (SOS) which are 52 and 18.4%, respectively, of the total (Chang et al., 1990). This sharp melting point is related to consumer acceptance of chocolate. Immobilized lipases are used for the interesterification of palm oil mid-fraction which contains high concentrations of triacylglycerols with palmitic acid in the sn-1 and sn-3 positions and oleic acid in the sn-2 position. In the presence of free steric acid, interesterification of palm oil mid fraction with an sn-1,3 specific lipase results in triacylglycerol composition of POS and SOS which resembles cocoa butter and is used as cocoa butter equivalent in the confectionery industry. Scientists at Unilever describe the production of cocoa butter substitutes via a fixed-bed reactor using a 1,3 specific lipase from vegetable oil and various fatty acids such as myristic, palmitic and/or stearic acids (Macrae and How, 1988, 1999). Obviously, this is another example where the enzymatic reaction is preferred to the random chemical interesterification reaction. Chang et al. (1990) and Bloomer et al. (1990) also describe the production of cocoa butter using immobilized lipases.

4.2.3 Production of modified triacylglycerols Triacylglycerols can be modified to provide improved nutritional or functional properties via chemical interesterification or with immobilized lipases. Since the chemical reaction lacks specificity, offers little or no control over the positional distribution of fatty acids, and by-products are also formed, the use of lipases is preferred. Structured lipids are tailor-made fats and oils with improved nutritional or physical properties owing to the incorporation of new fatty acids or a change in the position of existing fatty acids on the glycerol backbone (Osborn and Akoh, 2002). Medium chain or medium-long-medium chain triacylglycerols (MCT or MLM) that contain short or medium chain fatty acids at the sn-1 and sn-3 positions and long chain fatty acids at the sn-2 position are transported to the human liver after consumption and are metabolized to provide energy without being deposited in adipose tissue (Megremis, 1991; Osborn and Akoh, 2002). MCT are used to treat patients with fat-absorption abnormalities. The synthesis of structured lipids using immobilized lipases has been reported and if the enzyme is a sn-1,3 specific

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Novel enzyme technology for food applications

lipase, MCTs can be produced by the interesterification of short chain fatty acids. For example, Ko et al. (2003) synthesized structured lipids containing high alphalinolenic acid by interesterification of perilla oil and caprylic acid, Zhou et al. (2001) incorporated caproic acid into rapseed oil and Jennings and Akoh (2000) incorporated capric acid into rice brain oil. The enrichment of triacylglycerols by incorporating healthy fatty acids can be done to enhance the fat/oil nutritionally. For example, several researchers (Kim et al., 2001a; McNeil et al., 1999) have incorporated conjugated linoleic acid into tricaprylin oil or palm oil. Yang et al. (2003) investigated the interesterification of lard with soy fatty acids to produce a human milk fat substitute. Schmid et al. (1999) described an improved synthesis of 1,3-oleoyl-2-palmitoylglycerol (OPO) for use in infant formulas using immobilized sn-1,3 specific lipases. OPO is the major ingredient in a commercial product, Betapol, for infant formulas. There have been several publications on the modification of butterfat to improve its nutritional content by lowering the amount of saturated fatty acids present and by the incorporation of healthy fatty acids catalysed by immobilized lipases. Ronne et al. (2005) used commercially available immobilized lipases to catalyse the interesterification of butterfat with rapeseed oil to reduce the concentration of saturated fatty acids. Kim et al. (2002) incorporated α-linolenic acid purified from perilla oil into butterfat, while Balcao et al. (1998) and Oba and Witholt (1994) incorporated oleic acid, and Garcia et al. (2000) incorporated conjugated linoleic acid into butterfat. Other publications on improving the physical characteristics of butterfat by interesterification by immobilized enzymes have been reported (Rousseau and Marangoni, 1999; Lee and Swaisgood, 1997; Bornaz et al., 1994).

4.2.4 Modification of phospholipids Phospholipases are used in the food industry to modify egg yolk and soy phospholipids. The enzyme-modified products containing lyso-lecithin have enhanced emulsifying properties. Novozymes produces several phospholipases including two microbial phospholipases (Lecitase Novo and Lecitase Ultra) that do not require calcium and are typically used in the soluble form (www.novozymes. com). They are classified as phospholipase A1 to yield the corresponding lyso-1phospholipid plus free fatty acid. Acyl migration will result in conversion to the more stable lyso-2-phospholipids which is the same result obtained with phospholipase A2 (www.novozymes.com). Methods for the immobilization of phospholipase A2 (PPLA2, EC 3.1.1.4) for use in food are few. Kim et al. (2001b) described a method for the production of egg yolk lysolecithin with PPLA2 immobilized in an alginate–silicate sol–gel matrix and found the enzyme activity was very low in hydrophilic solvents compared with organic solvents and that the activity improved with the addition of 10 mM CaCl2. Nam and Walsh (2005) demonstrated the activity of covalently immobilized bovine pancreas PPLA2 in liquid eggs by measuring an increase in free fatty acid content.

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67

4.2.5 Production of diacylglycerols The Japan based Kao Corp has launched a new diacylglycerol (DAG) product under the name Healthy Econa Cooking Oil which is made enzymatically from natural oil using immobilized lipases. This product is marketed as a healthy oil since DAG aids in the maintenance or loss of weight and fat mass, may lower the level of cholesterol in the body and may help maintain healthy triacylglycerol levels (reviewed in Pszczola, 2001; Nagao et al., 2000). DAG is digested and absorbed in the small intestine and is consumed as energy without resynthesizing into a neutral fat like conventional oil. As a result, it reduces the level of neutral fat in the blood compared with conventional oil (Matsuo and Tokimitsu, 2001). A variety of Healthy Econa salad and cooking oils are sold in Japan and also used in processed products such as canned tuna fish, margarine and bread (www.novozymes.com). The US counterpart of Healthy Econa Cooking Oil is EnovaTM oil produced by Archer Daniels Midland (Decatur, Il). The DAG composition of EnovaTM oil is 20–65% weight oleic acid, 15–65% weight linoleic acid and less than 15% weight linolenic acid (http://www.enovaoil.com/ food/specs.asp). There are a variety of procedures for the synthesis of DAG using immobilized lipases. The glycerolysis reaction involves the use of glycerol and oils. An example is the synthesis of DAG from rapeseed or sunflower oil catalysed by Novozym 435 (Novozymes) yielding 60 wt% DAG which was increased to over 90 wt% after deodorization and distillation (Kristensen et al., 2005). The glycerolysis reaction using glycerol and free fatty acids can also result in DAG (Weber and Mukherjee, 2004; Lo et al., 2004; Watanabe et al., 2003.). For example, Lo et al. (2004) catalysed DAG synthesis from corn oil fatty acids and glycerol using Lipozyme RM IM for yields of 70%. The interesterification of oils in the presence of monoacylglycerols (MAG) can also lead to DAG. Weber and Mukherjee (2004) synthesized DAG using rapeseed oil, commercial preparations of MAG and Lipozyme RM IM (Novozymes) with a 60–70% yield. Archer Daniels Midland produces EnovaTM oil in Decatur, Il for the ADM-Kao joint venture. The process uses a combination of soy and canola oils as starting materials. The fatty acids are first cleaved from the oils, then cold crystallized to reduce the concentration of saturated fatty acids to very low levels. This product contains 3.5–4% saturated fatty acids. The fatty acids are then combined with glycerol in the presence of a 1,3 specific immobilized lipase to produce the 1,3-diacylglycerol. The reaction mixture contains some 1-monoacylglycerols and unreacted fatty acids. These are removed by molecular distillation and recycled. The purified 1,3-diacylglycerol at that point is slightly greater than 80%. It will equilibrate with the 1,2-diacylglycerol over time so that 70% of the diacylglycerol present remains in the 1,3 form (personal communication Mark Matlock, Sr. Vice Present Food Research ADM Co).

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4.3

Novel enzyme technology for food applications

Immobilized enzyme technology for modification of carbohydrates

4.3.1 Production of high-fructose corn syrup The production of high-fructose corn syrup (HFCS) involves the use of immobilized D-glucose/xylose isomerase (D-xylose ketol isomerase; EC 5.3.1.5). This represents the major use of immobilized enzymes for food processing. Sucrose derived from sugar beet (40%) and sugar cane (60%) was the main sweetener until the mid-1970s. The use of starch to produce HFCS for use as a sweetener was prompted by the lack of supply after the Cuban revolution in 1958. Initial use of glucose isomerase for HFCS production in the US in 1967 was done commercially by Clinton Corn Processing. The immobilized form of the enzyme was available in 1974 (reviewed in Bhosale et al., 1996). Since 1980, most major starch-processing companies have been using the immobilized form of glucose isomerase. Production of HFCS-42 in the USA was 3 342 000 tonnes and production of HFCS-55 was 5 000 000 tonnes for a total of 8 342 000 tonnes dry weight in 2005 (USDA). The enzymatic production of HFCS is an example where the use of enzymes is more desirable than the use of chemicals. The production of non-metabolizable sugars, colored products and reduced sweetness occur with the chemical conversion of starch to HFCS. The use of enzymes allows the production of HFCS under ambient pH and temperature, where fewer side products are formed and a higher fructose concentration is achieved. The production of HFCS generally begins with a 40% solution of insoluble corn starch that is adjusted to pH 6.0; then calcium and a thermostable α-amylase (commonly EC 3.2.1 from Bacillus lichenformis) are added. The solution is gelatinized at 105 °C for 5–8 min followed by cooling to 95 °C for 2 h for liquefaction. The α-amylase is an endo-acting enzyme that produces low dextrose equivalent (DE) maltodextrins. This enzyme requires a pH of 6.0 or above and the presence of calcium, which later needs to be removed via ion-exchange chromatography (Crabb and Shetty, 1999). Saccharification of the maltodextrins to form glucose syrup (32–34% dry solids) is carried out after adjusting the pH to 2 at 60 °C for 36–48 h in the presence of glucoamylase (EC 3.2.1.3) and pullulanase (EC 3.2.1.41). Glucoamylase is exoacting; it produces glucose from the non-reducing end of maltodextrins and is generally purified from Aspergillus awamori and Aspergillus niger strains. Pullulanase hydrolyses the α1-6 branch points and is generally derived from bacterial sources such as Klebsiella pneumoniae and Bacillus acidopullulyticus. The production of HFCS from glucose syrup requires the use of ion-exchange chromatography to remove calcium which inhibits glucose isomerase. Prior to isomerization, the glucose solution is adjusted to pH 7.8 and magnesium is added. The solution is then allowed to flow through an immobilized glucose isomerase column kept at approximately 58 °C. The flow rate and temperature are controlled to yield approximately 42–45% fructose. The fructose/glucose syrup is fractioned using moving-bed cation-exchange chromatography to produce 90% fructose syrup, which is blended with the 42% fructose syrup to yield a 55% fructose syrup

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(Swaisgood, 2004). Operating at a higher temperature favors fructose production, but most commercially available glucose isomerases are inactivated at temperatures greater than 60 °C (Crabb and Shetty, 1999). Also the KM value for glucose is a limiting factor in the isomerization step. For example, the KM value for glucose isomerase from Bacillus coagulans for D-glucose is over 15 times that for D-xylose (Wong, 1995). It is necessary to use the immobilized form of this enzyme owing to its cost. The cell-free enzymes are typically immobilized via adsorption onto anion-exchange resins and may be crosslinked with glutaraldehyde (Bhosole et al., 1996). In addition, the use of whole cell immobilization is more commonly done which includes crosslinking heat-treated cells with glutaraldehyde or adsorption of heat-treated cells onto anion-exchange resins. Production of high-fructose corn syrup is a multi-step process which includes step (1) pH 6–6.5, 95–105 °C, step (2) pH 4.5, 58–62 °C and step (3) pH 7.0–8.5, 55–60 °C. Identification of improved enzymes for the production of HFCS that would allow the process to flow without the need to change the pH, temperature or metal requirements would be beneficial. Current research into the identification of enzymes from alternative sources or genetic engineering of the currently used enzymes is continuing. Specifically, identification of a glucose isomerase that has a lower pH optimum (Kaneko et al., 2000), is not inhibited by the presence of calcium and has a higher thermostability (Bandlish et al., 2002) would simplify the process and yield higher fructose concentrations (Crabb and Shetty, 1999; Bhosale et al., 1996). Commercially available immobilized glucose isomerases for the production of high-fructose corn syrup include Novozyme Sweetzyme R from Novozyme, the Gensweet family from Genencor International and SWETASE® from Nagase Co. Sources of commercial glucose isomerases include Actinoplanes missousriensis, B. coagulans and Streptomyces rubiginosus (Bhosale et al., 1996). Specifically for Novozymes Sweetzyme IT, the isomerization reaction is stopped at a yield of 45% fructose and has a half life of about 200 days (www.novozymes.com).

4.3.2 Synthesis of functional oligosaccharides A growing area of interest is in the development of immobilization technologies for the synthesis of isomalto-, xylo-, fructo- and inulo-oligosaccharides for use in foods. These sugars can act as soluble dietary fibers that are also prebiotics, stimulating the growth of probiotic microorganisms such as Bifidobacterium spp. in the colon and Lactobacillus spp. in the gut. The degree of polymerization of functional oligosaccharides is between 2 and 10 saccharides. Oligosaccharides derived from starch include malto- and isomalto-oligosaccharides, isomaltose, cyclodextrins and trehalose. Sucrose-derived oligosaccharides include fructo-oligosaccharides, isomaltulose, and glycosylsucrose. Lactose-derived oligosaccharides include galacto-oligosaccharides, lactosucrose, lactulose and lactitol (Nakakuki, 2002). Enzymes used for the production of functional oligosaccharides belong to two groups. The glycosidases (EC 3.2) mediate hydrolytic cleavage of glycosidic bonds. The glycosyltransferases or transglycosylases (EC 2.4) are capable of transferring glycosyl groups (Table 4.2).

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Enzymes used for functional oligosaccharide production

Trivial names

Systematic name

Raw material Functional oligosaccharide

Levansucrase (betafructosyltransferase) Inulosucrase Beta-galactosidase Beta-xylanase Beta -fructofuranosidase

Sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase EC 2.4.1.10

Sucrose

Fructo-

Sucrose:2,1-beta-D-fructan 1-beta-D-fructosyltransferase EC 2.4.1.9 beta-D-Galactoside galactohydrolase EC 3.2.1.23 1,4-beta-D-Xylan xylanohydrolase EC 3.2.1.8 beta-D-Fructofuranoside fructohydrolase EC 3.2.1.26

Sucrose Lactose Corncob Sucrose Lactose Starch

FructoGalactoXyloLacto-fructo Isomalto-

Sucrose Inulin Sucrose

Isomaltulose (palatinose) Inulo Isomalto-

Beta-amylase Pullulanase Transglucosidase (Alpha-glucosidase) Isomaltulose synthase Endoinulinase (inulinase) Dextransucrase

1,4-alpha-D-Glucan maltohydrolase EC3.2.1.2 Pullulan alpha-1,6-glucanohydrolase EC 3.2.1.41 alpha-D-Glucoside glucohydrolase EC 2.4.1.20 Sucrose glucosylmutase EC 5.4.99.11 2,1-beta-D-Fructan fructanohydrolase EC 3.2.1.7 Sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase EC2.4.1.5

Novel enzyme technology for food applications

Table 4.2

Immobilized enzyme technology for food applications

71

They are typically microbial enzymes and are used to produce functional oligosaccharides via immobilized purified enzyme, entrapped microbial cells, or conventional batch reactions. Sugar can be converted to isomaltulose (palatinose) and the by-product trehalulose using isomaltulose synthase (EC 5.4.99.11). Isomaltulose is a low-calorie reducing sugar found naturally in honey. It has several characteristics that are advantageous compared with sucrose including stability in acid solutions, promoting bifidobacteria growth in the human intestine and it is non-cariogenic. Cheetham et al. (1982) described the first use of immobilized Erwinia rhaponica cells for the production of isomaltulose. Isomaltulose is produced using immobilized cell reactors using either Protaminobacter rubrum, E. rhaponica or Serratia plymuthica cells (Cheetham, 1987). Fructo-oligosaccharides (FOS) are produced from sucrose by the transfructosylation action of fungal beta-fructofuranosidase. Recent research using an immobilized form of the purified enzyme (Nishizawa et al., 2000; Tanriseven and Aslan, 2005) or immobilized Aureobasidium pullulans cells in alginate (Shin et al., 2004) has been described. FOS are nondigestible sweeteners, which are utilized by intestinal bifidobacteria. The continuous production of fructo-oligosaccharides using fructosyltransferase immobilized by adsorption onto a non-ionic ion exchange resin was reported by Yun et al. (1995). In this system, sucrose was used as the substrate for the synthesis of fructo-oligosaccharides using a column operated at 50 °C continuously for 30 days with an 8% loss of activity. Other investigators are also developing continuous systems for FOS production (Shin et al., 2004) using ceramic membranes. Inulin is another source of functional oligosaccharides, which is found in garlic, asparagus root, Jerusalem artichoke, dahlia tubers and chicory roots. Inulin consists of linear β1-2 linked fructose molecules which, when hydrolyzed, yield fructose syrups or oligofructose (inulo-oligosaccharides). Production of inulooligosaccharides can be done with either the immobilized endoinulinase (typically EC 3.2.1.7 produced by A. niger) (Rocha et al., 2006 and references therein; Nakamura et al., 2001; Yun et al., 2000) or whole cells (Barranco-Florido et al., 2001; Yun et al., 1997). For example, the production of inulo-oligosaccharides from inulin derived from dahlia tubers was done with immobilized endoinulinase EC3.2.1.7 from A. niger (Nakamura et al., 2001) and the products were preferentially utilized by Bifidobacterium spp. but not by Escherichia coli or Clostridium perfringens. Recent reports on the continuous production of isomalto-oligosaccharides (IMO) using immobilized dextransucrase (EC 2.4.1.5) from Leuconostoc mesenteroides include Reischwitz et al. (1995), Tanriseven and Dogan (2002) and Berensmeier et al. (2004). These reports immobilized the purified dextransucrase in alginate beads or fibres for the conversion of sucrose into IMO. Other examples of immobilized enzymes for carbohydrate hydrolysis or novel carbohydrate synthesis include the use of pectinases for fruit juice clarification (Sartoglu et al., 2001; Demir et al., 2001; Carrin et al., 2000) and immobilized cells for xylitol production from sugarcane bagasse (Santos et al., 2005a, b). Xylo-

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Novel enzyme technology for food applications

oligosaccharides can be formed from lignocellulosic materials. In a recent study (Ai et al., 2005), xylo-oligosaccharides were produced from corncob powder using a xylanase purified from Streptomyces olivaceoviridis E-86 immobilized onto Eudragit S-100.

4.3.3 Lactose hydrolysis Lactose is the major sugar found naturally in milk. It is composed of glucose and galactose and can be hydrolyzed by β-galactosidase (EC 3.2.1.23). Hydrolysis is beneficial in some cases because of lactose intolerance and the resulting hydrolyzed sugar is sweeter. Milk (pH 6.7) and whey (either sweet or acid which are byproducts of cheese manufacture) are the common substrates for the enzymatic hydrolysis of lactose. Various enzymes are used for the various dairy products owing to the pH differences. For example, β-galactosidase from A. niger has a pH optimum of about 4.5 while that from Saccharomyces lactis has a pH optimum of about 7. β-Galactosidase can be immobilized for lactose hydrolysis although currently the soluble enzyme is more often used. The use of immobilized β-galactosidase for the hydrolysis of lactose in dairy products has been reviewed by Gekas and Lopez-Leiva (1985). Several industrial processes were in operation for lactose hydrolysis in milk or whey. These included the processes by Snamprogetti in Italy to reduce lactose in milk, by the Corning Glass Company in the UK, France and USA to hydrolyse lactose in whey, and by Valio Laboratory in Finland to hydrolyse lactose in whey (Gekas and LopezLeiva, 1985). The greatest problems associated with immobilized β-galactosidase for lactose hydrolysis are contamination from microbial growth and cost (Swaisgood, 2004). The Snamprogetti process was used by Industrial Centrale Latteria di Milano in Italy. This system used enzyme entrapped in cellulose triacetate to reduce the lactose content in milk via a batch process to yield 9.1 tonnes/day lactosehydrolyzed milk (Gekas and Lopez-Leiva, 1985; Swaisgood, 2004). A similar process was used in Japan by Snow Brand milk to produce lactose-hydrolyzed milk but with an added sanitation step. The bioreactor was immersed in a solution of 10% glycerol at 10 °C to prevent microbial growth. The Corning Glass process was used by ULN Condi (France), Dairy Crest (UK) and Kroger (USA). This process used A. niger β-galactosidase covalently bound to silica beads in a fixed-bed reactor to hydrolyse lactose in acid whey and ultrafiltration permeate (Gekas and Lopez-Leiva, 1985). The Valio Laboratory process was used industrially by Keymenlaakso Dairy (Finland). β-Galactosidase from A. niger was adsorbed to phenol formaldehyde resin (Duolite ES-762) for whey and ultrafiltration permeate processing in a fixed bed reactor. The lactose-hydrolyzed whey product was used as a syrup in dairy and confectionery products. The typical reactor had a half life of 20 months and a productivity of 2000 kg dry matter per kg enzyme (Swaisgood, 2004). There are some recent publications on the immobilization of β-galactosidase for lactose hydrolysis (Zhou and Chen, 2001; Maciunska et al., 2000) or the use of

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calcium alginate immobilized cells for lactose hydrolysis in milk (Jordao et al., 2001). Petzelbauer et al. (2002) has demonstrated the use of two hyperthermostable β-galactosidases immobilized onto Eupergit C, chitosan and controlled pore glass for the hydrolysis of lactose in whey and milk at 70 °C.

4.3.4 Tagatose production D-Tagatose is a naturally occurring monosaccharide, which can be found naturally in small amounts in dairy products and it is 92% as sweet as sucrose with only 38% of the calories. D-Tagatose has generally recognized as safe (GRAS) status in the USA as a sweetener for use in foods. It can be produced from galactose via isomerization under alkaline conditions with a metal hydroxide (Beadle et al., 1992). D-Tagatose can be purified from the mixture of D- and L-tagatose by crystallization. The chemical processing method does result in by-product formation and generation of chemical waste. Tagatose can also be formed from galactose using L-arabinose isomerase (EC 5.3.1.4) either directly immobilized onto a support or via the immobilization of cells (for example Kim et al., 2003; Jung et al., 2005). In addition, a recent patent describes a process of preparing D-tagatose from galactose using an immobilized form of a thermostable L-arabinose isomerase enzyme derived from Thermotoga neapolitana (Pyun et al., 2005). The activity of the immobilized form of this thermostable L-arabinose isomerase was over 80% after a 20-day heat treatment. In 1996, MD/Arla Foods acquired the rights to produce and commercialize GaioR tagatose for foods and beverages from Spherix Inc. GaioR tagatose is produced by SweetGredients KG which is a joint venture between Nordzucker AG and Arla Foods Ingredients AMBA. Spherix also produces a tagatose product under the brand name of Naturlose. Valio has entered into an agreement with Nordzucker AG to provide the immobilized β-galactosidase technology for tagatose production (Swaisgood, 2004).

4.4

Immobilized enzyme technology protein modification

4.4.1 Production of protein hydrolysates There has been some renewed research on the use of immobilized proteases for production of bioactive peptides, hydrolysates for nutritional supplements and hypoallergenic infant formulas, or to change the functionality of protein systems. The degree of hydrolysis varies from low hydrolysis (2–5 kDa peptides) to improve functional properties of proteins to extensive hydrolysis (<2 kDa peptides or free amino acids) for nutritional supplements and hypoallergenic foods. The use of the immobilized form of the enzyme has several advantages including reuse, no need for heat or acid treatment in downstream processing which may change the functional and/or nutritional content of the product, increased enzyme thermal stability (Tardioli et al., 2003) and reduced autolysis. Drawbacks include a

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decrease in activity over time caused either by autolysis or by competition from the newly formed peptides (Sousa et al., 2004), mass transfer limitations and protein fouling of the matrix (Herbert and Dunnill, 1987). Despite the renewed interest in immobilized proteases, the industrial format of choice is still the soluble form owing to economics as well as the drawbacks listed above. By 1994, there were at least 100 medical foods on the market, many of which were formulated using protein hydrolysates from casein, soy, or wheat (Schmidl et al., 1994). This area includes hydrolysates for patients with Crohn’s disease, ulcerative colitis, short bowel syndrome, food allergies and for infant formula. Numerous publications describe the use of multiple immobilized enzymes for the complete hydrolysis of food proteins for the determination of protein digestibility, which is directly related to protein quality (Porter et al., 1984, Church et al., 1984; Thresher et al., 1989). This immobilized digestive enzyme assay (IDEA) system generally consisted of two bioreactors; one bioreactor contained immobilized pepsin at acidic pH to simulate the stomach environment while the second contained a mixture of immobilized trypsin, chymotrypsin and intestinal mucosal peptidases at pH 7 to simulate the small intestine environment. This IDEA system could also be used to produce free amino acids for use in medical foods. Recently, Pedroche et al. (2004) used a similar series of enzymatic hydrolysis steps to achieve a casein-hydrolyzed product with a high ratio of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) which has the potential use as a medical food for patients suffering from hepatic encephalophathies, tyrosinemia and phenylketonuria. The immobilized enzymes used consisted of trypsin, αchymotrypsin and carboxypeptidase A individually immobilized to agarose. A gel filtration step was used to separate hydrolysate fractions enriched in a high ratio of BCAA/AAA. Previous work has investigated the use of immobilized proteases to hydrolyse casein (Ge et al., 1996) and predigest soybean protein (Ge and Zhang, 1993) for use in infant formulas or as a medical food. Bioactive peptides have been identified in protein hydrolysates from milk, fish, corn, eggs and cereals (reviewed in Kitts and Weiler, 2003). Ticu et al. (2005) investigated the production of various bioactive peptides from hemoglobin hydrolysis using immobilized porcine pepsin. In addition, immobilized trypsin has been used to liberate phosphopeptides from casein (Lorenzen and Schlimme, 1995; Park et al., 1998).

4.4.2 Modifying protein functionality Enhancing the functionality of food proteins by limited hydrolysis with soluble proteases is reviewed in Panyam and Kilara (1996). There are several examples of the use of immobilized proteases for limited proteolysis of food proteins for enhanced functionality. Huang et al. (1996) evaluated the β-lactoglobulin hydrolysates derived from limited proteolysis (a 2.4% degree of hydrolysis) with immobilized trypsin. The authors demonstrated an enhanced emulsification activity index of the hydrolysates compared with the intact protein and stressed the importance of limited, controlled hydrolysis to achieve peptides with enhanced

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functionality, hence the immobilized form of the enzyme is optimum. A similar study by Chen et al. (1994) compared the gelation properties of native βlactoglobulin to a β-lactoglobulin hydrolysate prepared by limited proteolysis via immobilized trypsin. The partially hydrolyzed β-lactoglobulin showed a lower gel point and gelled more rapidly than the native protein. This concept of changing a protein’s functionality with immobilized enzymes has expanded from limited proteolysis with immobilized proteases to crosslinking with transglutaminase (Wilcox and Swaisgood 2002; Truong et al., 2004). Microbial transglutaminase (EC 2.3.2.13) is a calcium-independent transglutaminase that catalyses protein crosslinking through an acyl transfer reaction involving a proteinbound glutaminyl residue (acyl donor) and primary amines (acyl acceptors) such as lysine. Wilcox and Swaisgood (2002) characterized the functional changes associated with whey protein (8% solution) after exposure to immobilized transglutaminase. The crosslinking reaction only occurred in the presence of a reducing agent (dithiothreitol or sodium bisulfite) which the authors hypothesized was necessary to partially unfold the whey proteins. The intrinsic viscosity increased, the gelation temperature decreased, and stronger, more brittle gels were formed after heating with an increase in exposure of whey protein to immobilized transglutaminase. Other work by Truong et al. (2004) showed a decrease in gel strength of whey protein treated with immobilized transglutaminase which the authors proposed may have been caused by the degree of whey protein crosslinking. The former study investigated the functional properties of whey protein with limited crosslinking with immobilized transglutaminase while the later study investigated the changes in functional properties with extensive crosslinking. Extensive crosslinking of whey proteins may have inhibited protein unfolding and network formation which is necessary for strong gel formation (Truong et al., 2004). Unfortunately the immobilized enzyme matrix lost 50–70% of its activity which may have been due to clogging of the matrix pores and/or the formation of a monolayer of protein on the matrix surface (Wilcox and Swaisgood 2002; Truong et al., 2004). A novel system for the production of hydrolyzed/polymerized whey proteins was developed by combining the technologies described above (Wilcox et al., 2002). This system involved the use of a streptavidin–trypsin fusion protein and a streptavidin–transglutaminase fusion protein individually immobilized onto biotinylated controlled-pore glass for first, limited proteolysis followed by limited crosslinking of whey protein. As observed by Wilcox and Swaisgood (2002), the whey protein treated by limited proteolysis followed by limited crosslinking resulted in an increase in intrinsic viscosity, a decrease in gelation temperature and the formation of stronger, more brittle gels.

4.5

Immobilized enzyme technology for production of flavor compounds

4.5.1 Ester flavor synthesis Fatty acid esters can be synthesized by immobilized lipases. The reaction generally

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involves esterification of a carboxylic acid (C4 or longer fatty acids) with an alcohol in non-aqueous (that is, heptane or hexane) or low water environments (Bruno et al., 2004; Chiang et al., 2003). Various esters can be synthesized for application as flavors or surfactants. Flavor compounds are typically short chain fatty acids and alcohols such as ethyl butyrate (pineapple or strawberry flavor), methyl butyrate (pineapple or apple flavor), butyl butyrate and isobutyl isobutyrate (pineapple flavor), isoamyl isovalerate (apple flavor) and isoamyl acetate/butyrate (banana flavor). Ester production can be done by reacting a fatty acid and alcohol at high temperature in the presence of a metal catalyst. This can lead to undesirable side products, hence the use of immobilized lipases offers synthesis under milder conditions for the production of natural flavors. The parameters affecting the activities of lipase in esterification reactions include the reaction time, temperature, added water content and acyl donor (Yahya et al., 1998). Several researchers have investigated the synthesis of isoamyl acetate, which has a demand of 67 tonnes annually in the USA alone (Welsh et al., 1989). Most reports use commercially available immobilized lipases in organic solvents (Romero et al., 2005; Krishna et al., 2000; Krishna et al., 2001a) although there are reports of iosamyl acetate synthesis in solvent-free systems (Ghamgui et al., 2006; Guvenc et al., 2002) which alleviates the problems of product separation, toxicity and inflammability of the organic solutions. Other examples of the use of immobilized lipases for flavor ester production via esterification of an alcohol and fatty acid in organic solutions include the production of butyl butyrate from butyric acid and butanol (Soares et al., 2005; Kumar and Rao, 2004), isoamyl butyrate from butyric acid and isoamyl alcohol (Krishna et al.,1999), isoamyl isovalerate from isovaleric acid and isoamyl alcohol (Chowdary et al., 2002: Chowdary et al., 2000), isoamyl isobutyrate from isobutyric acid and isoamyl alcohol (Krishna et al., 2001b) and isobutyl isobutyrate from isobutyric acid and isobutyl alcohol (Hamsaveni et al., 2001). For example, Chiang et al. (2003) used Lipozyme IM (Novozymes Rhizomucor miehei) to catalyse a reaction between triacetin as the acyl donor and cis-3-hexen-1-ol in hexane to form a hexyl ester, which has a fruity odor.

4.5.2 Aspartame production Aspartame can by synthesized chemically from aspartic acid and phenylalanine but this does result in the formation of an optical isomer that has a bitter taste. Thermolysin (EC 3.4.24.27) is an endopeptidase that catalyses the hydrolysis of peptide bonds containing hydrophobic amino acid residues. Thermolysin can also catalyse the reverse reaction resulting in the synthesis of peptides such as the precursor of the artificial sweetener, aspartame (L-Asp-L-Phe methyl ester, reviewed in Inouye, 2003). The commercial source of the enzyme is derived from Bacillus thermoproteolyticus and is available as ThermolaseTM from Daiwa Kasei Co. (Osaka, Japan) (Oyama et al., 1987; Swaisgood, 2004; Ager et al., 1998). The advantages to the enzymatic synthesis of aspartame include the fact that the enzyme only recognizes the L isomer of the phenylalanine and the β-carboxyl

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group of aspartate does not require protection and deprotection to prevent formation of the bitter isomer (Swaisgood, 2003). In the enzymatic reaction, the N-protected-aspartame derivative produced is insoluble in a biphasic system of water and organic solvent (ethyl acetate or tert-amyl alcohol) and thus shifts the equilibrium to favor synthesis of the peptide. The protecting group can then be removed either chemically or biologically to yield aspartame (Ager et al., 1998). Scientists at Toya Soda Co. (Oyama et al., 1987; Swaisgood, 2003) developed a process using immobilized ThermolaseTM to condense carbobenzoxyL-aspartate and D,L-phenylalanine methyl ester. In fact, Oyama et al. (1981) first discuss the use of immobilized thermolysin for aspartame synthesis as early as 1981.

4.6

Future trends

Numerous publications over the past few years have focused on the development of immobilized enzymes for future commercial use, yet at this time there are few successful examples of immobilized enzymes for food processing despite the advantages associated with immobilized enzyme processing. The immobilized form of glucose isomerase is used for the production of high-fructose corn syrup and immobilized lipases are used for production of diacylglycerols and trans-free fats and oils. Despite the numerous benefits associated with immobilized enzymes, the economics of the system outweighs most other benefits. Changes caused by food and drug regulations and the worldwide concern about chemical waste (green chemistry) may spur the use of enzymes in general, and perhaps the immobilized form in the future. For example, the use of immobilized lipases coincided with the USA FDA regulation requiring manufacturers to list trans fatty acids or trans fat on the nutrition facts panel of foods beginning January 1, 2006. Advances in molecular biology, genomics and microbial biodiversity may facilitate the use of immobilized enzymes by decreasing the cost associated with the process. For example, developing recombinant enzyme fusion proteins which contain a protein moiety for selective immobilization coupled to the catalytic enzyme would allow selective immobilization directly from lysed cells, thereby by-passing the purification step. This type of immobilization method also allows for affinity adsorption for oriented immobilization which limits the factors that may cause a loss of enzyme activity with immobilization which include chemical modification of essential amino acid residues and steric hindrance by the matrix to access. Examples of recombinant enzyme fusions for food application include a streptavidin–β-galactosidase fusion (Walsh and Swaisgood, 1994), streptavidin– lipase fusion (Lee and Swaisgood, 1997), streptavidin–trypsin fusion (Clare et al., 2001), streptavidin–keratinase fusion (Wang et al., 2003), and streptavidin– transglutaminase fusion (Valentine et al., 1998). In addition to streptavidin fusion proteins, other examples include a cellulose binding domain-lipase and β-glucosidase fusions (Hwang et al., 2004; Ong et al., 1991) and the starch binding domain-β-galactosidase fusion (Dalmia and Nikolov, 1994).

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Running immobilized enzyme bioreactors at high temperatures has several advantages including a decrease in viscosity, an increase in diffusion coefficients and less microbial growth, which reduces the cleaning frequency. Identification of extremophilic microorganisms and the characterization of their enzymes for potential food use may expand the use of immobilized enzymes and simplify the processing of foods. Enzymes from extreme thermophilic (optimum growth 70– 80 °C) and hyperthermophilic (optimum growth 85–100 °C) are being identified and characterized (reviewed in Niehaus et al., 1999). For example, the gene encoding an extracullular α-amylase from Pyrococcal furiosus has been cloned and expressed in Bacillus subtilis and E. coli. This α-amylase displays a temperature optimum of 100 °C and is active at 130 °C without the metal requirement (reviewed in Niehaus et al., 1999). In addition thermostable pullulanase, cellulase, proteases (reviewed in Niehaus et al., 1999) and β-galactosidases (Petzelbauer et al., 2002) have been identified. Many excellent reviews on the use of immobilized enzymes for food processing have been written, mainly by Harold Swaisgood (Swaisgood, 2004; Swaisgood, 2003; Swaisgood, 1991; Swaisgood and Horton, 1989; Swaisgood, 1985). This author has addressed the factors that determine the economics as well as the use of immobilized enzyme for food processing for over 20 years. These references can guide the reader into determining the economics of the system, which is a key for commercial success. The time may not be right yet for the commercial success of immobilized enzymes other than discussed above, but global concerns about chemical waste and government regulations may provide the impetus for future success.

4.7

References

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Niehaus F, C Bertoldo, M Kähler and G Antranikian (1999). ‘Extremophiles as a source of novel enzymes for industrial application’. Appl Microbiol Biotechnol, 51(6), 711–729 Nishizawa K, M Nakajima and H Nabetani (2000). ‘A forced-flow membrane reactor for transfructosylation using ceramic membrane’. Biotechnol Bioeng, 68(1), 92–7. Oba, T and B Witholt (1994). ‘Interesterification of milk fat with oleic acid catalyzed by immobilized Rhizopus oryzae lipase’. J Dairy Sci, 77, 1790–1797. Ong, E, NR Gilkes, RC Miller Jr, RAJ Warren and DG Kilburn (1991). ‘Enzymatic immobilization using a cellulose binding domain of a Cellulomonas fimi exoglucanase’. Enzyme Microb Technol, 13, 59–65. Osborn, HT and CC Akoh (2002). ‘Structured lipids – novel fats with medical, nutraceutical, and food applications’. Comp Rev Food Sci Food Safety, 3, 93–103. Oyama, K, S Nishimura, Y Nonaka, K Kihara and T Hashimoto (1981). ‘Synthesis of an aspartame precursor by immobilized thermolysin in an organic solvent’. J Org Chem, 46, 5241–5242. Oyama, K, S Irino and N Hagi (1987). ‘Production of aspartame by immobilized thermolase’. Methods Enzymol, 136, 503–516. Panyam D, A Kilara (1996). ‘Enhancing the functionality of food proteins by enzymatic modification’. Trends Food Sci Technol, 7, 120–125. Park, O, HE Swaisgood and JC Allen (1998). ‘Calcium binding of phosphopeptides derived from hydrolysis of alpha s-casein or beta-casein using immobilized trypsin’. J Dairy Sci, 81, 2850–2857. Pedroche, J, MM Yust, H Lqari, J Giron-Calle, J Vioque, M Alaiz and F Millian (2004). ‘Production and characterization of casein hydrolysates with a high amino acid Fischer’s ratio using immobilized proteases’. Int Dairy J, 14, 527–533. Petzelbauer, I, B Kuhn, B Splechtna, KD Kulbe and B Nidetzky (2002). ‘Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. IV Immobilization of two thermostable beta-galactosidases and optimization of a packed-bed reactor for lactose conversion’. Biotechnol Bioeng, 77, 619–631. Porter, DH, HE Swaisgood and GL Catignani (1984). ‘Characterization of an immobilized digestive enzyme system for determination of protein digestibility’. J Agric Food Chem, 32, 334–339. Pszczola, DE (2001). ‘From soybeans to spaghetti: the broadening use of enzymes’. Food Technol, 55(11), 54–62. Pyun, YR, BC Kim, HS Lee, DW Lee and YH Lee (2005). Thermostable L-arabinose Isomrease and Process for Preparing D-Tagatose, United States Patent No 6,933,138. Reischwitz, A, KD Reh and K Buchholz (1995). ‘Unconventional immobilization of dextransucrase with alginate’. Enzyme Microb Technol, 17, 457–61. Rocha JR, R Catana, BS Ferreira, JMS Cabral and P Fernandes (2006). ‘Design and charactreisation of an enzyme system for inulin hydrolysis’. Food Chem, 95, 77–82. Romero, MD, L Calvo, C Alba, A Daneshfar and HS Ghaziaskar (2005). ‘Enzymatic synthesis of isoamyl acetate with immobilized Candida Antarctica lipase in n-hexane’. Enzyme Microb Technol, 37, 42–48. Ronne, TH, T Yang, H Mu, C Jacobsen and X Xu (2005). ‘Enzymatic interesterification of butterfat with rapseed oil in a continuous packed bed reactor’. J Agric Food Chem, 53, 5617–5624. Rousseau, D and AG Marangoni (1999). ‘The effects of interesterification on physical and sensory attributes of butterfat and butterfat-canola oil spreads’. Food Res Int, 31(5), 381– 388. Santos JC, SI Mussatto, MA Cunha and SS Silva SS (2005a). ‘Variables that affect xylitol production from sugarcane bagasse hydrolysate in a zeolite fluidized bed reactor’. Biotechnol Prog, 21(6), 1639–43. Santos, JC, SS Silva, SI Mussatto, W Carvalho and MA Cunha (2005b). ‘Immobilized cells cultivated in semi-continuous mode in a fluidized bed reactor for xylitol production from sugarcane bagasse’. World J Microb Biotechnol, 21, 531–535.

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Sartoglu K, N Demir, J Acar and M Mutlu (2001). ‘The use of commercial pectinase in the fruit juice industry, part 2: Determination of the kinetic behaviour of immobilized commercial pectinase’. J Food Eng, 47, 271–274. Schmid, U, UT Bornscheuer, MM Soumanou, GP McNeil and RD Schmid (1999). ‘Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis’. Biotechnol Bioeng, 64(6), 678–674. Schmidl, MK, SL Taylor and JA Nordlee (1994). ‘Use of hydrolysate-based products in special medical diets’. Food Technol, 48(10), 77–85 Shin, HT, KM Park, KH Kang, DJ Oh, SW Lee, SY Baig and JH Lee (2004). ‘Novel method for cell immobilization and its application for production of oligosaccharides from sucrose’. Lett Appl Microb, 38, 1760179. Soares, CM, HF de Castro, JE Itako, FF de Moraes and GM Zanin (2005). ‘Characterization of sol-gel bioencapsulates for ester hydrolysis and synthesis’. Appl Biochem Biotechnol, 121–124, 845–859. Sousa, Jr., R, GP Lopes, PW Tardioli, RLC Giordano, PIF Almeida and RC Giordano (2004). ‘Kinetic model for whey protein hydrolysis by alcalase multipoint-immobilized on agarose gel particles’. Brazilian J Chem Eng, 21(2), 147–153. Swaisgood, HE (1985). ‘Immobilized enzymes in the food industry’, in Laskin AI and CA Park (eds), Applications of Isolated Enzymes and Immobilized Cells to Biotechnology, Benjamin/Cummings, Menlo Park, CA, 1–24. Swaisgood, HE (1991). ‘Immobilized enzymes: applications to bioprocessing of food’, in Fox PF (ed), Food Enzymology, Vol. 2, Elsevier Applied Science Publishers, London, 309–341. Swaisgood, HE (2003). ‘Use of immobilized enzymes in the food industry’, in Whitaker JR, AGI Vorgen and DWS Wong (eds), Handbook of Food Enzymology, Marcel Dekker, New York, 185–236. Swaisgood, HE (2004). ‘The use of immobilized enzymes to improve functionality’, in RY Yada (ed), Proteins in Food Processing, Woodhead Publishing Limited, Cambridge England, 608–630. Swaisgood, HE and HR Horton (1989). ‘Immobilized enzymes as processing aids or analytical tools’, in Whitaker JR and Sonnet PE (eds), Biocatalysts in Agricultural Biotechnology, ACS Symp Series 389, Americal Chemical Society, Washington DC, 242–261. Tanriseven, A and S Dogan (2002). ‘Production of isomalto-oligosaccharides using dextransucrase immobilized in alginate fibres’. Process Biochem, 37, 1111–1115. Tanriseven A and Y Aslan (2005). ‘Immobilization of Pectinex Ultra SP-L to produce fructooligosaccharides’. Enzyme Microb Technol, 36, 550–554. Tardioli, PW, J Pedroche, RLC Giordano, R Fernandez-Lafuente and JM Guisan (2003). ‘Hydrolysis of proteins by immobilized-stabilized alcalase-glyoxyl agarose’. Biotechnol Prog, 19, 352–360. Thresher, WC, HE Swaisgood and GL Catignani (1984). ‘Digestibilities of the protein in various foods as determined in vitro by an immobilized digestive enzyme assay (IDEA)’. Plant Foods Human Nutrition, 39(1), 59–65. Ticu, L-L, D Vercaigne-Marko, R Froidevaux, A Huma, V Artenie and D Guillochon (2005). ‘Use of protease-modified-alumina complex to design a continuous stirred tank reactor for producing bioactive hydrolysates’. Process Biochem, 40, 2841–2848. Truong, V-D, DA Clare, GL Catignani and HE Swaisgood (2004). ‘Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase’. J Agric Food Chem, 52, 1170–1176. Valentine, VW, Clare DA and HE Swaisgood (1998). ‘Engineering a protein recombinase: Construction and characterization of heterologous fusions of streptavidin and transglutaminase’. FASEB J, 12, 520. Walsh, MK and HE Swaisgood (1994). ‘An E. coli plasmid vector system for production of streptavidin fusion proteins: expression and bioselective adsorption of streptavidin-betagalactosidase’. Biotech Bioeng, 44, 1348–1354.

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Wang, J-J, HE Swaisgood and JCH Shih (2003). ‘Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems’. Biotechnol Bioeng, 81(4), 421–429. Watanabe, T, M Shimizu, M Sugiura, M Sato, J Kohori, N Yamada and K Nakanishi (2003). ‘Optimization of reaction conditions for the production of DAG using immobilized 1,3regiospecific lipase lipozyme RM 1M’. JAOCS, 80(2), 1201–1207. Weber, N and KD Mukherjee (2004). ‘Solvent-free lipase-catalyzed preparation of diacylglycerols’. J Agric Food Chem, 52, 5347–5353. Welsh, FW, Murray WD and Williams RE (1989). ‘Microbiological and enzymatic production of flavor and fragrance chemicals’. Crit Rev Biotechnol, 9, 105–169. White CA and JF Kennedy (1980). ‘Popular matrices for enzyme and other immobilizations’. Enzyme Microb Technol, 2, 82–90. Wilcox, CP and HE Swaisgood (2002). ‘Modification of the rheological properties of whey protein isolate through the use of an immobilized microbial transglutaminase’. J Agric Food Chem, 50, 5546–5551. Wilcox, CP, DA Clare, VW Valentine and HE Swaisgood (2002). ‘Immobilization and utilization of the recombinant fusion proteins trypsin–streptavidin and streptavidin– transglutaminase for modification of whey protein isolate functionality’. J Agric Food Chem, 50, 3723–3730. Wiseman, A and L Woods (2004). ‘Problems still inherent in food-industry biocatalyst sustainable-deployment’. Trends Food Sci Technol, 15, 276–279. Wong, DWS (1995). Food Enzymes Structure and Mechanism, Chapman and Hall, Albany, NY. Yahya ARM, WA Anderson and M Moo-Young (1998). ‘Ester synthesis in lipase-catalyzed reactions – Characterization of Candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles’. Enzyme Microbial Technol, 23(7), 438–450. Yang, T, X Xu, C He and L Li (2003). ‘Lipase-catalyzed modification of lard to produce human milk fat substitutes’. Food Chem, 80, 473–481. Yun, JW, SC Kang and SK Song (1995). ‘Continuous production of fructooligosaccharides from sucrose by immobilized fructosyltransferase’. Biotechnol Techniques, 9(11), 805– 808. Yun, JW, DH Kim, BW Kim and SK Song (1997). ‘Comparison of sugar compositions between inulo- and fructo-oligosaccharides produced by different enzyme forms’. Biotechnol Lett, 19(6), 553–556. Yun, JW, JP Park, CH Song, CY Lee, JH Kim and SK Song (2000). ‘Continuous production of inulo-oligosaccharides from chicory juice by immobilized endoinulinase’. Bioprocess Biosystems Engineer, 22(3), 189–194. Zhou D, X Xu, H Mu, C-E Hoy and J Alder-Nissen (2001). ‘Synthesis of structured triacylglycerols containing caproic acid by lipase-catalyzed acidolysis: optimization by response surface methodology’. J Agric Food Chem, 49, 5771–5777. Zhou, QZK and XD Chen (2001). ‘Immobilization of beta-galactosidase on graphite surface by glutaraldehyde’. J. Food Engineer, 48, 69–74.

5 Consumer attitudes towards novel enzyme technologies in food processing Helle Søndergaard, Klaus G. Grunert and Joachim Scholderer, MAPP, University of Aarhus, Denmark

5.1

Introduction

Consumers are usually unaware of the technical details of food processing and certainly most consumers are unaware of the use of enzymes. However, consumers may form attitudes to food processing technologies, including the use of enzymes, when they are confronted with information about them, for example when stakeholders and pressure groups debate such issues in the media. Such attitudes formed on the spot may be negative and they may prevent consumers from buying products where these technologies have been used. The use of gene technology in certain enzyme technologies makes enzymes particularly vulnerable to such effects. This chapter deals with how consumers form attitudes to novel enzyme technologies and with the effects these attitudes may have on consumers’ intentions of buying foods that are processed using these technologies. The central issue is how attitudes towards novel enzyme technologies are formed – we cannot expect consumers to have attitudes to technologies they have never been confronted with before. Specifically, we look at how various types of information affect attitude formation, and we look at how the experience of tangible product benefits, resulting from the use of enzymes, has an impact on attitude formation. The chapter starts with a description of our theoretical approach to studying the consumer attitudes towards new technologies in the food industry. Then, design and results of two major studies investigating these issues are reported. Finally, implications and future trends are discussed.

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Enzymes have been a natural part of food production for centuries, for example in the traditional production of wine and cheese, but novel technologies are opening up new possibilities for using enzymes to enhance the structural properties of food products. Enzyme extraction from plants or microbes for use in large-scale food processing usually requires the use of gene technology. Therefore, it is in the interest of scientists, food producers and regulators to know how consumers form attitudes towards the use of novel enzyme technologies in food processing. Although it is still not clear how the labelling of enzymes from genetically modified (GM) systems will be regulated in the future, consumers will sooner or later be confronted by the existence of these technologies in food processing.

5.2

Theoretical approaches to how consumers form attitudes to new food production technologies

Previous research has shown that consumers form attitudes towards new food production technologies in two prototypical ways (see Scholderer and Frewer, 2003). On the one hand, consumers base their attitudes on factual information about the new technology or on direct experience of products that have been produced using the technology. This type of attitude formation can generically be called a bottom-up or a formative process. This is the kind of attitude formation process that scientists and food engineers usually assume; it suggests that people will form a positive attitude towards a technology if they are informed about its positive consequences or can experience its advantages. It is also consistent with those parts of the attitude literature that focus on reasoned, ‘rational’ processes of attitude formation and change (McGuire, 1985). But consumers can also form attitudes in other ways, especially in cases where the attitude object is as yet unfamiliar to them. They can, for example, view new food processing technologies as instances of more abstract classes of attitude objects, for example as a general category of unfamiliar and unwanted technologies, and base their attitude towards the novel technology on the generalised attitude towards this broader category (Prislin et al., 1998; Scholderer, 2004; Shook et al., in press). Attitudes formed in this way are not based on information about the new attitude object, but emerge from a process in which the new object is evaluated by means of a series of congruity–incongruity tests relating it to more general socio-political attitude objects such as the environment, technological progress in general, or industrial food production. This type of attitude formation can generically be called a top-down process. Figure 5.1 illustrates the two different routes. The route on the left-hand side of Fig. 5.1 illustrates top-down information processing where the formation of an attitude towards a new technology (in the present case, enzyme technology) is determined by consumers’ pre-existing attitudes towards other, more general socio-political issues. The route on the right-hand side of Fig. 5.1 illustrates bottom-up information processing where information and direct product experience influence consumer attitudes towards the enzyme technology.

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General socio-political attitudes

Overall technology evaluation

Specific risk/benefit evaluations

Attitudinal inference

Overall technology evaluation

Specific risk/benefit evaluations

Consumer information

Fig. 5.1

Risk/benefit trade off

Direct product experience

Two ways of attitude formation (based on Scholderer et al., 2000).

Both types of process may operate simultaneously in any given situation, but will usually do so to a different extent. Research into consumer attitudes to GM foods has indeed shown that both processes operate. Among the general sociopolitical attitudes which have been shown to predict attitudes to new technologies are, for example, attitudes towards technological progress (Bredahl, 2001; Frewer et al., 1994; Hamstra, 1991; Sparks et al., 1995), attitudes towards environment and nature (Bredahl, 2001; Frewer et al., 1997; Hamstra, 1995; Siegrist, 1998), attitudes towards industrial food production (Beckmann et al., 2001) and trust in the institutions that regulate emerging technologies and manage their risks (Hoban et al., 1992; Siegrist, 1999, 2000). But empirical research has also shown that direct experience with high-quality GM products can override pre-existing attitudes (Grunert et al., 2004; Lähteenmäki et al., 2002), turning a hitherto top-down attitude into a bottom-up attitude. 5.2.1 Impact of attitudes on buying behaviour The way in which an attitude towards a production technology may affect intentions to buy a product is often explained in terms of rational choice models such as the theory of reasoned action (Ajzen and Fishbein, 1980). In this type of model, an attitude towards a product is defined as a weighted sum of beliefs about the positive and negative attributes of the product (which may include aspects of the technology used). Behaviour is seen as a function of behavioural intentions, which in turn depend on the attitude and the perceived social pressure to buy or not buy the product in question. In a purchase situation, consumers may trade off different attributes against each other which are associated with the product. When purchasing a product produced by means of technologies that consumers are

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relatively aware of (such as GM), the attitude towards the production method may influence purchase intentions in addition to the usual attributes such as the perceived costs (monetary and other) and benefits of the product.

5.3

Studies of consumer attitudes to enzyme technologies

In the following section, the results of two consumer studies are reported that were carried out simultaneously in Finland, Germany and Italy in 2003 and 2005, respectively. The aim of the studies was to assess consumer attitudes towards novel enzyme technologies and consumer preferences for products processed by means of these technologies. A total of 1200 respondents participated in the first study and 810 participated in the second study. 5.3.1 Formation of attitudes to enzyme technologies In Study 1, the first objective was to assess the overall level of consumers’ attitudes towards different enzyme production technologies. Participants received a brief general introduction to enzymes and then answered a set of attitude scales about four enzyme production technologies, including a non-GM plant technology, a non-GM microbe technology, a GM plant technology and a GM microbe technology. As shown in Fig. 5.2, consumers had relatively neutral attitudes to enzymes ■

Italy

Germany

■

■

Finland

7

Attitude (7-point scale)

6

5 ■

■ ■ ■

4

■ ■ ■

■ ■

■ ■

3

■

■ ■

■

2

1 Attitude to Attitude to enzymes in non-GM plant food method production

Attitude to non-GM microbe method

Attitude to GM plant method

Attitude to GM microbe method

Fig. 5.2 Consumer attitudes towards different enzyme technologies.

Consumer attitudes towards novel enzyme technologies in food processing

Fig. 5.3

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Causal effects of general socio-political attitudes on attitudes towards enzyme production by means of GM plants (standardised path coefficients).

in general. However, consumers felt significantly more positive about non-GM enzyme technologies than about GM technologies. Specifically, consumers were most positive about the non-GM plant enzyme technology. Figure 5.3 shows the results of an analysis investigating how different types of general socio-political attitudes are linked to the more specific attitudes to enzyme production methods, here for the production method involving a GM plant system (the numbers are standardised path coefficients, estimated based on the German data). A general direction of causality can be observed that proceeds from the abstract to the concrete; attitudes towards environment and nature and towards technological progress, affect consumers’ general attitude towards industrial food production, which in turn affects their attitude towards GM in food production, which then affects the attitude towards its specific application in enzyme production. Consumers’ confidence in their own judgements and trust in the relevant actors in the food chain were mediators of these relationships. The results clearly show that consumers understand specific technologies used in the production of enzymes as instances of a much broader class of technologies and that their attitudes towards a specific technology are relatively a simple and straightforward derivations from their general attitudes towards the broader class of technologies.

5.3.2 Effects of information on consumers’ attitudes It is tempting to expect that, when consumers have no knowledge about a new technology, informing them about the benefits of this technology will influence their attitudes towards it. In the general approach outlined above, this was defined as an example of the bottom-up route to attitude formation. However, previous research has shown that consumer attitudes towards products or technologies involving GM can be highly resistant to all forms of information (e.g. Grunert et al., 2003; Scholderer and Frewer, 2003). In Study 1, we therefore investigated what effects different types of information material would have on consumers’ attitudes towards novel enzyme technologies.

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Press release The German consumer association’s citizens’ jury says enzymes from plants should be available for industrial food production in Germany Monday, 04 August 2003 The consumer association Bundesverband has run a citizens’ jury on enzymes in food production with the purpose of testing public opinion about enzymes from genetically modified plants, exploring public concerns and testing their validity, and informing Bundesverbands thinking about the issue. A jury of 15 members of the public considered the question: ‘Should enzymes from GM plants be available to use in food production in Germany?’ Citizens’ jury members were recruited at random in Hannover, reflecting the local population. They heard evidence from a wide cross-section of experts in the field, and were able to question the experts and ask for additional evidence, before formulating a response. The jury was also asked to explain the reasoning behind their decision, including any conditions or recommendations. Among others, the jury included an accountant, two students, a housewife, taxi driver, driving instructor, and a minister of religion. Bundesverbands citizens’ jury decided on the afternoon of 4 August 2003 that enzymes from GM plants should be available to use in Germany, although a sizeable minority (6 out of 15) disagreed, believing that Germany is not yet ready for GM enzymes. Exhibit 1 Excerpt from one of the press releases used in Study 1.

In order to study the effect of information on attitude formation experimentally, a press release was designed where the main message was a recommendation to use enzyme technologies in food production. This press release was then experimentally manipulated, varying the enzyme technology, the sender (i.e. the institution governing the technology assessment process) and the type of public participation in the technology assessment process. A control group which received no such press release was also included in the study. The press release was designed to reflect the national actors so that respondents would not feel confused. An example is shown in Exhibit 1. It was expected that the type of press release, that is, whether it had been sent out by a consumer association or a food manufacturer, whether the views of the public had been accommodated in the form of a citizens’ jury or an expert workshop, or what kind of enzyme technology it was dealing with, would affect the direction and extent of change in consumers’ attitudes towards enzymes. However, the analysis showed that none of the experimental factors had substantial effects, not even the enzyme technology that consumers had been informed about. Although initially surprising, these results are in line with earlier research on the effects of information on attitudes towards GM (see above).

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5.3.3

Effect of enzyme technology on intention to buy particular food products Furthermore, we estimated the impact that clear labelling of enzyme technologies on a product would have on consumers’ buying intentions. Consumers were asked to indicate their preferences for a number of product profiles. Various attributes were combined according to a factorial design in such a way that their effects on preference could be separated by means of conjoint analysis (Green and Srinivasan, 1990; Louvière, 1994). In this study, two different types of benefit, three different price levels, and four different enzyme production methods were included in the conjoint design. Three products categories (bread, pasta and ice cream) were used in the study. Respondents were shown 16 product profiles generated from the design. The four enzyme technologies were described in detail immediately before the conjoint task. Intentions to buy the product were measured by seven-point scales with end points labelled ‘I will definitely not buy this product’ and ‘I will definitely buy this product’. The results in Table 5.1 indicate that, in this particular situation, enzyme technology was the most important determinant of consumer preferences. The effect of price was half as strong as the effect of enzyme technology, whilst product and process benefits had only weak effects. The partial utilities, representing the effects of specific factor levels on preference, show that among the enzyme technologies, the plant method was clearly the most liked method, whereas the two GM technologies were least preferred. There were no great differences between the three product categories in terms of the utilities associated with the enzyme production methods, indicating that the production method had the same effect on purchase intentions. The results obtained in this study suggest that neither of the benefits nor a 25% price reduction could make consumers ‘ignore’ that the bread, ice cream and pasta had been produced using GM enzymes. Table 5.1 Factor

Conjoint analysis results (relative importance and part-worth estimates) Level

Enzyme production method Relative importance (%) Plants Microbes GM plants GM microbes Product benefit Relative importance (%) Present Not present Process benefit Relative importance (%) Present Not present Price Relative importance (%) Low (–25%) Average High (+25%)

Bread

Ice cream

Pasta

59.1 1.24 0.34 –0.79 –0.79 10.3 –0.01 0.01 7.7 –0.02 0.02 22.8 0.23 0.02 –0.26

58.7 1.23 0.33 –0.84 –0.72 9.7 0.03 –0.03 8.3 0.04 –0.04 23.3 0.27 0.01 –0.28

62.8 1.35 0.31 –0.86 –0.81 8.8 0.04 –0.04 7.5 0.03 –0.03 20.9 0.18 –0.04 –0.18

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It has to be said though that the somewhat artificial experimental situation has probably blown the effect of enzyme technology on consumers’ preferences out of proportion. In a real buying situation, where consumers are known to pay relatively little attention to the ingredient list on a product, the effects are likely to be much weaker.

5.3.4 Effects of direct product experience on attitudes The aim of the second consumer study was to assess the effects of direct product experience on the formation and change of consumer attitudes towards enzymes. The experimental design included three conditions. In the first condition, respondents tasted two number-coded product samples and then received written information about the enzyme technology used in the processing of one of these products. In the second condition, respondents received the written information first and then tasted the product samples. In the third condition, respondents received only information and did not participate in the product tasting. Three products categories were used in this study (bread, margarine, yoghurt). Respondents were told that the two product samples they tasted (e.g., two samples of bread) were a conventionally processed product and a product processed by means of enzyme technology. It was important that respondents experienced clear product differences when tasting the two product samples. Therefore, in order to determine which product samples to use in the study, descriptive sensory evaluations of different versions of the three products had been carried out beforehand (as an example, the results for the yoghurt samples are presented in Fig. 5.4). The sensory panel evaluated four to five samples from each category and, based on the results, two samples from each category were chosen for inclusion in the consumer preference test. In the preference test, consumers were asked to evaluate each product in terms of appearance, texture and taste. Subsequently, respondents were asked to indicate their overall liking of the products, their buying intentions and which of the two products they preferred. The results showed that buying intentions were higher for all three enzyme products than for the conventional products. Furthermore, when asked which of the two products consumers preferred, a majority chose the enzyme product (Fig. 5.5). Preferences for the enzyme product were highest for yoghurt, which was in line with the expectations as the yoghurt samples had shown the largest differences in the descriptive sensory analysis. Most importantly, preferences for the enzyme products did not depend on the type of enzyme technology that had been used, even if the technology involved genetic modification. Apparently, respondents preferred the products that had been improved by means of enzymes technologies. The key question following from this is if the direct positive experience also resulted in a more positive attitude to the use of enzymes in general, and to the use of enzymes from GM systems in particular. Figure 5.6 shows that this is indeed the case: attitudes were significantly more positive among those respondents who had actually tried products that involved the use of enzymes. Thus, direct product experience can counteract spontaneous

Consumer attitudes towards novel enzyme technologies in food processing

Fig. 5.4

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Results from descriptive sensory analysis of yoghurt.

Consumers using novel enzyme product (%)

100

80

60

40

20

0 Bread

Fig. 5.5

Margarine

Yoghurt

Percentage of consumers choosing the enzyme product depending on enzyme technology applied.

94

Novel enzyme technology for food applications No product tasting

Product tasting

Consumers using novel enzyme product (%)

0.6

0.5

0.4

0.3

0.2

0.1

0.0 GM technology in general Enzymes from GM systems Attitude towards

Fig. 5.6

Attitude change relative to control population and effect of product tasting.

scepticism about GM-related technologies in consumers, while just communicating product benefits cannot. Figure 5.6 also shows that direct product experience even resulted in a more positive attitude to the use of genetic modification in general.

5.4

Implications of consumer attitudes to enzyme technologies

Results from the consumer studies reported in this chapter show that attitudes towards the use of enzymes in food production in general are fairly neutral and that attitudes towards enzymes specifically produced by means of gene technology are slightly more negative. In the absence of product experience, consumers form attitudes towards enzymes by relating enzyme production systems to more general attitude objects such as technological progress, nature and the environment, and industrial food production. Giving consumers more information on enzymes, their production and use did not affect their attitudes, irrespective of the particular communication that was

Consumer attitudes towards novel enzyme technologies in food processing

95

used in the experimental treatment (i.e. which source, how exactly the public was involved in the technology assessment process and which enzyme production method the communication was about). Among the four enzyme technologies investigated here, respondents developed the most positive attitudes to a non-GM plant technology. GM-based technologies were less favoured. The results also showed that in the formation of purchase intentions for products where these enzymes have been used, negative attitudes towards GM enzyme production methods clearly outweighed product and process benefits when consumers were made explicitly aware that GM methods had been used in processing. In this particular study, not even a 25% price reduction could make consumers ignore the perceived disadvantages of products employing GM technologies. The situation changes when consumers obtain actual experience with real products. In Study 2, consumers tasted samples from three different product categories (bread, yoghurt and margarine). Each condition involved a base product and a product that had been improved in its sensory properties by means of enzyme technologies. Consumers were told either before or after the tasting that the improved product had been manufactured using enzymes produced by a certain technology. Consumers who had tasted the products developed more positive attitudes towards the use of GM technology in food production in general and in enzyme production compared with a control group that did not taste the products.

5.5

Future trends

The results in this chapter suggest that sensory product experience can override the otherwise prevalent mechanisms of attitude formation based on general sociopolitical attitudes, and lead consumers to develop more positive attitudes towards enzyme technologies, in particular those technologies that involve genetic modification. This presupposes, though, that the use of these technologies leads to products that consumers will actually perceive as superior. Hence, it is imperative for the food industry to focus on novel enzyme technologies that result in noticeably higher eating quality, high enough to be clearly perceived by consumers. Ideally, the product development strategy should be coupled with launch strategies focusing on point-of-sale promotions and the distribution of free product samples, enabling consumers to experience superior product quality.

5.6

Sources of further information and advice

For further details on the results from the first consumer study see Søndergaard et al. (2005). For more general information on consumer attitudes to genetic modification in food production see Grunert et al. (2003) and Grunert et al. (2004).

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Novel enzyme technology for food applications

Acknowledgements

The two studies reported in this chapter have been carried out with financial support from the Commission of the European Communities, specific RTD programme ‘Quality of Life and Management of Living Resources’, proposal number QLK1-2002-02208 ‘Novel crosslinking enzymes and their consumer acceptance for structure engineering of foods’ (CROSSENZ). It does not reflect its views and in no way anticipates the Commission’s future policy in this area.

5.8

References

Ajzen, I. and Fishbein, M. (1980). Understanding Attitudes and Predicting Social Behavior, Prentice Hall, Englewood Cliffs, NJ. Beckmann, S. C., Brokmose, S. and Lind, R. L. (2001). Danske forbrugere og økologiske fødevarer, Handelshøjskolens Forlag, København. Bredahl, L. (2001). ‘Determinants of consumer attitudes and purchase intentions with regard to genetically modified foods: Results of a cross-national survey’. Journal of Consumer Policy, 24, 23–61. Frewer, L. J., Shepherd, R. and Sparks, P. (1994). ‘Biotechnology and food production: Knowledge and perceived risk’. British Food Journal, 96, 26–32. Frewer, L. J., Howard, C. and Shepherd, R. (1997). ‘Public concerns about general and specific applications of genetic engineering: Risk, benefit and ethics’. Science, Technology and Human Values, 22, 98–124. Green, P. E. and Srinivasan, V. (1990). ‘Conjoint analysis in marketing: New developments with implications for research and practice’. Journal of Marketing, 54(4), 3–19. Grunert, K. G., Bredahl, L. and Scholderer, J. (2003). ‘Four questions on European consumers’ attitudes to the use of genetic modification in food production’. Innovative Food Science and Emerging Technologies, 4, 435–445. Grunert, K. G., Bech-Larsen, T., Lähteenmäki, L., Ueland, Ø. and Åström, A. (2004). ‘Attitudes towards the use of GMOs in food production and their impact on buying intention: The role of positive sensory experience’. Agribusiness, 20, 95–107. Hamstra, A. M. (1991). ‘Biotechnology in foodstuffs’. Towards a Model of Consumer Acceptance, The SWOKA Institute, The Hague. Hamstra, A. M. (1995). Consumer Acceptance of Model for Food Biotechnology. Final report, The SWOKA Institute, The Hague Hoban, T., Woodrum, E. and Czaja, R. (1992). ‘Public opposition to genetic engineering’. Rural Sociology, 57, 476–493. Lähteenmäki, L., Grunert, K. G., Ueland, Ø., Åström, A. and Bech-Larsen, T. (2002). ‘Acceptability of genetically modified cheese presented as real product alternative’. Food Quality and Preference, 13, 523–533. Louviere, J. J. (1994). ‘Conjoint analysis’, in R. E. Bagozzi (ed.), Advanced Methods of Marketing Research, Blackwell, Cambridge. McGuire, W. J. (1985). ‘Attitudes and attitude change’, in G. Lindzey and E. Aronson (eds.), The Handbook of Social Psychology, Randon House, New York, 3rd edition, Vol. 2, 233– 346. Prislin, R., Wood, W. and Pool, G. J. (1998). ‘Structural consistency and the deduction of novel from existing attitudes. Journal of Experimental Social Psychology, 34, 66–89. Scholderer, J. (2004). Consumer Attitudes Towards Genetically Modified Foods in Europe: Structure and Changeability, Potsdam University Publishers, Potsdam. Scholderer, J. and Frewer, L. J. (2003). ‘The biotechnology communication paradox: Experimental evidence and the need for a new strategy’. Journal of Consumer Policy, 26,

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125–157. Scholderer, J., Bredahl, L. and Frewer, L. (2000). ‘Ill-founded models of consumer choice in communication about food biotechnology’, in F. van Raaij (ed.), Marketing Communications in the New millennium: New media and new approaches, Erasmus University, Rotterdam, 29–152. Shook, N. J., Fazio, R. H. and Eiser, J. R. (in press). ‘Attitude generalization: Similarity, valence, and extremity’. Journal of Experimental Social Psychology. Siegrist, M. (1998). ‘Belief in gene technology: The influence of environmental attitudes and gender’. Personality and Individual Differences, 24, 861–866. Siegrist, M. (1999). ‘A causal model explaining the perception and acceptance of genetic engineering’. Journal of Applied Social Psychology, 29, 2093–2106. Siegrist, M. (2000). ‘The influence of trust and perceptions of risks and benefits on the acceptance of gene technology’. Risk Analysis, 20, 195–204. Søndergaard, H. A., Grunert, K. G. and Scholderer J. (2005) ‘Consumer attitudes to enzymes in food production’. Trends in Food Science and Technology, 16, 466–474. Sparks, P., Shepherd, R. and Frewer, L.J. (1995). ‘Assessing and structuring attitudes towards the use of gene technology in food production: The role of perceived ethical obligation’. Journal of Basic and Applied Social Psychology, 16, 267–285.

Part II Novel enzyme technology for food applications

6 Using crosslinking enzymes to improve textural and other properties of food Johanna Buchert, Emilia Selinheimo, Kristiina Kruus, Maija-Liisa Mattinen, Raija Lantto and Karin Autio, VTT, Finland

6.1

Introduction

Food texture plays a major role in food product quality. The rheological properties of a food are determined by the number of weak and strong physical interactions (hydrophobic, hydrogen bonding, electrostatic) and the permanent covalent bonds, crosslinks, present in the food matrix. Covalent crosslinks make the major contribution to the firmness of food matrices. Crosslinks can be introduced to a food matrix by chemical, enzymatic and physical means as reviewed by Singh (1991) and Gerrard (2002). Enzymatic crosslinking of food biopolymers is an attractive option owing to the specificity of enzymes and mild reaction conditions. Both food proteins and carbohydrates can be crosslinked by enzymes. Crosslinking can occur via aromatic groups present in proteins or carbohydrates or through certain amino acid moieties present in proteins. Crosslinking can be a result of direct enzymatic catalysis of crosslink formation, or occur indirectly by enzymatic production of a crosslinking agent, such as H2O2 or lipid-derived radicals, which in turn are able to oxidize reactive structures with subsequent crosslink formation. Proteins have several reactive groups for crosslinking enzymes, such as glutamine, lysine, tyrosine and cysteine residues. The reactions obtained are dependent on the type of enzyme used, the accessibility of the target reactive groups in the biopolymer and on process conditions used. In the case of carbohydrates, only feruloylated polysaccharides such as arabinoxylans and pectins can form crosslinks. Potential enzymes for food structure engineering are summarized in Table 6.1.

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Table 6.1

Summary of potential crosslinking enzymes for food structure engineering

Crosslinking Enzyme mechanism

Direct

Indirect

Reaction

Acting on protein (target amino acid)

Transglut- Formation of Glutamine aminase isopeptide Lysine linkage Peroxidase Oxidation of Tyrosine aromatic groups to radicals Laccase Oxidation of Tyrosine aromatic groups Cysteine to radicals Tyrosinase Oxidation of tyroTyrosine sine to dopaquinone Glutathione * Cysteine oxidase Sulphydryl * Cysteine oxidase LipoxyProduction of genase hydroperoxide radical from unsaturated fatty acids Glucose Production of H2O2 oxidase in conjunction with glucose oxidation Hexose Production of H2O2 in oxidase conjunction with hexose oxidation

Acting on Commercial ferulic acid status in containing food carbohydrate processing C

x

R

x

R

R R R C

C

C

*Exact reaction mechanism is unresolved. C = commercially available. R = available as research enzyme.

The benefits of crosslinking enzymes are highly dependent on application. It is possible to improve the strength of weak flours, containing low quality protein or fibre, improve dough handling properties and baking quality. Crosslinking can also be exploited in dairy products in order to prevent syneresis or to make the soft texture firmer. A potential for making low fat fermented milk products with sensory properties identical to those of products with normal fat content has been demonstrated. In the meat industry crosslinking enzymes are exploited in strengthening the structure of processed products such as fermented sausages and ham, as well as poultry meat products which generally have a weaker texture than beef and pork. In these applications enzymes are applied to improve texture particularly in products with low salt and fat content. Another application in the meat sector is restructuring of fresh meat with the aid of enzymes thus increasing the value of the products.

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Commercial exploitation of crosslinking enzymes in food processing started with the discovery of microbial transglutaminase from Streptoverticillium mobaraense (as reviewed by Yokoyama et al., 2004). Transglutaminase-based crosslinking technology is well patented and the markets are dominated by the Japanese company Ajinomoto. In addition to transglutaminase, commercial enzyme products that are able to create indirect crosslinking of food proteins are in the market. These enzymes include glucose oxidase and hexose oxidase and their crosslinking mechanism is based on hydrogen peroxide, which is produced in reactions catalysed by these enzymes.

6.2

Types of crosslinking enzymes

6.2.1 Transglutaminase Transglutaminase (TG, EC 2.3.2.13) is an acyltransferase, which catalyses acyltransfer reactions between a γ-carboxyamine group of a peptide-bound glutamyl residue (acyl-donor) and a primary amino group (acyl acceptor) of various substrates including the ε-amino group of lysyl residues in certain proteins. When the amine substrates are not available, TG catalyses deamination of glutamyl residues. During the reaction, water molecules are used as acyl acceptors. In addition to protein crosslinking, TGs can modify proteins by means of amine incorporation and deamination (Yokoyama et al., 2004). TGs have been reported to crosslink many food proteins as reviewed by, for example Zhu et al. (1995), Motoki and Seguro (1998), Nielsen (1995) and Kuraishi et al. (2001). TG-catalysed reactions lead to inter- or intramolecular crosslinking in proteins depending on whether the glutamyl and lysyl residues are located on the same protein molecule or on two different ones. The covalent linkage formed between lysyl and glutamyl residues is an ε−(γ-glutamyl)lysine isopeptide bond. TGs are widely distributed enzymes found in various animal tissues and body fluids, fish, birds, invertebrates, amphibians, plants and microbes. They are involved in several biological functions including blood clotting, wound healing, epidermal keratinization and in a number of human disease states (for reviews see Griffin et al., 2002; Yokoyama et al., 2004). The best characterized TG is the mammalian plasma protein, a coagulating Factor XIII (Folk and Finlayson, 1977; Folk, 1980). Discovery of microbial TGs from Streptomyces and Streptoverticillium species in late 1980s (Yokoyama et al., 2004) enabled fast development of TG for various food applications. Properties of TGs vary considerably depending on the source. Both guinea pig liver and Streptomyces mobaraensis (former Streptoverticillum mobaraense) TGs are monomeric proteins with molecular weights of 75 kDa and 38 kDa, respectively (Ando et al., 1989). In contrast to mammalian TGs the Streptomyces TGs are independent of Ca2+. S. mobaraense TG is a secreted protein, which is activated by proteolytic processing (Pasternack et al., 1998) The enzyme shows pH optimum between 5 and 8, and has substantial activity at pH 4–9 (Ando et al., 1989). The

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enzyme retains full activity in 40 °C for 10 min but is totally inactivated within a few minutes at 70 °C. 6.2.2 Oxidative enzymes Enzymatic crosslinking by an oxidative mechanism is also an interesting possibility to generate crosslinks in a food matrix. Various oxidoreductases including tyrosinases, laccases, peroxidases and sulphydryl oxidases are reported to crosslink either carbohydrate or protein matrix. These enzymes have been tested in only a few applications, mainly because of the limited availability of the enzymes. In addition, enzymes which create indirect crosslinking to food proteins are in the market. These enzymes include glucose oxidase and hexose oxidase or lipoxygenase. Their crosslinking mechanism is based on either hydrogen peroxide formation or formation of lipid-derived radicals. Tyrosinases Tyrosinases (EC 1.14.18.1) are capable of oxidizing peptide-bound tyrosine residues to the corresponding quinones, which can further react non-enzymatically with, for example free sulphydryl and amino groups resulting in formation of tyrosine–cysteine and tyrosine–lysine crosslinks, respectively (Ito et al., 1984). Quinones can also be coupled together and form tyrosine–tyrosine linkages. Tyrosinases are copper-containing proteins, they contain two type-3 copper atoms in their active site to shuttle electrons from the substrate to molecular oxygen, which is a terminal electron acceptor. Tyrosinases are ubiquitous enzymes and can be found in microbes, plants and higher animals (Lerch, 1983). The physiological role of tyrosinases is related to melanin and eumelanin synthesis. In fruits and vegetables, tyrosinase is responsible for enzymatic browning reactions and, in mammals, for pigmentation. In fungi, the role of tyrosinase is correlated with cell differentiation, spore formation, virulence and pathogenesis. Studies with Neurospora crassa have shown that the enzyme is completely absent in the vegetative stage, however, under stress conditions high levels of the enzyme are induced (Lerch, 1983). The result suggests that tyrosinases are not essential to the metabolism of the fungi, but improve the survival and competence of the fungi (for a review see Claus and Decker, 2006; Halaouli et al., 2006). The best known and characterized tyrosinases are of mammalian origin. The most extensively investigated fungal tyrosinases both from a structural and functional point of view are from Agaricus bisporus (Wichers et al., 1996; Seo et al., 2003) and N. crassa (Lerch, 1983). Also a few bacterial tyrosinases have been reported, of which Streptomyces tyrosinases are the most thoroughly characterized (Ito and Oda, 2000; Katz and Betancourt, 1988; Lerch and Ettlinger, 1972; Matoba et al., 2006). In addition, tyrosinases have been disclosed, for example from Trichoderma reesei (Selinheimo et al., 2006b), Aspergillus (Bull and Carter, 1973), Ascovaginospora (Abdel-Raheem and Shearer, 2002), Trametes (Tomsovsky and Homolka, 2004), Pycnoporus species (Halaouli et al., 2005) and Marinomonas (Lopez-Serrano et al., 2000). Both intra- and extra-cellular tyrosinases have been reported.

Using crosslinking enzymes to improve properties of food

Fig. 6.1

105

Oxidation of tyrosine by tyrosinase.

Besides tyrosine, tyrosinases are capable of oxidizing various mono- and diphenols like p-cresol, catechol and L-Dopa. In tyrosinase-catalysed reactions monophenols are first hydroxylated to the ortho position (cresolase or monophenol oxidase activity) and further oxidized to quinones (catechol oxidase or diphenol oxidase activity) as shown in Fig. 6.1 (Lerch, 1988). Many microbial and plant-derived tyrosinases have their pH optima in a slightly acidic pH range (Zawistowski et al., 1991), although some very alkaline tyrosinases have also been recently reported, for example T. reesei and Thermomicrobium roseum tyrosinase, which have their pH optimum at 9 and 9.5, respectively (Selinheimo et al., 2006b; Kong et al., 2000). In general tyrosinases are not very thermostable enzymes, they often lose their activity within a few minutes at 70– 90 °C (Zawistowski et al., 1991). Post-translational processing of tyrosinases is reported for many fungal tyrosinases. The intracellular tyrosinases from N. crassa (Kupper et al., 1989; van Gelder et al., 1997), Agaricus (Espin et al., 1999) and Pycnoporus species (Halaouli et al., 2005), and extracellular tyrosinase from T. reesei (Selinheimo et al., 2006b) have an additional C-terminal domain which is proteolytically released from the catalytic domain. It has been postulated that the function of the C-terminal domain is to retain the enzyme inactive until the activity is needed (Kupper et al., 1989). Plant tyrosinases cause unfavourable browning of fruits and vegetables (Nicolas et al., 1994; Whitaker and Lee, 1995) and thus several natural and non-toxic tyrosinase inhibitors such as various glycyl dipeptides have been developed (Girelli et al., 2004; Vámos-Vigyázó, 1995). Although very interesting enzymes for enzymatic crosslinking, tyrosinases are currently not commercially available for large scale applications. Sigma and Fluka sell crude Agaricus tyrosinase for research purposes. Laccases Laccases (EC 1.10.3.2) have been shown to crosslink pentosans via ferulic acid residues (Figueroa-Espinoza and Rouau, 1998.). The ability of laccases to form crosslinks in proteins and peptides has also been reported (Færgemand et al., 1998b; Yamaguchi, 2000; Mattinen et al., 2005). Laccases are copper-containing enzymes. They contain four copper atoms at their active site and use molecular oxygen as a terminal electron acceptor. Laccases have surprisingly broad substrate specificity: they are capable of oxidizing various phenolic compounds, for example diphenols, polyphenols, different substituted phenols, diamines, aromatic amines, benzenethiols and even some inorganic compounds such as iodine (for a

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Fig. 6.2

Laccase-catalysed oxidation of a diphenol.

review see Thurston, 1994). Compared with tyrosinases the laccase-catalysed reactions are different: whereas tyrosinase-catalysed crosslinking is based on quinone formation, laccase-catalysed crosslinking is based on free radicals and their further reactions. Laccases oxidize their substrates with a one electron removal mechanism (Kersten et al., 1990; Thurston, 1994). The unstable radicals undergo further non-enzymatic reactions including polymerization, hydration and disproportionation. The radicals may also be further oxidized by the enzyme. Figure 6.2 shows the schematic laccase-catalysed oxidation of a p-diphenol. Laccases are common enzymes in nature; they are found widely in plants and fungi as well as in some bacteria and insects. The majority of laccases characterized so far have been derived from fungi, especially from white-rot fungi, which are efficient lignin degraders. Well-known laccase producers include Trametes, Pleurotus, Coprinus, Myceliophthora, Melanocarpus, Phlebia, Pycnoporus, Rhizoctonia and Schizophyllum (for review see Xu, 1996). It is well recognized that laccases are involved in both polymerization and depolymerization processes of lignin. The plant origin laccases are reported to have an important role in wound response and lignin biosynthesis (Thurston, 1994), whereas in fungi they are involved in lignin degradation, as well as in several other functions including pigmentation, fruiting body formation, sporulation and pathogenesis (Thurston, 1994; Leatham and Stahmann, 1981). Many laccases have their pH optima in the acidic pH range, although neutral and alkaline laccases have also been reported (Kiiskinen et al., 2002; Martins et al., 2002). The pH optima of laccases are not only affected by the enzyme, but also by the substrate. Increasing pH decreases the redox potential of the phenolic substrates and makes the substrate more susceptible to oxidation. In contrast to their activity, the stability of laccases is in general better at alkaline pH values (Xu, 1996; Chefetz et al., 1998). Temperature stabilities of laccases vary considerably, depending on the source of the enzyme. In general fungal laccases are stable at 30–60 °C and rapidly lose their activity at temperatures above 60 °C. The most thermostable laccases are from bacterial origin. The half life of the Streptomyces lavendulae enzyme is reported to be 100 min at 70 °C (Suzuki et al., 2003) and for Bacillus subtilis CotA, 112 min at 80 °C (Martins et al., 2002). The molecular size of laccases varies between 40 and 100 kDa and they occur as monomers. The enzymes are structurally similar. The laccase monomer contains

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three so-called cupredoxin-like domains, which are all needed for the catalytic activity of the enzyme. The copper atoms are organized into a mononuclear and trinuclear centre. Presently there are several complete laccase structures available (Bertrand et al., 2002; Piontek et al., 2002; Hakulinen et al., 2002). Although intensively studied, the detailed mechanism, with which laccases oxidize their substrates, is not fully understood. Currently, laccases are commercially available only for non-food applications. Novozymes provides fungal laccases mainly for textile applications. Peroxidases Peroxidases are a diverse group of oxidoreductases, which use hydrogen peroxide as an electron acceptor for oxidation of a wide variety of substrates. As a result of oxidation, a radical is generated and the radicals formed can further react with other substrates. It has been hypothesized that peroxidases affect the gluten network by crosslinking of gluten proteins or by attaching arabinoxylans to gluten proteins via ferulic acid moieties and lysine, tyrosine or cysteine residues (Hilhorst et al.,1999). On the other hand, peroxidases have been reported to be able to crosslink selected proteins such as gelatine and casein effectively (Matheis and Whitaker, 1984). Since peroxidases are common plant enzymes, they also work as endogenous enzymes, for example in flour. Peroxidases have been discovered in various plants and prokaryotic and eukaryotic microbes as well as in mammalian cells. The best characterized plant peroxidase is from horseradish, from which more than 40 isoenzymes have been isolated (Shih et al., 1971). In plants, peroxidases have various physiological roles in for example, degradation and synthesis of lignin in cell walls (Fry, 1979), in the defence mechanism and in cell damage (Vance et al., 1980). The secreted fungal peroxidases lignin peroxidase, manganese-dependent peroxidase and versatile peroxidases are related to lignin degradation (for a review see Martinez, 2002). Sulphydryl oxidases Sulphydryl oxidases (SOX) can generate crosslinks via disulphide bridges between two cysteine residues. The nomenclature is somewhat confusing since sulphydryloxidase can refer to glutathione oxidase (GTOX, EC 1.8.3.3) or thiol oxidases (EC 1.8.3.2). Glutathione oxidase reduces molecular oxygen to hydrogen peroxide (Kusakabe et al., 1982) and thiol oxidase to water (Aurbach and Jakoby, 1962). Glutathione oxidase is a flavoprotein catalysing oxidation of glutathione and cysteine, as well as other thiol group containing compounds, for example dithiothreitol (DTT) and β-mercaptoethanol. Glutathione oxidases have been isolated from rat seminal vesicles and fungi, for example Aspergillus (de la Motte and Wagner, 1987), Penicillium (Kusakabe et al., 1982) and Saccharomyces, (Gerber et al., 2001). Thiol oxidases are enzymes that oxidize small molecular weight thiol compounds. They use either metal ions or FAD (flavin adenine dinucleotide) for catalysis, and they have been isolated, for example from Saccaharomyces and mammalian tissues (Gerber et al., 2001). SOXs can exist either as intracellular or secreted enzymes. SOXs have a role in

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protein folding processes (Kadokura et al., 2003). SOXs are commercially available only for research purposes. It is still under discussion whether SOXs have any affinity to peptide- or protein-bound thiol groups, therefore the crosslinking ability of this enzyme in protein matrix needs to be proven. The synergistic effect of SOX with glucose oxidase has been claimed in baking (Haarasilta et al., 1991). Lipoxygenases Lipoxygenases (LOX, EC 1.13.11.12) catalyse oxidation of polyunsaturated fatty acids, which contain cis,cis-1,4-pentadiene structures (for example linoleic, linolenic and arachidonic acid). Molecular oxygen acts as a terminal electron acceptor in lipoxygenase-catalysed reactions. The primary products are free hydroperoxy radicals, which further generate hydroxy acids and hydroperoxides. Therefore lipoxygenases are also capable of modifying proteins indirectly. The crosslinking ability of lipoxygenases has been attributed to both the free radical oxidation of free thiol groups to generate disulphide bonds and to the generation of reactive crosslinking molecules, for example malondialdehydes (for a review see Matheis and Whitaker, 1987; Gardner, 2003). Lipoxygenases have been derived from many plants, mammals and recently also from both eukaryotic and prokaryotic microbes. The best known and characterized lipoxygenases are from soya (Gardner, 1989). Soya produces three lipoxygenase isoenzymes, which differ in their biochemical properties (Galliard and Chan, 1980). Lipoxygenases have also been reported from fungi Fusarium oxysporym (Matsuda et al., 1978), Penicillium camemberti (Perraud and Kermasha, 2000), Geotrichum candidum (Perraud et al., 1999), and a thermophilic fungus Thermomyces lanuginosus (Li et al., 2001). Many microbial lipoxygenases show their pH optima in a neutral and alkaline pH range. Thermostability of the enzymes is not very good, except with the Th. lanuginosus lipoxygenase, which has half lives at 60 and 70 °C of 20 and 7 min, respectively (Li et al., 2001). Depending on the source of the enzyme, microbial lipoxygenases vary in their substrate specificity and end-product pattern. Lipoxygenases are iron-containing proteins. They contain one iron molecule in their active site. The plant-derived lipoxygenases are monomers and typically 90– 110 kDa large molecules. The three-dimensional structure of soya lipoxygenase I has been solved in high resolution (Minor et al., 1996). Lipoxygenases also play an important role in flavour generation and off-flavour formation. They are present as endogenous enzymes in plant raw materials. Many microbes are reported to produce lipoxygenases, however, the production levels are very low and the enzyme is often intracellular. It is a great challenge to discover stable lipoxygenases and to produce the enzyme in high amount for food applications. Currently the commercial availability of lipoxygenases is limited. Enriched soya flour lipoxygenase is available and it is used for bleaching of carotenoid pigments of wheat flour and to improve baking quality (Nicolas, 1978). Glucose and hexose oxidases Glucose oxidase (EC 1.1.3.4) catalyses oxidation of glucose at the C1 position

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with concomitant consumption of oxygen to provide glucono-δ-lactone and hydrogen peroxide (See eqn [1]). In aqueous systems the lactones will hydrolyse to the corresponding acid. The formed hydrogen peroxide reacts with free thiol groups, which are oxidized to disulphides. The crosslinking ability of glucose oxidase is thus based on in situ hydrogen peroxide formation in baking applications. Glucose oxidase has been proposed in disulphide bond formation in gluten and ferulic acid dimerization of arabinoxylan (Vemulapalli et al., 1998; Vemulapalli and Hoseney, 1998). Furthermore the role of glucose oxidase in baking has been explained by activation of endogenous peroxidases. β-D-glucose + O2 → D-glucono-1,5-lactone + H2O2

[1]

Glucose oxidases are predominantly produced by fungi. The best characterized glucose oxidases are produced by species from Aspergillus and Penicillium. The Aspergillus glucose oxidase is intracellular and located in peroxisomes (van Dijken and Veenhuis, 1980), the Penicillium glucose oxidase is a secreted enzyme (Pronk et al., 1989). Glucose oxidases are large proteins; the size of fungal glucose oxidases is 140–160 kDa. The enzyme is a dimer containing two identical subunits. Each subunit has one FAD molecule. Glucose oxidase is commercially available from various suppliers. All commercial glucose oxidases are from Aspergillus. Both Aspergillus and Penicillium glucose oxidases have their pH optima in a relatively broad pH range of 4.5–7.5. The enzymes are stabilized by excess glucose. Aspergillus and Penicillium glucose oxidases stay active for a short time at 60 °C. Hexose oxidase (EC 1.1.3.5) has many similarities to glucose oxidase although its substrate specificity range is much broader. Hexose oxidase is capable of oxidizing a number of mono- and oligosaccharides to the corresponding lactones with a concomitant production of hydrogen peroxide. Hexose oxidase is produced by marine algal species Chondrus crispus, Euthora cristata, and Iridophycus flaccidum (Sullivan and Ikawa, 1973; Bean and Hassid, 1956). The bacterium Malleomyces pseudomallei (Dowling and Levine, 1956) and developing citrus fruits (Bean et al., 1961) have also been reported to produce an enzyme capable of oxidizing various saccharides. Hexose oxidase from C. crispus is currently commercially available. The enzyme is a copper-containing glycoprotein with molecular a weight of 130 kDa as approximated by gel filtration (Sullivan and Ikawa, 1973). The open reading frame of the isolated cDNA corresponded to a polypeptide of MW 62 kDa suggesting thus that the protein is a homodimer (Hansen and Stougaard, 1997). C. crispus hexose oxidase has the highest affinity to the monosaccharides D-glucose and D-galactose and for the disaccharides cellobiose and maltose (Poulsen and Bak Høstrup, 1998). In the baking experiments the enzyme increased dough strength and bread volume more efficiently than glucose oxidase.

6.3

Application of crosslinking enzymes in baking and pasta manufacture

Wheat proteins are unique among the cereal proteins in their ability to form a

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viscoelastic, extensible dough which is able to retain gas during proofing and baking. Although other cereal grains, for instance oat or rye, contain very similar proteins, their gas holding capacity is limited (Dobraszczyk et al., 2001). In addition to proteins, arabinoxylans have a significant role in baking especially in rye flour, but also in wheat. Arabinoxylans consist of a linear (1→4)-β-Dxylopyranosyl backbone, in which substitution patterns and degree of polymerization vary depending on its origin (Bengtsson et al., 1992; Hoffman et al., 1991; Oscarsson et al., 1996). Arabinose is the major substituent present and it is bound to backbone xyloses by (1→3)- and/or (1→2)-glycosidic linkages (Gruppen et al., 1993; Bengtsson et al., 1992; Hoffmann et al., 1991). Some of the xylan-linked arabinoses are esterified to ferulic acid, forming a linkage to the O-5 position of the arabinose (Smith and Harley, 1983). Arabinose-bound ferulic acid has also been found to form dimers, such as 8-0-4´, 8-5´ and 5-5´, in plant cell wall structures, referring to crosslinking of the polysaccharides (Figueroa-Espinoza and Rouau, 1998; Dervilly et al., 2000). Hydrolytic enzymes, that is xylanases and amylases are widely used as baking aids to increase the bread volume and reduce the staling rate of the crumb (Cauvain and Chamberlain, 1988; Hilhorst et al., 1999; Tenkanen et al., 2000). The drawback with the use of xylanases is impaired dough handling as the dough becomes sticky. Crosslinking enzymes can potentially counteract this negative effect. Crosslinking enzymes also have potential in frozen dough baking and baking with weak flours. Owing to the complex composition of dough matrix, the crosslinking enzymes can act on either proteins or arabinoxylans, or even catalyse conjugate formation between these biopolymers. TG has been elucidated in several cereal-based food applications, as the enzyme modifies the functional properties of products via aggregation and polymerization of gluten proteins. TG-tailored products with improved textural properties, elasticity, water holding capacity, firmness and heat stability (Zhu et al., 1995; Motoki and Seguro, 1998; Collar and Bollaín, 2004, 2005) have been reported. The possibilities for utilization of TG in cereal applications are summarized in Table 6.2.

6.3.1 Use of transglutaminase (TG) in baking TG has been widely investigated in baking and currently several TG-based enzyme products are on the market for baking purposes. TG is reported to crosslink highmolecular weight glutenins to large insoluble polymers (Gerrard et al., 2000, 2001; Rosell et al., 2003; Larré et al., 2000). Crosslinking of α-gliadins (Bauer et al., 2003a), water-extractable albumins and globulins (Gerrard et al., 2000, 2001, Siu et al., 2002) has also been detected. In normal fresh baking TG is reported to decrease dough extensibility and increase dough resistance to extension which will hinder the growth of air bubbles in the dough resulting in a decrease in volume of the bread (Gerrard et al., 1998; Bauer et al., 2003b, Autio et al., 2005). It has been also shown that TG will increase water absorption of dough and therefore larger amounts of water should be added. According to light microscopy, the protein network was accumulated on strong

Table 6.2

Transglutaminase-aided crosslinking in cereal applications

Application

Effect

Improved stability, texture and volume Improved preservation and lift of puff pastry. Improved volume and staling properties of yeasted croissants Rebuilding the structure of dough Improved texture Formation of protein network; improved loaf volume, crumb characteristics and bread appearance Cake manufacturing Improved texture and volume Noodles and pasta manufacturing Improved texture; prevention of texture deterioration

Substitution or reduction of ingredients or oxidizing improvers Pan breadmaking Modifying wheat flour during milling Manufacture of fat-rich bakery products Gluten formulation Yeasted wheat flour doughs

Increased or maintained loaf volume Improved crumb strength, retarded staling Improved elasticity and smoothness of processed noodles, bread and pastries Formation of porous or multilayered structure with enhanced organoleptic properties Improved gluten functionality after protease digestion or acid hydrolysis Improved the resistance in a manner comparable to traditional oxidizing improvers

Gerrard, 2002; Poza, 2002 Gerrard et al., 2000 Gerrard et al., 2000; Gerrard et al., 2001 Köksel et al., 2001; Caballero et al., 2005 Gerrard, 2002; Poza, 2002 Moore et al., 2006 Ashikawa et al., 1990 Soeda et al., 1993; Sakamoto et al., 1996; Kuraishi et al., 2001; Yamazaki and Nishimura, 2002; Wu and Corke, 2005 Lindsay and Skerritt, 1999; Gerrard et al., 1998; Motoki and Seguro, 1998 Gerrard et al., 1998; Collar and Bollaín, 2005 Yamazaki and Soeda, 1998 Kuraishi et al., 1997b Babiker et al., 1996 Gottmann and Sproessler, 1992; Motoki and Seguro, 1998

Using crosslinking enzymes to improve properties of food

Frozen, laminated doughs Pastry products Croissant doughs Low-quality flour baking High fibre baking Gluten free baking

References

111

112

Novel enzyme technology for food applications

protein fibres owing to TG (Autio et al., 2005). Addition of TG also increased crumb firmness (Gerrard et al., 1998). Several studies have shown that TG makes a very weak gluten into a very strong one and it can improve the baking quality of dough containing weak protein (Larré et al., 2000; Autio et al., 2005). TG treatment is also reported to increase the specific volume of breads baked from organic flour only, when additional water (+10% of farinogram absorption) and a small enzyme dosage was used (Autio et al., 2005). TG has been shown to have positive effects on the quality of pastry and croissant doughs (Gerrard et al., 2000, 2001). TG treatment has also been evaluated in oat baking, but was not found to have positive impact on the specific volume of the oat breads or on the sensory profiles (Salmenkallio-Martilla et al., 2004). TG treatment is reported to affect positively the quality of rice flour breads (Gujral and Rosell, 2004b). Use of transglutaminase (TG) in pasta manufacture The structure of pasta (and noodles) is largely determined by a well-organized protein network constituted mainly from gluten proteins. Although there are only a few reports about TG-treated pasta products, according to Kuraishi et al. (2001), TG is widely used in pasta (and noodle) processing especially in Japan. For instance, improved texture and cooking stability have been reported in TG-treated cooked noodles and pasta (Sakamoto et al., 1996; Kuraishi et al., 2001; Yamazaki and Nishimura, 2002; Wu and Corke, 2005). As a result of TG-strengthened protein structure, starch will be also fixed in the protein network and the release of starch amylose, for example into the boiling water, could be reduced and the surface of product becomes less sticky. It has also been suggested that TG treatment enables utilization of lower-grade flours for production of good quality pasta and noodles (Sakamoto et al., 1996).

6.3.2 Oxidative enzymes as crosslinking agents in cereal applications Oxidative enzymes are potential tools in cereal processing as they can form covalent linkages on proteins and certain carbohydrates and also act on lipids. Subsequent changes in the rheological properties of gluten, dough and bread, and flavour and colour of products, are the most ordinary implications, as summarized in Table 6.3. The mechanism of oxidative enzymes in cereals is complicated as the enzymes may induce linkages in/between polysaccharides, in/between proteins and between polysaccharides and proteins. For instance, laccase, peroxidase, tyrosinase, and also glucose oxidase and hexose oxidase indirectly by H2O2 production, are able to catalyse oxidation of phenolics present in both proteins and polysaccharides. Thereby, through crosslinking of proteins and/or polysaccharides, various positive effects in cereal product applications can be achieved as illustrated in Table 6.3. In addition, combining oxidative enzymes with hydrolytic enzymes, such as pentosanases, has been reported to result in improved characteristics of baked products (Primo-Martín et al., 2003).

Table 6.3

Oxidative enzymes as crosslinking agents in cereal applications Main substrate in bread flour

Effects

References

Lipoxygenase

Polyunsaturated fatty acids (FA)

Hosenay et al., 1980; Nicolas and Potus, 2000; Martinez-Anaya, 1996

Glucose oxidase

β-D-glucose

Peroxidase

Phenolic compounds, especially FA

Improved mixing tolerance of dough and dough-strengthening, bleaching of bread crumb, flavour-effect Dough strengthening, better crumb and larger volume of product, improvement of the breadmaking quality of rice flour Improved dough handling, loaf volume and crumb structure of bread

Laccase

Phenolic compounds, especially FA

Tyrosinase

Protein tyrosyl residues, phenolic compounds of cell wall structures, not FA Mono- and oligosaccharides

Hexose oxidase

Sulphhydryl oxidase Protein cysteinyl residues

Improved dough handling, loaf volume and crumb structure of bread Protein/AX network strengthening. Increased accessibility of iron of polyphenol-containing products Resistance to stretching, increased dough strength and increased bread volume Gluten strengthening, preventing over-mixing, flavour (undesirable?)

Vemulapalli and Hoseney, 1998; Dunnewind et al., 2002; Gujral and Rosell, 2004a Dunnewind et al., 2002; FigueroaEspinoza and Rouau, 1998; FigueroaEspinoza et al., 1999b Si, 1994; Labat et al., 2000 and 2001 Hoseney and Faubion, 1981; Matuschek and Svanberg, 2005 Stougaard and Hansen, 2002; Poulsen and Bak Høstrup, 1998 Hammer et al., 1990; Verbakel et al., 1996; Martinez-Anaya, 1996

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Enzyme

113

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Crosslinking by glucose oxidases is hypothesized to take place via H2O2 production. It has been suggested that glucose oxidases participate in both protein network strengthening, especially via disulpide bonding and in oxidative gelation of arabinoxylan (AX) (Vemulapalli et al., 1998; Vemulapalli and Hoseney, 1998; Vroemen, 2003). Effects similar to those of glucose oxidases have been observed with hexose oxidase (Poulsen and Bak Høstrup, 1998). Peroxidases are proposed to promote crosslinking of the protein network and arabinoxylans separately in dough (Figueroa-Espinoza and Rouau, 1998, Labat et al., 2001), whereas laccases are reported to crosslink mainly AX matrix in doughs through dimerization of the esterified ferulic acid (FA) (Figueroa-Espinoza and Rouau, 1998; FigueroaEspinoza et al., 1998; 1999a; Labat et al., 2000; Vemulapalli et al., 1998). Arabinoxylan gels and wheat doughs treated by laccase have also been found to have poor stability properties, probably owing to radical catalysed depolymerization (Carvajal-Milan et al., 2005b; Selinheimo et al., 2006a). Piber and Koehler (2005) detected covalent crosslinking between feruloylated AX and protein tyrosine residues in wheat and rye, apparently caused by endogenous oxidative enzymes in grains. Oudgenoeg et al. (2001) showed that FA can be linked to tyrosine-containing peptides by peroxidase. Laccase was also found to oxidize tyrosine containing peptides, with consequent peptide polymerization (Mattinen et al., 2005). Lipoxygenases have been used in the baking industry since 1930 for bleaching flour to produce white bread crumb. The bleaching action is thought to be coupled to oxidation of pigments and unsaturated fatty acids by atmospheric oxygen (Hammer, 1993). The mechanism of LOX in structural bread improvement is suggested to involve the formation of disulphide bonds between gluten molecules (Nicolas and Potus, 2000).

6.4

Application of crosslinking enzymes in meat and fish processing

The high cost of meat production requires technologies to utilize the meat raw material efficiently. One solution is restructuring or reshaping a value-added meat product from lower-value parts of fresh meat by glueing the parts together, by crosslinking enzymes. As fat and salt contribute to many technological and sensory properties of meat products, their reduction in the products is not straightforward. Crosslinking enzymes have shown potential in maintaining the textural properties of meat products, particularly those that have a low salt content. The ability of TG to crosslink different meat proteins has been common knowledge for over two decades. Industrial application of TG in meat binding started less than a decade ago when microbial TGs were launched (Nonaka et al., 1989). TGs from different origins have been reported to differ in their crosslinking capacity towards myofibrillar proteins. Mammalian TGs (Kahn and Cohen, 1981; De Backer-Royer et al., 1992; Tseng et al., 2002) tend to modify in addition to myosin also actin,

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while microbial TGs originating from Streptomyces have only a limited activity on actin. Ca2+-dependency is another feature that draws a line between mammalian and microbial TGs. In addition to myosin and actin, troponin T has also been found to be modified by microbial TG (Lantto et al., 2005; Ramirez-Suarez et al., 2005). Interestingly, laccase which has been suggested to create an isodityrosine bond in tyrosine-containing peptides (Mattinen et al., 2005), was found on SDS-PAGE to react with myosin and troponin T but not actin (Lantto et al., 2005). Studies of the effects of different crosslinking enzymes on meat protein systems are summarized in Table 6.4. Owing to efficient crosslinking of myosin, TG is capable of affecting viscoelastic properties, texture, and water holding capacity of meat protein gels. The type and quantity of meat proteins involved, as well as solubility, pH, temperature, the amount and type of salts in the system and heating/processing conditions used affect gelation (Asghar et al., 1983; Xiong, 1994; Lesiów and Xiong, 2001a, b). Generally observed changes that are caused by adding TG to a meat system are improved binding of fresh meat pieces together (Nielsen et al., 1995; Kuraishi et al., 1997a), heat-induced gelation (Ramirez-Suarez et al., 2001; RamirezSuarez and Xiong, 2002, Ramirez-Suarez et al., 2005; Lantto et al., 2005) and strengthening of the gel (Dondero et al., 2006; Lantto et al., 2006; Tsai et al., 1996). In addition to TG, tyrosinase and laccase have also been tested in processing of pork and chicken proteins (Lantto et al., 2005, 2006). Chicken breast myofibrillar protein suspension was gelled as a function of TG dosage and NaCl concentration (Lantto et al., 2005). Laccase was able to improve gel formation only slightly when a high NaCl concentration (0.6 M NaCl) was used, indicating that fully solubilized protein is needed as substrate. Furthermore, too high dosages of laccase led to protein fragmentation and decreased gel strength. Similar effects have been observed in laccase treated wheat dough (Selinheimo et al., 2006a).

6.4.1 Heat-treated meat products TG has been proposed as an alternative binding agent to reduce the need for NaCl addition in processed meat products (Kuraishi et al., 1997a). Although the positive effects of TG on texture are evident, TG has not turned out to be a generally accepted salt replacer, as the reported results on its role in water holding differ greatly. Kilic (2003) found that cooking loss of liquid from chicken döner kebab (2% salt) was not altered compared with the control whether TG was used as such or together with caseinate. According to Pietrasik and Jarmoluk (2003) and Jarmoluk and Pietrasik (2003) TG had no effect on cooking loss of liquid from pork muscle gels with 2% curing salt. On the other hand, positive effects of TG on water-holding properties have been reported in low-salt (1%) meat balls (Tseng et al., 2000) and beef gels with 2% salt (Pietrasik and Li-Chan, 2002a; Pietrasik, 2003). Discrepancies in the reports of the effects of TG on water holding are likely to be due to different TGs and dosages used, different meat raw materials, different

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Effects of crosslinking enzymes on meat proteins and technological properties of meat systems

Enzyme

Source

Meat substrate

Effects

Reference

TG

Bovine plasma Guinea pig liver

Polymerization of myosin Polymerization of actomyosin

Kurth and Rogers, 1984 Akamittath and Ball, 1992, Kim et al., 1993 Tseng et al., 2002

Pig plasma

Purified beef myosin Actomyosin from turkey or beef Chicken myofibrillar proteins Chicken meat balls

S. mobaraense S. ladakanum

Rabbit actin Mackerel surimi

Ajinomoto

Pork homogenate

Activa TG-TI, Ajinomoto

Bovine cardiac myofibrillar protein

TG purified from Activa WM product

Chicken myofibrillar protein

Pig plasma

Polymerization of myosin and actin Increased strength and cooking yield Intramolecular bond in actin Gel strength improved, elasticity decreased Gel formation during heating increased, flat transition phase at 50–60 °C decreased, gel firmness increased Gel formation (G´) improved at 70 °C and in the transition at 55.8–56.9 °C. Gel formation improved as a function of TG dosage and NaCl concentration

Tseng et al., 2000 Eli-Berchoer et al., 2000 Tsai et al., 1996 Lantto et al., 2006

Ramirez-Suarez et al., 2001

Lantto et al., 2005

Novel enzyme technology for food applications

Table 6.4

Ajinomoto

Ajinomoto Ajinomoto Ajinomoto Ajinomoto

Human placental factor XIIIa Tyrosinase Laccase

Mushroom Purified from Trametes hirsuta

Increased strength of cooked gel, decreased cooking yield Beef Polymerization of myosin Ground chicken breast Improved emulsion stability, muscle increased water uptake ability Ground chicken meat Increased hardness of cooked gel Restructured pork shoulder Increased consistency of cooked gel, no effect on cooking yield Pork batter Increased hardness, decreased cooking loss Ground beef No effect on cooking or purge losses, water-holding capacity of unheated gels and hardness of cooked gels improved Beef myofibrillar proteins Polymerization of myosin, partial polymerization of actin Pork homogenate Gel formation during heating was not affected, gel firmness increased Chicken myofibrillar Gel formation improved at 0.6 M protein NaCl, high laccase dosages decreased gel formation

Dondero et al., 2006 Aktas and Kiliç, 2005 Ruiz-Carrascal and Regenstein, 2002 Trespalacios and Pla, 2007 Dimitrakopoulou et al., 2005 Pietrasik and Li-Chan, 2002b Pietrasik and Li-Chan, 2002a

De Backer-Royer et al., 1992 Lantto et al., 2006 Lantto et al., 2005

Using crosslinking enzymes to improve properties of food

Microbial TG Ajinomoto

Beef

117

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amounts of NaCl, other salts and ingredients, and different meat gel preparation conditions. Inconsistent research reports clearly show that the role of TG on water holding is not as clear as it is regarding texture improvement. In a typical sausage process, TG is added to the meat mixture together with water, salt and other ingredients after comminuting of the meat, or in the case of ham, before or during tumbling. These production phases are carried out at cold temperatures. TG is active when cold (>2 °C), but by definition much less so than in its optimal temperature range of 40–60 °C. The activity of TG increases during the cooking phase until the internal temperature rises above 70 °C at which temperature TG is no longer active.

6.4.2 Restructured fresh meat products High quality meat products with moderate prices demanded by consumers are the driving force to restructure low-value cuts and meat pieces to palatable steaks resembling intact meat, thus maximizing the efficiency of use per carcass. Traditionally salt and phosphates have been used with heat treatment to bind meat pieces together. TG has also been successfully used for binding fresh meat pieces together and fresh meat binding is still the main application of TG in the meat sector. TG has been observed to improve meat protein gel formation without or with only low levels of added NaCl and phosphates (Wijngaards and Paardekooper, 1988; Nielsen et al., 1995; Kuraishi et al., 1997a). Kuraishi et al. (1997a,b) observed that restructured meat products that are traditionally prepared using NaCl and phosphates to promote extraction of proteins can be prepared without added salt using TG and that binding strength was further enhanced when caseinate was added to the system. Caseinate has been found to be an excellent extender in TG-aided restructuring. Caseinate is an excellent substrate for microbial TG (Færgemand et al., 1998a) and it polymerizes during the enzymatic reaction, turns viscous and thus acts like a glue binding meat pieces together. In addition to meat, this TG catalysed restructuring technology can be applied also in fish products (Ramírez et al., 2002; Uresti et al., 2004).

6.5

Application of crosslinking enzymes in dairy applications

Bovine milk contains approximately 3.5% protein. The milk proteins can be fractionated into two groups: caseins are insoluble at their isoelectric point at pH 4.6 and represent about 80% of the total protein; the remaining 20% are whey proteins, which are soluble at pH 4.6. The whey proteins in turn include βlactoglobulin, α-lactalbumin, bovine serum albumin (BSA), immunoglobulins and several minor proteins. Caseins are mainly located in micelles, the diameter of which ranges from 50 to 300 nm. The protein content of the micelles is about 94%, the remaining 6% consists of calcium and phosphate and lower levels of

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magnesium and citrate, which all stabilize the micelle structure. Sodium caseinate is a mixture of αs1-, αs2-, β- and κ-casein macromolecules of which β-casein is the major component. The main whey proteins are globular proteins with intramolecular disulphide bonds stabilizing their structure. β-Lactoglobulin represents approximately 50% of the whey protein fraction and α-lactalbumin 20%. The whey proteins are relatively heat labile; heating at 90ºC for 10 min causes complete denaturation (Fox and McSweeney, 1998). The use of TG in dairy applications has been intensively studied (Lorenzen et al., 1998; Nonaka et al., 1992; Dickinson and Yamamato, 1996; Færgemand et al., 1997a, b, 1998a, b; Özrenk, 2006). Caseinate is considered to be a very good substrate for TG because of the flexible and open tertiary structure as the α- and βcasein monomers lack disulphide bonds (Færgemand et al., 1999a; Lorenzen et al., 1998). Only a small number of crosslinking sites are necessary for complete oligomerization of caseinates (Lorenzen et al., 1998). Of the different caseins, the β- and κ-caseins are reported to have greater reactivity than α-casein with TG (Ikura et al., 1980; Færgemand et al., 1999a). β- and κ-caseins were crosslinked more than αs1 mainly because κ-casein and some β-casein are located on the surface of the micelle (Smiddy et al., 2006). In their native form, the globular whey proteins, α-lactoglobulin and β-lactalbumin, are poor substrates for TG (Lorenzen and Neve, 2003; Sharma et al., 2001). The globular structure can, however, be unfolded by chemical reduction (with DTT, cysteine, 2-mercaptoethanol), by alkaline pH, heating, or high pressure treatment whereafter the protein can form gels with TG (Coussons et al., 1992; Traoré and Meunier, 1992; Færgemand et al., 1997b; Lee et al., 2002; Eissa and Khan, 2006; Kang et al., 2003). High pressure treatment with subsequent TG treatment has also been reported in formation of hetero-oligomers of β-casein and β-lactoglobulin (Lauber et al., 2003). Use of a Ca2+ independent TG is essential for extensive crosslinking of β-lactoglobulin, as the presence of just 5 mM Ca2+ limits the formation of covalent crosslinks (Færgemand and Qvist, 1999). TG is reported to induce only minor crosslinking of unheated milk proteins, whereas after heat treatment a dramatic increase in crosslinking has been observed. It has been shown that bovine milk serum contains a heat labile low molecular weight inhibitor of TG whose presence can be counteracted by increasing the dosage of TG (De Jong et al., 2003). Either the preheating inactivates the natural inhibitor or increases the crosslinking of whey proteins due to unfolding. The TGinduced crosslinking efficiency can be affected by optimizing the preheating conditions (Rodriquez-Nogales, 2006). Preheating skim milk at 85 °C for 15 min enhanced the susceptibility of milk proteins towards crosslinking (Sharma et al., 2001). Crosslinking enzymes can be used in many types of dairy products to improve texture and other technological properties. The effect of TG on technological properties of dairy products is summarized in Table 6.5. Oxidative enzymes are also an interesting alternative for crosslinking of different milk proteins. Tyrosinase, laccase and peroxidase are reported to induce at least partial crosslinking of whey proteins (Thalmann and Lötzbeyer, 2002; Færgemand et al., 1998b).

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Effects of transglutaminase on the technological properties of dairy products

Application

Milk

Effect

References

Yoghurts, set type and stirred Preheated milk

Increased firmness or viscosity, less syneresis

Lorenzen et al., 2002b; Lorenzen and Neve, 2003

Low fat yoghurts

Preheated milk

Stabilization against syneresis, increased viscosity

Færgemand et al., 1999b; Ozer et al., 2006

Goat milk yoghurt

Spray-dried goat milk

Improved consistency, decreased whey separation, improved survivability of probiotic bacteria

Farnsworth et al., 2005

Quark

Preheated milk

Creamier texture and less grainy

Lorenzen et al., 2002a

Increasing stability of milk

Preheated milk

Sullivan et al., 2002; Smiddy et al., 2006

Cheese

Clotted milk

Stabilization of casein micelles against various treatments, such as UHT Improved incorporation of whey proteins

Han and Spradlin 2000; Han et al., 2003

Novel enzyme technology for food applications

Table 6.5

Using crosslinking enzymes to improve properties of food

121

6.5.1 Set and stirred yoghurts In yoghurt manufacturing the use of crosslinking enzymes allows either replacement of additional protein or polysaccharides or reduction of dry matter content without changing texture and water-binding properties. The crosslinking enzyme is added before or concomitantly with the starter (Neve et al., 2001; Lorenzen et al., 2002b). Crosslinking of milk proteins by TG with subsequent heat inactivation is reported to have a slight growth-slowing effect on yoghurt starter bacteria (Lorenzen et al., 1999, Ozer et al., 2006). According to Neve et al. (2001) no delay in growth occurs when TG and starter bacteria are applied simultaneously. It has also been suggested that starter bacteria with good ability to produce aroma compounds should be used in conjunction with crosslinking enzymes (Ozer et al., 2006). According to Lorenzen et al. (1999) yoghurt manufactured from TG treated milk had decreased post-acidification during storage, lower syneresis and milder taste. With optimized TG dosages non-fat yoghurts with improved physical and sensory properties could be obtained enabling non-fat yoghurt production without the need for extra protein or stabilizer (Ozer et al., 2006). Improved survivability of probiotic cultures has also been reported in set yoghurts after TG treatment (Neve et al., 2001). Crosslinking technology can also be applied to quark manufacture. Quark prepared from TG-treated skim milk was less grainy than control products, but the enzyme made the product smoother and creamier (Lorenzen et al., 2002a).

6.5.2 Increasing stability of milk against various treatments The stability of milk against coagulation at sterilization temperatures has commercial importance in the manufacture of UHT milk and milk-containing products, such as concentrated milk. Sullivan et al. (2002) found increased heat stability of preheated (70–90 °C for 1 min) and TG-treated (24 h at 6 °C) milk at pH values 6.6 to 7.3 compared with unheated TG-treated milk. The stability of casein micelles against high pressure and against heating in the presence of ethanol can also be increased by TG (Sullivan et al., 2002; Smiddy et al., 2006).

6.5.3 Crosslinking enzymes in cheese manufacture TG is widely patented in cheese applications, mainly for incorporation of whey proteins into caseinates with subsequent improvement in yield. The patents differ mainly in which order TG is added to the process (Han and Spradlin, 2000; Han et al., 2003). The effect of TG treatment of milk on renneting properties has been studied (Lorenzen, 2000). According to Cozzolino et al. (2003) milk coagulation time was dependent on the addition stage of TG. TG addition preceeding or simultaneously with the rennet addition decreased the coagulation (Cozzolino et al., 2003). Optimal TG treatment could, however, be used to improve the firmness of the cheese. Furthermore, the addition of TG after clotting and curd cutting could also be used to improve the cheese yield as some whey proteins were incorporated into the cheese matrix (Cozzolino et al., 2003).

122

6.6

Novel enzyme technology for food applications

Other applications of crosslinking enzymes in food manufacture

Crosslinking enzymes have been used to improve the technological or nutritional properties of protein-based food ingredients or ferulic acid-containing carbohydrates such as sugar beet pectins. TG can be applied to improve the gelling properties of proteins, for example fish gelatin (Fernández-Díaz et al., 2001; Kolodziejska et al., 2004), wheat gluten (Wang et al., 2007) or soy protein isolate (Tang et al., 2006). The emulsification or foaming properties of protein-based systems can be improved by TG treatment (Dickinson, 1997; Færgemand et al., 1998a; Sharma et al., 2002; Liu and Damodaran, 1999). The quality attributes of tofu can also be enhanced by TG treatment (Tang et al., 2007). Mariniello et al. (2003) and Chambi and Grosso (2006) have exploited TG to produce edible protein-based films with improved properties. Microbial TG has also been used to incorporate lysine and lysine dipeptides covalently into casein with potential improvement in the nutritional value of casein (Nonaka et al., 1996). Enzymatic crosslinking with peroxidase and laccases has been applied to improve the gelling of sugar beet pectin or arabinoxylan (Guillon and Thibault, 1990; Kuuva et al., 2003; Carvajal-Millan et al., 2005a). Laccase and peroxidase can oxidize ferulic acid groups in pectins or xylans and as a result of the radical mediated process a covalent bond is formed between ferulic acid residues (Micard and Thibault, 1999; Carvajal-Millan et al., 2005a). A hydrated network is subsequently formed. Hetero-crosslinking of proteins with polysaccharides or low molecular weight components has also been attempted with the aim of forming new macromolecular structures with totally novel properties. For example soluble protein–polysaccharide conjugates or hetero protein–protein conjugates, such as ovomucin-αs1-casein conjugate obtained with TG are reported to have improved emulsifying properties (Dickinson, 1993; Kato et al., 1991). Peroxidases are reported to form covalent protein–carbohydrate conjugates between gluten or β-casein and feroylated arabinoxylans (Hilhorst et al., 1999; Boeriu et al., 2004). Tyrosinase has been used to graft casein peptides to chitosan (Aberg et al., 2004). Gelatine–catechin conjugate was synthesized by the laccase-catalysed oxidation of catechin in the presence of gelatin resulting in increased antioxidative properties of the conjugate compared to unconjugated catechin (Chung et al., 2003).

6.7

Analysing the chemistry of crosslinks formed by enzymes

Development of novel enzyme-aided crosslinking concepts for food applications requires profound understanding of the chemistry of the crosslinks formed, of the enzymatic and non-enzymatic reaction mechanisms and kinetics, and so on. Therefore for mechanistic analyses, experimental data from different analytical methods need to be combined. Owing to the complexity of food matrices some

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123

mechanistic studies are typically carried out using single model proteins or even lower molecular weight model compounds such as peptides. The primary enzymecatalysed reactions can be followed by measuring consumption of the cosubstrate, that is O2 (laccase, tyrosinase) or H2O2 (peroxidase) or formation of the co-product (TG), for example NH3 (Preininger et al., 1994; Zhou et al., 1997; Levitzki, 1970). Radical-forming reactions can be monitored by an electron paramagnetic resonance (EPR) spectroscopy. The formation of the novel covalent bonds can be confirmed by various mass spectrometric (MS) techniques and the chemical nature of the crosslinks can be analysed by Fourier transform infrared (FTIR) spectroscopy. Particularly in the food matrix, the formation of crosslinks needs to be elucidated by various analytical techniques using different polymers as models. Then the crosslinks formed in food polymers need to be isolated by, for example, enzymatic or acid hydrolysis whereafter the crosslinked fragments must be separated. Then the crosslinked pieces can identified using various spectroscopic techniques (Lee et al., 2002; Schäfer et al., 2005; Takasaki et al., 2005; Rodriguez-Mateos et al., 2006; Hanft and Koehler, 2005).

6.7.1

Types of crosslink formed by oxidative enzymes in protein crosslinking Of the oxidative enzymes, laccase and peroxidase react via radical formation and tyrosinase reacts via ortho-quinone formation. Thus, the reaction mechanisms of crosslink formation are different in these cases. In protein matrix, the orthoquinones formed are prone to react non-enzymatically with other amino acid side chains such as free sulphydryl (-SH) and amino groups (-NH2), resulting in the formation of tyrosine–cysteine and tyrosine–lysine crosslinks, respectively (Ito and Prota, 1976; Ito et al., 1984; Burzio, 2000; Marumo and Waite, 1986; Takasaki and Kawakishi, 1997). Quinones can also be coupled together and form dityrosine linkages (Jee et al., 2000; Bertazzo et al., 1999a, b; Thalmann and Lötzbeyer, 2002; Takasaki et al., 2001). Laccase-catalysed crosslinking is inititated by formation of free radicals, which in turn react further non-enzymatically. Laccase-catalysed reactions on proteins and peptides are poorly understood. However, some studies show crosslinking abilities of these enzymes on proteinous matrices in foods (Figueroa-Espinoza et al., 1999a, Si and Sørensen, 1993; Yamaguchi, 2000; Dickinson, 1997; Færgemand et al., 1998b; Lantto et al., 2005). The covalent linkage connecting the model peptides was found to be mostly an ether bond (isodityrosine bond) whereas only small amounts of dityrosine bonds were detected in the reaction products (Mattinen et al., 2005). Also disulphide bonds have been found in laccase-catalysed reactions on proteins and the oxidation of -SH groups was found to be accelerated by addition of phenolic acid (Figueroa-Espinoza et al., 1999a). Peroxidases also primarily oxidize tyrosine residues to the corresponding radical in the presence of hydrogen peroxide. The radicals formed have been detected by EPR (Steffensen et al., in press). Tyrosine-containing peptides have been oxidatively crosslinked by horseradish peroxidase in the presence of

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Novel enzyme technology for food applications

hydrogen peroxide resulting in production of peptide oligomers from dimers to pentamers (Michon et al., 1997, 1999; Oudgenoeg et al., 2001, 2002). Isodityrosine and dityrosine linkages were formed in those reactions.

6.7.2

Types of crosslink formed by oxidative enzymes in feruloylated carbohydrates In plant cell wall structures, crosslinking reactions may occur via photochemical reactions or by endogenous enzymes through radical coupling (Micard and Thibault, 1999). The degree of ferulate-coupling can be modified remarkably by exogenous enzymes and the crosslinking property of ferulic acids in pectins and arabinoxylans is widely exploited in tailoring the characteristics of pectin- and cereal-derived foods (Micard and Thibault, 1999). In arabinoxylans and pectins, distribution of ferulate coupling consists predominantly of diferulate bonds. Ferulic acids are known to dehydrodimerize via oxidative mechanism, resulting mainly in 8-5, 8-O-4, 8-8 and 5-5 linkages (Schooneveld-Bergmans et al., 1999; Oosterveld et al., 1997, 2000; Figueroa-Espinoza and Rouau, 1998; Dervilly et al., 2000; Bunzel et al., 2001). In pectins isolated from sugar beet pulp, dehydrodimers 8-8 and 8-O-4 are predominant and oxidative crosslinking with hydrogen peroxide/peroxidase has been found to result mainly in an increase of the 8-5 and 8-O-4 dehydrodimers (Oosterveld et al., 1997, 2000). It has also been suggested by Fry et al. (2000) that radical polymerization of ferulates would not stop at the dimeric stage and, recently, dehydrotriferulic acids have been characterized, indicating that higher ferulate oligomers are also involved in crosslinking of cell wall polysaccharides. The presence of 5–5/8–O–4-coupled and 8–O–4/8–O–4-coupled ferulic acid dehydrotrimers and 8–8(cyclic), 8–O–4-dehydrotriferulic acid in the arabinoxylan network structure has been identified (Hatfield et al., 1999; Funk et al., 2005; Bunzel et al., 2005; Allerdings et al., 2005). The crosslinking mechanisms of feruloylated carbohydrates and proteins has been studied using model substrates. The ability of laccase to create a crosslink between FA and small tyrosine-containing peptides has been shown by Mattinen et al. (2005). The ability of peroxidase to crosslink FA to small tyrosine-containing peptides was shown by Oudgenoug et al. in 2001 and 2002. However the detailed reaction mechanisms are not yet fully understood.

6.8

Effect of biopolymer crosslinking on nutritional properties of food

Crosslinking of food biopolymers is expected to affect the nutritional value of the food material with possible impacts on digestibility, fibre degradation by gut microbes, availability of essential amino acids or even on allergenicity. The impact can be either positive or negative, but thus far, these issues have not been very widely studied and more research is clearly needed. However, it should be

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emphasized that natural crosslinking is occurring constantly in raw materials owing to different physical processes or certain endogenous enzymes. Thus, crosslinked structures have invariably been a part of human nutrition. Dietary fibres, the non-digestible polysaccharides such as arabinoxylans, occur naturally in many foods and are related to health-positive physiological actions in the small and large intestine (for a review see Tungland and Meyer, 2002). There is only a little information available concerning the microbiological and physiological consequences of modified or crosslinked AX breakdown in the large bowel. Hopkins et al. (2003) investigated the breakdown of crosslinked AX in children’s intestinal microbiotas. The authors found that there was a variation in the metabolism of these polysaccharides by colonic microbiotas, the ferulate crosslinking reducing the rate of AX fermentation. If the enterobacterial metabolism is excluded, bacterial metabolism was not markedly affected by the crosslinking. By modifying the quantity and quality of protein crosslinks during food processing, the functional properties of food proteins can be changed. Crosslinking of proteins by the Maillard reaction during food processing, under a variety of conditions, is well established (Bristow and Isaacs, 1999; Fayle and Gerrard, 2002). Crosslinks are usually generated via bridging of the protein-bound amines by carbonyl-containing compounds and a decrease in nutritional quality has been reported caused by these reactions (Friedman, 1996; Fayle and Gerrard, 2002; Miller and Gerrard, 2005). However, not too much is known about the extent of Maillard or other covalent crosslinking in processed foods, its impact on food quality, especially texture and digestibility. Whether covalent crosslinks in the protein matrix affect gastric emptying time by influencing transport kinetics during the gastric and intestinal phases of digestion has to be studied further. The digestibility of the TG derived isopeptide bond and subsequently the nutritional availability of lysine in the isopeptide bond has also been discussed. Finot et al. (1978) reported that the lysine derivative of the isopeptide was absorbed unchanged in the intestine and is hydrolyzed in vivo. As the isopeptide bond is known to be cleaved by the human enzymes γ-glutamylamine cyclotransferase, and γ-glutamyl transpeptidase, the released lysine could thus be utilized in the body (Seguro et al., 1996). Experiments showed that no significant differences in food intake and overall health were observed among rats fed the intact or TGcrosslinked casein diets, suggesting that the isopeptide moiety in crosslinked caseins is digested. Some reports on the impact of crosslinking enzymes on allerginicity of proteins have also been published. TG has been able to reduce the immunogenic and allergenic properties of soy and wheat proteins (Babiker et al., 1998; Watanabe et al., 1994). Similar results have been reported with peroxidase on roasted peanut proteins (Chung et al., 2004). Recently, TG has been associated with celiac disease and hypotheses about the possibility of TG acting on gliadin proteins in dough with the subsequent generation of the epitope associated with the celiac response have arisen.

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Deamidation and crosslinking of gliadin peptides by TGs and its relation to celiac disease have been studied by Skovbjerg et al. (2004). The authors concluded that intestinal TG is responsible for generation of the active deamidated epitope. There has also been speculation about whether microbial TG creates similar types of products (Gerrard and Sutton, 2005). So far, no experimental data has been produced supporting this assumption.

6.9

Conclusions

Sensory perception of texture plays an important role in different types of foods, for example baking, meat and dairy products. The significance of food texture has further been increased with the trend towards low fat products and ‘natural’ additive-free products. Enzymes provide specific and natural means for tailoring food structure. By the use of enzymes it will be possible to transform inherently available food components into functional ingredients during food processing and manufacturing. Enzymatic crosslinking in the food matrix can occur via proteins or certain carbohydrates. The type of enzyme used affects the chemistry of the crosslink formed and subsequently the structure of biopolymer network of the food product. Among potential enzymes for protein crosslinking are TG and various oxidative enzymes, such as laccase, tyrosinase or peroxidase. These enzymes have different modes of action and thus the chemistry of crosslinking varies. Protein and carbohydrate crosslinking initiated by these enzymes is an efficient process, resulting in the formation of protein–protein, carbohydrate–carbohydrate or even protein–carbohydrate adducts. The course of the reactions is primarily determined by the conformations of the substrate molecules as well as the accessibility of the target amino acid side chains or phenolic groups. With flexible proteins or carbohydrates both intra- and intermolecular modifications are feasible, whereas in the case of globular proteins formation of intermolecular linkages is mainly expected. Commercial applications are currently based on TG as a crosslinking agent. However, interesting research results on crosslinking of food biopolymers with other types of enzymes have also been reported. Thus, it is expected that the number of commercially available crosslinking enzymes for food structure engineering will increase in the future.

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7 Enzymatically modified whey protein and other protein-based fat replacers Jacek Leman, University of Warmia and Mazury in Olsztyn, Poland

7.1

Introduction

Protein-based fat replacers belong to fat mimetics that imitate one or more of the organoleptic and physical functions of fat in food, but do not replace fat on a oneto-one basis (Akoh, 1998). They provide 4.2–16.7 kJ g–1 and are used in macronutrient amounts, usually up to 3% w/w. Protein-based fat replacers retain the biological value of the protein used. Because they are not suitable for use in heated foods, in which they impart grittiness, protein-based fat replacers are satisfactorily used only in products consumed at low temperatures (up to 15 °C). Because of recent concerns about diet and health, protein-based fat replacers have facilitated the development of reduced-fat foods (Akoh, 1998; Giese, 1994, 1996; Glicksman, 1991). Proteins act as a fat mimetic owing to their unique functional properties. A variety of protein sources, including milk, whey, soybean, wheat gluten, gelatin, fish and egg are used to produce protein-based fat replacers (Roller and Jones, 1996). Casein is the principal protein of milk, used in foods in the form of acid casein or its sodium, potassium, calcium and magnesium salts, called caseinates. Whey proteins, with β-lactoglobulin and α-lactalbumin as the major fractions, and soybean proteins, with glycinin and β-conglycinin as the major fractions, are widely used in foods in several forms, the most common of which are protein concentrates and protein isolates. The protein isolates have a higher protein concentration (above 90%) and less impurities, such as lipid, sugar and minerals, than protein concentrates (50–57%). These protein products also differ in their

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degree of denaturation and aggregation owing to different protein sources and the various steps used in production. It is also the protein functional properties that allow a particular protein structure to be produced by a microparticulation process. This process yields small spheroidal protein particles 0.1 to 3 µm in diameter, which are perceived to be creamy and smooth in the mouth, in contrast to smaller particles which are perceived to be watery and particles with larger diameters that are perceived to be powdery (Zunft and Ragotzky, 1997). A microparticulated protein may thus be defined as an engineered protein aggregate able to match the textural and structural properties of emulsified fat (Sanchez et al., 1997). Microparticulated protein-based fat replacers, containing milk and/or egg or whey protein are commercially produced by either heat-induced gelation or aggregation and crosslinking under shear stress of whey proteins (Kulozik et al., 2003; de la Fuente et al., 2002; Vardhanabhuti and Foegeding, 1999; Spiegel, 1999; Miller, 1994). Microparticulated proteins are also produced commercially by enzymatic crosslinking of casein, whey protein, gelatin and fish protein (Novo Nordisk A/S, 1995). In the light of recent developments, the in-mouth microparticulation of gelled emulsion particles can also be considered and this will be discussed in the section on fat replacer applications. The technologies of production of the fat replacers are protected by numerous patents (Nielsen, 1995). Developments in the technology of protein microparticles and their functional properties have been reviewed (Sanchez and Paquin, 1997). The functional properties of proteins are those physicochemical properties of proteins which affect their behaviour in food systems during preparation, processing, storage and consumption, and contribute to the quality and organoleptic attributes of food systems (Kinsella, 1982). The molecular basis of whey protein functionality has been reviewed (Holt, 2000; Bryant and McClements, 1998). The key functional properties of proteins that relate to fat mimicking properties basically include water binding and gelling abilities. As a fat replacer, the protein concentrates and isolates impart creaminess, smoothness, firmness and consistency, acting as gelling, surface active and water binding agents (Giese, 1994). Both the composition and degree of denaturation affect the functionality of these protein products (Fachin and Viotto, 2005; Ye and Singh, 2000; Vaghela and Kilara, 1996; Morr and Ha, 1993). One of the first attempts to employ milk protein concentrate in order to benefit from its functionality was proposed over 30 years ago by a Polish research group led by Professor Poznan´ski (Chojnowski et al., 1975). Since that time, there has been tremendous progress in this area. High pressure- or heatinduced aggregation of whey proteins and their thermal fractionation based on different reactivities of the individual proteins to heat are a means of producing highly functional products (Kulozik et al., 2003). An example of the latter is a whey protein isolate, in which β-lactoglobulin constitutes above 95% of the total protein content, having the ability to modify the gel elasticity (Orlien et al., 2006). Improved functionality of protein concentrates also results from the lipolysis of residual lipids (Ainsworth et al., 2000; Blecker et al., 1997, 2000). All differences in the functional properties of protein concentrates and pure proteins arise from their unique structure, which may be modified to match the

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functionality needed for specific applications (Vojdani and Whitaker, 1994). Although chemical methods may be used to modify proteins, heating and enzymatic modifications are most common for technical and regulatory reasons. Wellcontrolled enzymatic modification of the proteins has an advantage over thermal modification because it is more specific in modifying the protein molecular structure, as reflected in the different rheological properties of enzyme- and heatset protein gels (Doucet et al., 2001; Ju and Kilara, 1998a; Dickinson, 1997). The methods used to tailor the fat mimicking properties of proteins enzymatically are discussed in the following sections.

7.2

Enhancing the fat mimicking properties of proteins

The modification of the proteins to mimic the oral sensation of fat basically needs an improvement in the ability of protein to bind and hold water and to form gels (Giese, 1996; Nielsen, 1995). Emulsifying and foaming properties are also important. A thick interface, in the formation of which the proteins contribute to protein-stabilized emulsions and foams (Leman and Kinsella, 1989), gives a high fat perception (Haque, 1993). The interfacial concentration and composition may be manipulated to produce emulsions with different stabilities and properties (Sharma and Singh, 1998). Recent progress in knowledge of how the interface affects foam and emulsion behaviour has been reviewed by Wilde (2000). The flavour-binding ability of modified proteins deserves substantial consideration since, many of today’s low- and reduced-fat products do not meet the fatty sensation expectations of consumers because of the inability of microparticulated proteins to impart the taste and aroma of fat (Sanchez et al., 1997). The functions performed by protein-based fat replacers in foods are summarized in Table 7.1. Enzymatic modification of a protein for use as a fat replacer is carried out almost exclusively using proteases and transglutaminase. The potential of other enzymes, such as oxidases, isomerases and reductases has not yet been fully explored or recognized. The functional changes induced by enzymes are dependent first of all on enzyme specificity, extent of protein denaturation, protein and enzyme concentrations, pH, ionic strength and temperature (Kunst, 2003; Haertlé and Chobert, 1999). The enzyme-induced changes in the protein affect its physico-chemical Table 7.1

Function of protein-based fat replacers in food

Type of fat replacer

Function

Egg white Gelatin Soybean and whey protein concentrates and isolates Microparticulated proteins

Fat extender Texture and viscosity enhancer, smooth mouthfeel Texture and viscosity enhancer, creaminess, opacity, water/ foam/emulsion stabilizer Texture and viscosity enhancer, creaminess, opacity, clean flavour base, good flavour release Flavour and texture enhancer, mouthfeel, water binding

Protein-gum/starch blends

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properties such as solubility (required for expression of functional properties), surface activity (determining emulsification and foaming) and water binding and viscoelasticity (both of which determine gelation). Advances in modifying whey protein interfacial properties and gelation ability have been reviewed (Foegeding et al., 2002).

7.2.1 Proteolysis In order to modify the functional properties of proteins, a limited hydrolysis is generally carried out using mostly endopeptidases and proteases. Most frequently used enzymes include trypsin, chymotrypsin, pepsin, papain, bromelain, thermolysin and proteolytic preparations of microbial origin, such as Alcalase, Neutrase, pronase and Protamex. Limited hydrolysis is restricted to the cleavage of single or a few peptide bonds, located mostly on the protein molecule surface and thus easily available to an enzyme. The duration of the hydrolysis is relatively short (up to 2 h) at a temperature that is usually lower then the optimum temperature of the enzyme used. There is generally a low degree of hydrolysis, usually a few per cent. The functionality of the resulting proteolysates is dependent on enzyme specificity and generally on the degree of hydrolysis, although other factors such as hydrophobicity, molecular size and amphipathicity of proteolysates are important apart from degree of hydrolysis (Panyam and Kilara, 1996). Improvement in the protein solubilities upon limited hydrolysis is well-documented in the literature and is attributed to a decrease in the molecular weight and an increase in the number of exposed ionizable groups (van der Ven et al., 2001; Hettiarachchy and Kalapathy, 1997; Were et al., 1997; Panyam and Kilara, 1996). Along with the increased solubility, the emulsifying, foaming and gelling abilities of the proteins are modified to a different degree, depending on the hydrophobic– hydrophilic balance that is a derivative of protein and enzyme system (for example type of protein and enzyme, pH, ionic strength, temperature and duration of the process). Strategies that have been undertaken since the mid-1990s to control and tailor these abilities of proteins include the following approaches used alone or in combination: (i) protein pretreatments before hydrolysis by heat or high pressure, (ii) extensive hydrolysis, selective hydrolysis, and in-ethanol hydrolysis, (iii) fractionation of proteolysates to isolate functional peptides and (iv) modelling the protein gel structure. The first two groups of these strategies aim both to increase or decrease the susceptibility of the proteins to hydrolysis by affecting their degree of denaturation. An example might be either 300 MPa-pressurized or ethylated β-lactoglobulin that can readily be hydrolysed by pepsin, unlike the native protein (Stapelfeldt et al., 1996; Chobert et al., 1996). High-pressure-induced denaturation of the protein or proteolysis by various enzymes or their mutants creates opportunities for the formation of hydrolysates with novel peptide profiles, which are expected to have desired functionality (Knudsen et al., 2002; Haertlé and Chobert, 1999; Maynard et al., 1998; Heremans et al., 1997; Messens et al., 1997; Stapel-feldt et al., 1996). Depending on the hydrolysis conditions, tryptic

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hydrolysates of β-lactoglobulin differ in their interfacial, gelation and emulsionstabilizing properties (Caessens et al., 1999c; Agboola et al., 1998; Chen et al., 1994; Turgeon et al., 1992). Hydrolysates with peptide populations that differ in molecular size and functionality allow further fractionation, forming peptides that have enhanced emulsifying, foaming or gelling properties. Such peptides have been identified in β-lactoglobulin tryptic hydrolysate (Huang et al., 1996; Turgeon et al., 1992), plasmin hydrolysate (Caessens et al., 1999c) or Bacillus licheniformis protease hydrolysate (Otte et al., 1997a, 2000a). A combination of tryptic hydrolysis and ultrafiltration allows isolation of the peptide fractions from β-lactoglobulin with better emulsifying and foaming properties (Huang et al., 1996; Mutilangi et al., 1996; Turgeon et al., 1992). Research on nanofiltration in order to obtain a more efficient separation between the hydrolysate peptides is in progress (Pouliot et al., 1999, 2000). When whey protein concentrate and sodium caseinate were hydrolysed with 11 different commercially available enzymes to various degrees of hydrolysis (0.5–24%) to investigate correlations between the biochemical and emulsion properties of the hydrolysates, their emulsion-forming properties were generally independent of molecular weight distribution and degree of hydrolysis, in contrast to the emulsion-stabilizing properties which correlated with the molecular weight distribution (van der Ven et al., 2001). There is, however, much evidence that peptides larger than 2 kDa are beneficial for emulsion activity and stability (Caessens et al., 1999b; Agboola et al., 1998; Huang et al. 1996; Turgeon et al., 1992). According to the latest findings, amphiphilicity of peptides is more important for the interfacial and emulsifying properties of whey protein hydrolysates than molecular size and degree of hydrolysis (van der Ven et al., 2001; Rahali et al., 2000; Caessens et al., 1999a). Smaller peptides with a specific distribution of hydrophobic and hydrophilic amino acids might have enhanced interfacial properties (Luck et al., 2002; Singh and Dalgleish, 1998). The release of such peptides might be protein- and/or enzyme-dependent. The literature on the functional peptides of soybean protein is rather limited compared with that concerning whey protein peptides. Soybean proteins are hydrolysed with less specific proteases, such as pancreatin and papain which improves their emulsifying and foaming properties (Qi et al., 1997; Were et al., 1997). Selective proteolysis of soybean protein by pepsin and papain yields hydrolysates in which either glycinin or β-conglycinin are hydrolysed (Tsumura et al., 2004). Both hydrolysates have improved foaming, but not emulsifying properties, except at acidic pH 4, and differ in viscosity and gel forming ability (Tsumura et al., 2005). The reduced-β-conglycinin hydrolysate has a low viscosity and forms a harder gel than the reduced-glycinin hydrolysate. Selective enzymatic treatment may also be a useful approach to the modification of whey protein functional properties. Peptic hydrolysis of whey protein concentrate yields a hydrolysate with improved surface properties in which only native β-lactoglobulin remains unaffected (Konrad et al., 2005a, 2005b). Interestingly, two different optimum degrees of hydrolysis (1.0–1.5% and 5.8%) for both emulsification and foaming properties were found, thus contributing to contradictory findings about how the degree of hydrolysis affects the protein functionality.

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Proteolysis in hydro-ethanolic solution allows modification of the peptide composition, depending on the ethanol concentration (Chobert et al., 1996), opening up the possibility of forming functional peptides. Peptic hydrolysate of βlactoglobulin in 40% ethanol has better emulsifying properties than the native protein (Rahali et al., 2000), but neither tryptic nor peptic ethanolic proteolysis affects the foaming capacity of this protein (Rahali and Guéguen, 2000). Extensive hydrolysis of β lactoglobulin A by a serine protease from B. licheniformis to degrees ranging from 19 to 86% results in the best improved foamability at the highest degree of hydrolysis (Ipsen et al., 2001a), suggesting that high maximum values for interfacial elasticity and viscosity are not necessary prerequisities for formation of a voluminous and stable foam. Whey protein hydrolysates with a 10 or 20% degree of hydrolysis were also shown to have the maximum emulsifying capacity, compared with less or more extensively hydrolysed proteins (Singh and Dalgleish, 1998). Protein gelation may either be inhibited or promoted by proteolysis (Panyam and Kilara, 1996). Enzyme-induced gelation of proteins is explored for a relatively short time compared with highly advanced studies on heat-induced gelation (Foegeding et al., 2002; Panyam and Kilara, 1996). The mechanism of enzymeinduced gelation is not yet clear apart from the fact that proteins undergo denaturation and aggregation before the network formation (Foegeding et al., 2002; Clark et al., 2001; Doucet et al., 2001; Ipsen et al., 2000; Bryant and McClements, 1998; Panyam and Kilara, 1996). The nature of protein aggregates, network structure, water holding and rheological properties of the protein solution or gel are affected by the protein concentration, composition and denaturation degree as well as by ionic strength, pH and temperature (Doucet et al., 2001; Ipsen et al., 2000; Otte et al., 1999; Ju et al., 1997). Two approaches are used for enzymatic modification of the whey protein gel properties, that is (i) heat-induced gelation of the protein hydrolysates (Otte et al., 2000b, 1996a; Huang et al., 1999; Ju et al., 1995) and (ii) protease-induced gelation of native or heat-denatured whey protein (Ipsen et al., 2000; Otte et al., 2000a, 1999, 1997b, 1996b; Ju et al., 1997; Sato et al., 1995). Limited proteolysis by trypsin, papain, pronase and a protease from B. licheniformis of whey proteins effectively modifies the gelling ability and the physical properties of gels, influencing their structure and water binding properties (Ju et al., 1995; Sato et al., 1995; Chen et al., 1994). Gelling abilities may also be modified by selective or extensive hydrolysis of whey and soybean protein concentrates (Tsumura et al., 2005; Doucet et al., 2001; Stockmann et al., 2000). For example, reduced-β-conglycinin hydrolysate of soybean protein isolate retains more gel-forming ability than reduced-glycinin hydrolysate when mixed with meat protein (Tsumura et al., 2005). On the other hand, extensive hydrolysis (to a degree of 18%) of whey protein isolate with Alcalase 2.4L leads to the formation of strong elastic gels, similar to, but more stable over a wide range of temperature (10–65 °C) than heat-induced gels (Doucet et al., 2001). The serine protease from B. licheniformis with a different specificity to Alcalase 2.4L forms particulate gels at degrees of hydrolysis as low as 1.3% (Otte et al., 1996b).

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B. licheniformis protease-induced gelation has been most thoroughly studied (Ipsen et al., 2000). The minimum concentration required to form a gel of whey protein hydolysed by this protease at neutral pH is much lower (0.5–2.0% w/w) than the minimum concentration for thermal gelation (4–12% w/w). Proteaseinduced gelation is enhanced by increasing protein and enzyme concentrations, and by increasing temperature (Otte et al., 1999). Temperature affects both the mechanism of whey protein gelation and the gel structure (Ipsen et al., 2000; Otte et al., 1999). Lowering the pH in the range of seven to five and increasing the ionic strength up to 500 mM NaCl or 30 mM CaCl2 affects the gelation and the gel structure differently, depending on the degree of protein denaturation (Otte et al., 1999). Peptides with a molecular weight between 2 and 6 kDa, derived from β-lactoglobulin fragment f135–158, initiate aggregation through non-covalent interactions (Otte et al., 2000a, 1999). Whereas the protease-induced gels of β-lactoglobulin have a particulate microstructure consisting of irregular, spherical particles, approximately 100 nm in diameter (Otte et al., 2000b), the proteaseinduced gels of α-lactalbumin have a totally different character and a nanotubular microstructure, which is exceptional for food protein gels (Ipsen et al., 2001b,c). The formation of soluble protein aggregates (0.2–0.5 µm in diameter) from denatured protein is essential for network formation, creating the possibility of separating both processes, thus providing the means of modelling the protein gel properties through developing preaggregated protein products or protein gel powders (Ju and Kilara, 1998). Whey protein gel powder suitable for thickening and forming weak gel over a wide range of temperature and pH values has already been developed while manipulating heating time, pH and mineral type and content (Hudson et al., 2000). An attempt to obtain such products by combining heat treatment and enzymatic hydrolysis of whey protein by the B. licheniformis protease has also been described (Ipsen et al., 2000). Since the protease is heat resistant, varying the temperature for enzyme-induced gelation in the range of 40– 80 °C enables the whey protein gels with different rheological and microstructural properties to be obtained. Gels made at 50 or 60 °C have an open structure with large pores, whereas at higher temperatures a structure composed of smaller aggregates and pores is formed (Ipsen et al., 2000). Another possibility of producing protein aggregates emerges from the use of specific β-lactoglobulin peptides isolated from a tryptic hydrolysate of whey protein isolate (Sanchez et al., 1997). In this approach, whey protein microparticulates can potentially be produced through improved thermal aggregation and isoelectric precipitation of whey protein induced by β-lactoglobulin specific peptides.

7.2.2 Protein crosslinking Enzymatic crosslinking has received increasing attention during the last ten years as the method for both protein modification and food processing. For this purpose, microbial transglutaminase has mostly been used for economical reasons and Ca2+ independent catalytic property of the enzyme (Kuraishi et al., 2001; Motoki and Seguro, 1998; Lorenzen and Schlimme, 1998; Dickinson, 1997; Nielsen,

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1995; Zhu et al., 1995). Studies on oxidative crosslinking using oxidoreductases have only just been initiated (Thalman and Lötzbeyer, 2002; Færgemand et al., 1998a). Transglutaminases (EC 2.3.2.13) are a group of enzymes that catalyse the formation of covalent crosslinks between peptide-bound glutamine and various primary amines, including peptide-bound lysine, the latter generating ε-(γglutamyl)lysine crosslinks. In the absence of amines, transglutaminase catalyses the hydrolysis of the γ-carboxyamide group of the glutamine residue, resulting in deamidation. The potential of transglutaminase for deamidation has been studied mostly to modify wheat proteins. Transglutaminase-deamidated gliadins have improved solubility in the pH 5–9 and improved emulsion stabilizing properties (Chobert et al., 1996). Crosslinking by microbial transglutaminase has been investigated using whey protein, casein, soybean protein, fish protein as well as fractions of these proteins with the aim of improving their hydration, gelling and emulsifying properties, and also viscoelasticity and heat stability. The desired functionality of transglutaminase-modified proteins can be obtained by controlling the amount of enzyme and protein, reaction time, pH and temperature (Motoki and Seguro, 1998; Sakamoto et al., 1994). A direct result of transglutaminase action on proteins is increased viscosity of a protein solution, increased protein aggregation and polymerization, leading eventually to formation of a gel with improved water-holding capacity. The number of covalent crosslinks formed determines the gel properties, such as melting point, strength and elasticity, and can be controlled freely with the amount of transglutaminase, usually being in the range from 10 to 50 units/g protein at 50 °C (Lee et al., 1997; Sakamoto et al., 1994; Nonaka et al., 1992). The protein concentration needed for the enzymeinduced gelation varies from 1 to more than 8%, depending on the protein type, and is lower than that needed for heat-induced gelation. Among proteins, casein is a very good substrate for transglutaminase, whereas globular proteins stabilized with disulphide bonds, such as whey proteins, are poor substrates owing to a limiting number of binding sites or steric inaccessibility. Such proteins become accessible for transglutaminase after unfolding by, for example, reduction of disulphide bonds, increasing the pH or adsorption on an oil– water interface (Færgemand et al., 1997a, 1997b). Besides the protein type, the nature of heat-induced aggregates in protein also influences its gelation and the gel rheology (De la Fuente et al., 2002; Kang et al., 1994). In contrast to linearly linked more-or-less spherical aggregates of β-lactoglobulin held together mostly via disulphide bonds, and thus inaccessible to transglutaminase (De la Fuente et al., 2002), linear strands and branched strands of soybean glycinin soluble aggregates offer the possibility of controlling the rheological properties of the gels from, respectively, elastic hard gels to soft viscous gels (Kang et al., 1994). The viscoelastic and gelling properties of fish gelatin and casein considerably increase after crosslinking with transglutaminase (Gómez-Guillén et al., 2001; Færgemand and Qvist, 1997; Sakamoto et al., 1994). Combination of excessive proteolysis and transglutaminase crosslinking of the chymotryptic, peptic and papain proteolysates greatly improves emulsifying and

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foaming properties of wheat gluten and soy protein (Babiker, 2000; Babiker et al., 1996). The stability of a β-casein-stabilized emulsion is significantly improved after microbial transglutaminase-catalysed polymerization of β-casein (1% w/w protein, 4.25 units/g protein, pH 7.5, 37 °C) (Liu and Damodaran, 1999). Milk proteins (sodium caseinate and β-lactoglobulin) crosslinked with microbial transglutaminase before or after emulsification have improved emulsion stabilizing properties, depending on the degree of crosslinking (Færgemand et al., 1998a). A lower degree of crosslinking improves coalescence, whereas extensive crosslinking impairs the stability. The creaming stability of emulsions stabilized with milk proteins crosslinked after emulsification is improved even with extensive crosslinking owing to increased viscosity of continuous phase and modification of the interfacial film (Færgemand et al., 1997b, 1998b; Færgemand and Murray, 1998). Protein-stabilized emulsions can be gelled by microbial transglutaminase to form emulsion gels (Dickinson and Yamamoto, 1996; Dickinson, 1997; Matsumura et al., 1993). Whey protein-based emulsion gels are composite systems in which the dispersed lipid droplets serve as the filler phase and the proteins constitute the continuous matrix of the composite and, in addition, are adsorbed at the oil–water interface (Chen et al., 2000; Reiffers-Magnani et al., 1999). In protein-stabilized transglutaminase-induced emulsion gel, the covalent crosslinks are formed between the proteins adsorbed, not only on the same but also on different droplets. These covalent crosslinks may enhance the stability and rheology of the protein-stabilized emulsions and foams providing that intra-layer crosslinks and inter-droplet repulsion are properly balanced by adjusting the ionic strength and pH (Dickinson, 1997). The β-lactoglobulin-stabilized emulsion gel induced by microbial transglutaminase has predominantly particle gel character in contrast to a polymer gel character of a pure protein gel (Dickinson and Yamamoto, 1996). A combination of transglutaminase and heat treatment creates a possibility for producing whey protein-stabilized emulsion gels with strain-independent rheological properties over a wide range of deformations (Dickinson and Yamamoto, 1996). The strength of transglutaminase-induced whey protein gel and whey protein emulsion gel can be further modified by the addition of sodium caseinate or skim milk powder (Dickinson and Yamamoto, 1996). The elasticity and breaking strengths of the enzyme-induced gels are greater than those of heat-induced gels produced under similar conditions, reflecting the different nature of chemical forces involved in the gel formation, that is, permanent covalent bonds in transglutaminase-induced gels and breakable or deformable hydrophobic, hydrogen and electrostatic bonds in heat- or protease-induced gels (Dickinson, 1997). The emulsion gels formed by transglutaminase crosslinking offer a great opportunity for developing protein aggregates and microparticulates that combine various functional qualities, such as high water-holding capacity, emulsifying activity and heat stability. Transglutaminase also offers a possibilty for synthesizing the functional ingredients that have a combination of the desired properties or improved functionality, by forming protein hybrid aggregates, utilizing glycoproteins for this process (Dickinson, 1997).

Enzymatically modified whey protein and other protein-based fat replacers

7.3

149

Applications in low-fat foods

Low-fat products, recommended by dieticians for maintenance of health, differ in organoleptic quality from their full-fat counterparts, a consequence of which is poor consumer acceptance of such foods (ADA Reports, 2005; Sandrou and Arvanitoyannis, 2000). The use of fat replacers to reduce the total fat and energy contents of food is seen as having a great potential in the successful manufacture of low-fat products. In low-fat products, the use of fat replacers, however, leads to changes in the food texture that are often perceived by consumers as giving a less attractive mouthfeel (Wilkinson et al., 2000). Many protein-based fat replacers are on the market and their list is changing all the time. A large amount of information on the capabilities and advantages of the protein-based fat replacers comes, however, from the manufacturers’ literature rather than from basic research sources. The latter mostly describe microparticulated products known under the trade name SimplesseTM and controlled heat-denatured products with lesser or greater ability for aggregation known under the trade name Dairy-LoTM, both made from whey protein. To replace fat, a fat replacer should have not only a fat-like mouthfeel, but also a fat-similar functionality in affecting the appearance, taste, aroma and texture of food. Fat in food has multiple functions and its importance varies in different food systems (see Table 7.2). At present, there is no single fat replacer, including protein-based fat replacers, that contributes all of the desired sensory and functional qualities to all products. Protein blended with gums and food starch can be an effective option in some applications. The main criticism against protein-based fat replacers is that they affect the microstructure of food, are poorly melting, heat-unstable and mask or modify the Table 7.2

Application of some protein-based fat replacers

Food category

Function of fat

Type of fat replacer

Meat products

Contributes to juiciness and tenderness; carries flavour; absorbs frying-generated flavours; reduces sharpness of acid compounds Imparts smooth mouthfeel; affects meltability, viscosity, body, crystallinity, spreadability and palatability Inhibits formation of tough gluten strands; softens crumb; imparts tenderness; delays staling Emulsifies fat; stabilizes emulsion; imparts smooth mouthfeel and palatability; affects meltability and viscosity

Microparticulated proteins, soybean isolate, caseinates, protein blends

Dairy products

Baked products

Cooking and salad oils, salad dressings, soups, sauces, gravies

Microparticulated proteins, heat- or enzyme-denatured whey protein concentrates, protein blends Microparticulated proteins, heat- or enzyme-denatured whey protein concentrates, protein blends Microparticulated proteins, protein blends, protein concentrates

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Novel enzyme technology for food applications

flavour owing to irreversible flavour binding and increasing viscosity (Sanchez et al., 1997; Sanchez and Paquin, 1997; Lucca and Tepper, 1994). The production of low-fat products using protein-based fat replacers, which still possess the quality characteristics derived from the functional properties of fats, poses a serious technical challenge. Some of the most successful applications of protein-based fat replacers include dairy products, meat products, baked goods, mayonnaise- and margarine-type products, salad dressings, frozen desserts, soups and sauces (see Table 7. 2). Among those food products, generally less successful applications include cheese, yoghurt, ice-cream and bread, that is, the products in which texture has a tremendous impact on the flavour and overall sensory quality (Kulozik et al., 2003; de Roos, 2003). For cheese, fat contributes to the flavour, texture and meltability. Protein-based fat replacers appear to have limited (microparticulated protein) or no effect (heatdenatured protein) on improving the meltability of low-fat mozzarella cheese (McMahon et al., 1996) or low-fat soft cheeses (Kavas et al., 2004; Zalazar et al., 2002). The size and extent of protein microparticulation and interaction between the protein-based fat replacer and caseins affect the location of the fat replacers in the cheese structure; large particles (above 10 µm) increase the openness of the cheese structure in contrast to small particles (0.5–1.0 µm) embedded within the casein matrix (McMahon et al., 1996). The microstructure of low-fat cheddar cheese is also affected differently by microparticulated and heat-denatured proteins. Microparticulated protein, in contrast to heat-denatured proteins imparts discontinuity to the protein matrix, just as milk fat globules do in full-fat cheese, which results in softer low-fat cheese (Aryana and Haque, 2001). From the literature, it seems that microparticulated proteins fulfill the role of fat replacer better than heat-denatured ones which neither simulate the function of milk fat, despite increasing the water content, nor improve the protein matrix of low-fat cheese (Koca and Metin, 2004; Ma et al., 1997). Some studies suggest, however, that there is greater efficiency of heat-denatured than microparticulated proteins in the production of low-fat yoghurt (Lobatto-Calleros et al., 2004; Sandoval-Castilla et al., 2004; Yazici and Akgun, 2004; Kulozik et al., 2003). Low-fat yoghurt needs reinforcement of the protein network to build up the structure. Although, microparticulated protein becomes an integral part of the yoghurt microstructure (Tamime et al., 1995), the protein particles are larger than milk fat globules and thus ineffective in reinforcing the protein network and in preventing syneresis. In contrast, microparticulated whey protein used to replace half or total fat in an Egyptian set yoghurt produced from buffalo milk effectively reduced syneresis and increased viscosity and organoleptic scores (Kebary and Hussein, 1999). This may suggest that species-dependent milk composition might influence the fat replacer functionality. Low-fat ice-cream, as a complex colloidal system stabilized at both the air– water and oil–water interfaces (Goff, 2002), needs a fat replacer which, in addition to water binding, would combine emulsifying and foaming abilities, imparting viscoelasticity to those interfaces (Stanley et al., 1996). Microparticulated proteins and heat-denatured proteins impart different qualities to low-fat ice-creams, the

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former better mimicking milk fat, particularly in regard to flavour, colour and textural stability, but not thickness and mouthcoating, than the latter (Adapa et al., 2000; Prindiville et al., 2000). An interesting alternative to those protein fat mimetics might be the proteose–peptone whey fraction of bovine milk. This peptide fraction, a glycoprotein in nature, contains 10% w/w or more of peptides (Haque, 1993) and exhibits good emulsifying and foaming capabilities (Innocente et al., 2002). When used to substitute mono- and diglyceride emulsifiers in the preparation of ice cream, this peptide fraction promotes destabilization and coalescence of the fat globules, which is fundamental for the creation of the internal lattice that confers body and stability to the structure of ice cream, yet does not affect adversely the ice cream flavour (Innocente et al., 2002). Non-fat wheat breads containing 2.5–5.0% microparticulated whey protein have unsatisfactory quality characteristics compared with fat-containing breads because of weak gluten networks, resulting in more porous structure, low loaf volume and high crumb hardness (O’Brien et al., 2003). The textural characteristics of low-fat cookies may, however, be improved by microparticulated proteins, depending on the percentage of the fat replacer. Increasing the amount of microparticulated protein by up to 50% of the fat replacement results in more tender cookies with moderately increased brittleness (Zoulias et al., 2002). In meat products, traditionally used fat replacers, such as soybean protein isolate or caseinates, seem to lose their applicability to transglutaminase or transglutaminase-crosslinked proteins, offering tremendous possibilities for lowfat meat product production (Kuraishi et al., 2001; Motoki and Seguro, 1998). This holds good for low-fat yoghurt, cheese and ice-cream manufacture as well (Kuraishi et al., 2001; Faergeman et al., 1999; Lorenzen and Schlimme, 1998; Nielsen, 1995). Nevertheless, high quality functional whey protein concentrates with 80% protein may compete with the enzyme-modified products, as exemplified by palatable low-fat breakfast sausage, in which the whey protein concentrate acts as both a gelling agent and emulsifier (Wang et al., 2006). Texture perception, which actually influences the food acceptance by consumers, despite their being unaware of this (Wilkinson et al., 2000), is a subject of intensive multidisciplinary research (for example Gwartney et al., 2004; Malone et al., 2003a, 2003b; Weel et al., 2002) and is already having a considerable output (Wikinson et al., 2000; de Roos, 2003). Changes in the food microstructure upon use of protein-based fat replacers lead to altered flavour perception. Factors such flavour release, flavour binding and mouthcoating play a role (de Roos 1997, 2003; Fischer and Widder, 1997). In lowfat food, the intensity and release rate of flavour are increased owing to lowered mass transfer resistance and in-mouth flavour release is quicker compared with full-fat food (Leland, 1997). To slow the release of flavour, as demonstrated for lipophilic volatiles, gelled emulsion microparticles have recently been developed, based on mouth physiology and mouth material interactions (Malone et al., 2003a, 2003b; Lian et al., 2004). These microstructured gel particles (70-5000 µm) are heterogenous systems made up of a dispersion of oil droplets in a continuous gel matrix. By a suitable choice of a gelling agent, such as sodium caseinate, gelatin, gum or starch, the particle functionality differs and the release of flavour is

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controlled, depending on the particle size, oil phase volume and oil-water partition coefficient of the flavour (Malone et al., 2003b). Among the gelled emulsion particles, those based on gelatin, characterized by melting behaviour, have a protein concentration-dependent flavour release profile, melting rapidly at low protein content and slowly at high protein content. Gelatin–gum arabic emulsion particles control flavour release through differences in the gelatin bloom strengthdependent melting temperature and the particle sizes. Gelled emulsion particles slow the release of flavour independently of mouthfeel, not altering the food texture significantly (Malone et al., 2003a, 2003b). Whether such gelled emulsion particles will be able to slow food-contained flavour release, creating a chance to develop protein-based fat replacers of the second generation, or will only be a fat replacer adjuvant in some applications is in question. Previous solutions to the problem of flavour perception, mouthcoating and mouthfeel, such as flavour emulsification with lipid-like materials or incorporation of polysaccharides, slowing the flavour release much as fat does, appear hardly satisfactory and mostly applicable to low-fat formulations (Lucca and Tepper, 1994).

7.4

Future trends

The continuing trend towards a low-fat diet and recent advances in understanding of whey protein functionality will certainly promote further multidisciplinary research on protein-based fat replacers. Using enzymes to modify the protein functionality has been proved to be effective. Fundamental research is however needed regarding, first of all, the relation between process conditions and the hydrolysate properties and then the suitability of enzymes other than proteases for protein structure modification. Proteases with narrower specificities, that enable specific cleavage of the protein into a few large fragments are needed since they will allow the relation between the structure and functionality of proteins to be recognized better, and help to identify the functional peptides responsible for protein functionality. Specific peptides in whey protein hydrolysate able to produce functional protein aggregates and to modify whey protein thermal aggregation exemplify yet undiscovered possibilities for protein enzymatic modification. Another such spectacular example is protein gel-encapsulated oil droplet particles, able to instigate in-mouth microparticulation owing to their high meltability. Peptide aggregates with improved heat stability or oil-holding capacity so far have been virtually unexplored but may be enzymatically formed by amphipathic milk protein peptides. The employment of lipophilized peptides and glycoproteins also seems a promising area of future research for functional protein microstructures and aggregates. The functionality of protein fat mimetics has been examined mostly from the point of view of structure and its influence on sensory properties. Food system-dependent functionality or interactions of different microparticulated products with the food components have not been methodically studied and such knowledge is lacking.

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Damodaran S, Paraf A, Food Proteins and their Applications, Marcel Dekker, New York, Bassel, Hong Kong, 503–528. Sanchez C, Pouliot M, Gauthier S F and Paquin P (1997). ‘Thermal aggregation of whey protein isolate containing microparticulates or hydrolyzed whey proteins’, J Agric Food Chem, 45, 2384–2392. Sandoval-Castilla O, Lobato-Calleros C, Aguirre-Mandujano E and Vernon-Carter E J (2004). ‘Microstructure and texture of yogurt as influenced by fat replacers’, Int Dairy J, 14, 151–159. Sandrou D K and Arvanitoyannis I S (2000). ‘Low-fat/calorie foods: current state and perspectives’, Crit Rev Food Sci Nutr, 40(5), 427–447. Sato K, Nakamura M, Nishiya T, Kawanari M and Nakajima I (1995). ‘Preparation of a gel of partially heat-denatured whey protein by proteolytic digestion’, Milchwissenschaft, 50(7), 389–392. Sharma R and Singh H (1998). ‘Adsorption behaviour of commercial milk protein and milk powder products in low-fat emulsions’, Milchwissenschaft, 53(7), 373–377. Singh A M and Dalgleish D G (1998). ‘The emulsifying properties of hydrolyzates of whey proteins’, J Dairy Sci, 81, 918–924. Spiegel T (1999). ‘Whey protein aggregation under shear conditions – effects of lactose and heating temperature on aggregate size and structure’, Int J Food Sci Technol, 34, 523– 531. Stanley D W, Goff H D and Smith A K (1996). ‘Texture-structure relationship in foamed dairy emulsions’, Food Res Int, 29(1), 1–13. Stapelfeldt H, Petersen P H, Kristiansen K R, Qvist K B and Skibsted L H (1996). ‘Effect of high hydrostatic pressure on the enzymic hydrolysis of β-lactoglobulin B by trypsin, thermolysin and pepsin’, J Dairy Res, 63, 111–118. Stockmann R, Fielding J M and Smithers G W (2000). ‘Enzymatic hydrolysis of βlactoglobulin’, Aust J Dairy Technol, 55, 83. Tamime A Y, Kalab M, Muir D D and Barrantes E (1995). ‘The microstructure of set-style, natural yogurt made by substituting microparticulate whey protein for milk fat’, J Soc Dairy Technol, 48(4), 107–111. Thalmann C R and Lõtzbeyer T (2002). ‘Enzymatic cross-linking of proteins with tyrosinase’, Eur Food Res Technol, 214, 276–281. Tsumura K, Saito T, Kugimya W and Inouye K (2004). ‘Selective proteolysis of the glycinin and β-conglycinin fractions in a soy protein isolate by pepsin and papain with controlled pH and temperature’, J Food Sci, 69(5), 363–367. Tsumura K, Saito T, Tsuge K, Ashida H, Kugimiya W and Inouye K (2005). ‘Functional properties of soy protein hydrolysates obtained by selective proteolysis’, LebensmittelWissenschaft und Technologie, 38, 255–261. Turgeon S L, Gauthier S F, Mollé D and Léonil J (1992). ‘Interfacial properties of tryptic peptides of β-lactoglobulin’, J Agric Food Chem, 40, 669–675. Vaghela M N and Kilara A (1996). ‘Foaming and emulsifying properties of whey protein concentrates as affected by lipid composition’, J Food Sci, 61, 275–280. Van der Ven C, Gruppen H, de Bont D B A and Voragen A G J (2001). ‘Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics’, J Agric Food Chem, 49, 5005–5012. Vardhanabhuti B and Foegeding E A (1999). ‘Rheological properties and characterization of polymerized whey protein isolates’, J Agric Food Chem, 47, 3649–3655. Vojdani F and Whitaker J R (1994). ‘Chemical and enzymatic modification of proteins for improved functionality’, in Hettiarachchy N S and Ziegler G S, Protein Functionality in Food Systems, Marcel Dekker, New York, 261–310. Wang S-T, Fligner K and Mangino M E (2006). ‘Formulation of a palatable low-fat sausage using whey protein concentrate’, Milchwissenschaft, 61(1), 79–83. Weel K G C, Boelrijk A E M, Alting A C, van Mil P J J M, Burger J J, Gruppen H, Voragen A G J and Smit G (2002). ‘Flavor release and perception of flavored whey protein gels:

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perception is determined by texture rather than by release’, J Agric Food Chem, 50(18), 5149–5155. Were L, Hettiarachchy N S and Kalapathy U (1997). ‘Modified soy proteins with improved foaming and water hydration properties’, J Food Sci, 62(4), 821–823, 850. Wilde P J (2000). ‘Interfaces: their role in foam and emulsion behaviour’, Curr Opinion Coll Interface Sci, 5, 176–181. Wilkinson C, Dijksterhuis G B and Minekus M (2000). ‘From food structure to texture’, Trends Food Sci Technol, 11, 442–450. Yazici F and Akgun A (2004). ‘Effect of some protein based fat replacers on physical, chemical, textural, and sensory properties of strained yoghurt’, J Food Eng, 62(3), 245– 254. Ye A and Singh H (2000). ‘Influence of calcium chloride on the properties of emulsions stabilized by whey protein concentrate’, Food Hydrocolloids, 14, 337–346. Zalazar C A, Zalazar C S, Bernal S, Bertola N, Bevilacqua A and Zaritzky N (2002). ‘Effect of moisture level and fat replacer on physicochemical, rheological and sensory properties of low fat soft cheeses’, Int Dairy J, 12, 45–50. Zhu Y, Rinzema A, Tramper J and Bol J (1995). ‘Microbial transglutaminase – a review of its production and application in food processing’, Appl Microbiol Biotechnol, 44, 277– 282. Zoulias E I, Oreopoulou V and Tzia C (2002). ‘Textural properties of low-fat cookies containing carbohydrate- or protein-based fat replacers’, J Food Eng, 55, 337–342. Zunft H-J F and Ragotzky K (1997). ‘Strategies for substitution of dietary fat’, Fett/Lipids, 6, 204–213 (in German).

8 Enzymatic production of bioactive peptides from milk and whey proteins Paola A. Ortiz-Chao and Paula Jauregi, University of Reading, UK

8.1

Introduction

Much research worldwide is devoted to the isolation and identification of peptides from food proteins and, with the use of novel high-throughput proteomic techniques, an increasing number of peptides with a wider range of functionalities are likely to emerge. Most of these are being tested in in vitro studies and several studies support their bioactivity in vivo. However research on technologies that would enable the production of these bioactives is not developing at the same pace. Therefore, there is a clear need to develop innovative technologies that will allow the delivery of these functionalities. The aim of this chapter is to review current developments in enzyme technology and enzymatic processes for the production of bioactive peptides from milk proteins. Particular emphasis will be given to current developments in enzyme reactors and enzymatic processes with a view to identifying the main challenges and scope for future development in their scale-up and industrial application. Special attention will be given to the production of peptides from whey proteins; however most of these processes are also applicable to other food proteins. In recent years, much scientific interest has been focused on the study of the two major groups of proteins in milk – caseins and whey proteins – mainly because of the biological and physiological in vitro and in vivo effects that they have shown to exert in organisms. These properties are attributed to peptides encrypted in their primary amino acid sequence which can be released by enzymatic digestion or milk fermentation (Korhonen, 2002; Korhonen and Pihlanto, 2003) and are known

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Table 8.1 Summary of bioactive peptides derived from milk proteins. (FitzGerald and Meisel, 2003) Bioactive peptide

Protein precursor

Bioactivity

Casomorphins α-Lactorphin β-Lactorphin Lactoferroxins Casoxins Casokinins Lactokinins

αs1- and β-Caseins α-Lactalbumin β-Lactoglobulin Lactoferrin κ-Casein αs1- and β-Caseins α-Lactalbumin, β-lactoglobulin and serum albumin αs1-, β- and κ-Caseins Lactoferrin αs2-Casein αs1-Casein κ-Casein αs1-, αs2-, β- and κ-Caseins

Opioid agonist Opioid agonist Opioid agonist Opioid antagonist Opioid antagonist ACE-inhibitory ACE-inhibitory

Immunopeptides Lactoferricin Casocidicin Isradicin Casoplatelins Phosphopeptides

Immunomodulatory Antimicrobial Antimicrobial Antimicrobial Antithrombotic Mineral binding

as bioactive peptides. Although these peptides have been found in different animal and plant proteins, bovine milk proteins are, to date, the main source of foodderived bioactive peptides (Meisel, 2001). In general, bioactive peptides contain 2–20 amino acids per molecule and have been defined as specific protein fragments that have a positive impact on body function or condition and may ultimately influence health (Kitts and Weiler, 2003). These peptides have been produced using four different strategies (FitzGerald and Meisel, 2003; Meisel, 2001):

• in vitro digestion of milk proteins with both pure and crude proteinase and exopeptidase preparations

• in vivo digestion of milk proteins using gastrointestinal proteinases or peptidases • by the action of bacterial proteinase and peptidase activities during the generation of fermented milk products

• chemical synthesis of known bioactive peptides. Among the different functionalities found in casein and whey protein-derived peptides are opioid, antimicrobial, immunoregulatory, antihypertensive, antioxidant and antithrombotic effects (Clare and Swaisgood, 2000; Hernández-Ledesma et al., 2005; FitzGerald and Meisel, 2003). Table 8.1 gives some examples of peptides that have been found in milk proteins and their bioactivity.

8.2

Angiotensin I-converting enzyme inhibitory peptides

One of the most widely studied properties of bioactive peptides derived from milk proteins is their antihypertensive activity mediated by their ability to inhibit angiotensin I-converting enzyme. The angiotensin I-converting enzyme (ACE, peptidyldipeptide hydrolase, EC

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Fig. 8.1

Role of angiotensin I-converting enzyme in the renin–angiotensin, kallikrein– kinin and the immune systems. Adapted from Pihlanto-Leppälä (2001).

3.4.15.1) is a zinc- and chloride-dependent metallopeptidase (Sturrock et al., 2004). It is widely distributed in the body. It has been found in lungs, kidneys, blood, testes and other tissues; pulmonary arteries contain more ACE that any other vessels where it might have an important role in controlling the systemic blood pressure. Chloride appears to be an allosteric modifier of the enzyme (Erdös, 1975). It has been associated with the renin–angiotensin system, which regulates peripheral blood pressure and with the kallikrein–kinin and the immune systems (Fig. 8.1). ACE raises blood pressure by converting the decapeptide angiotensin I, released from angiotensinogen by renin, into the octapeptide and potent vasoconstrictor angiotensin II. The ACE cleaves a histidyl–leucine dipeptide from the C-terminal end of the angiotensin I (Erdös, 1975); this activation also stimulates the production of aldosterone, which regulates fluid and electrolyte homeostasis (Sturrock et al., 2004). This enzyme also degrades the vasodilatory agent bradykinin, releasing the C-terminal phenylalanylarginine (Petrillo and Ondetti, 1982). Besides its vasodilatory properties, bradykinin has cardioprotective properties promoting the formation of protective nitric oxide by the endothelium. Therefore, ACE-inhibitors derived from milk and whey proteins could exert an antihypertensive effect in the organism. Antihypertensive peptides have been isolated from αs1-casein, β-casein and κcasein and they have also been found in the whey proteins α-lactalbumin, β-lactoglobulin and bovine serum albumin (Abubakar et al., 1998; Chiba and Yoshikawa, 1991; FitzGerald et al., 2004; Gómez-Ruiz, et al., 2002; HernándezLedesma et al., 2002; Mullally et al., 1996; Pihlanto-Leppälä et al., 1998; Pihlanto-Leppäla et al., 2000). β-Lactoglobulin (β-Lg), the major protein in whey, has been shown to be a good precursor of ACE inhibitory peptides. These peptides have been mainly produced using pancreatic enzymes – pepsin, trypsin, elastase and chymotrypsin – however, enzymes from other bacterial and fungal sources have been used, such as

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163

thermolysin and proteinase K. Some of the peptides found have also been chemically synthesised. Table 8.2 shows a summary of the identified ACE inhibitory peptides from β-Lg hydrolysates. ACE inhibitory activity is measured in terms of IC50 which is defined as the concentration of peptide required to reduce the ACE inhibitory activity by 50%.

8.2.1 Structural implications of bioactive peptides on ACE inhibition Although some x-ray crystallography studies of the ACE have started to clarify the mechanism of action and three-dimensional design of this enzyme (Spyroulias et al., 2004; Sturrock et al., 2004), the structure–activity relationship of ACE inhibitors is not completely understood and peptides with a wide range of sequences have been produced from milk and many other food proteins. Several studies have been carried out with different ACE peptide substrates and inhibitors in order to elucidate which peptides would be stronger inhibitors and also to correlate their findings to a model structure. ACE displays both exopeptidase and endopeptidase activity and acts on a diverse range of substrates. The binding of the substrate to the enzyme is strongly influenced by the C-terminal tripeptide sequence of the substrate; these three amino acids have been shown to have a fundamental role in competitive binding to the active site of ACE (Cheung et al., 1980; Cushman and Cheung, 1971; Petrillo and Ondetti, 1982). The general structure of ACE substrates is R1-R2-R3-OH. The enzyme cleaves R1 from R2, where R1 can be a protected amino acid or peptide. Erdös (1975) reported that R3 should be an amino acid with a free carboxyl terminal, except glutamic acid, and that peptides that have proline in the R2 position are not cleaved. This is the reason why angiotensin II is not further hydrolysed by the ACE. These findings were later completed by Cheung et al. (1980), who also reported that enzyme binds weakly to substrates with terminal dicarboxylic amino acids (Glu, Asp) together with Pro in the R2 position, but that an antepenultimate aromatic amino acid enhances binding. ACE prefers substrates or competitive inhibitors that mainly have hydrophobic (aromatic or branched-chain aliphatic) amino acid residues at the three C-terminal positions (Cheung et al., 1980). The terminal sequence Trp-Ala-Pro is the most favourable (Cushman and Cheung, 1971). After testing different dipeptide substrates, Cheung et al. (1980) found that the most favourable COOH terminal amino acids are the aromatic acids, tryptophan, tyrosine and phenylalanine and the imino acid, proline. On the other hand, the most favourable NH2 terminal amino acids are the branched-chain aliphatic amino acids, valine and isoleucine; however, they pointed out that the relative contribution of the NH2 terminal amino acid of dipeptide inhibitors might not exactly reflect its behaviour on the penultimate position of a larger peptide. It has also been suggested that the mechanism of ACE inhibition between the inhibitor and the enzyme might depend on an anionic binding site different to the catalytic site, as a C-terminal lysine or arginine with a positive ε-amino group, seems to contribute as well to the inhibitory potency (Vermeirssen et al., 2004).

β-Lactoglobulin derived angiotension I-converting enzyme inhibitory peptides Sequencea

Treatment or origin

f(9-14) f(15–19) f(15–20) f(22–25) f(32–40) f(46–53)c f(58–61)c f(78–80)f f(81–83) f(94–100) f(102–103) f(102–105) f(103–105)c f(104–105) f(106–111) f(122–125)c f(142–146) f(142–148) f(143–148) f(146–148) f(146–149) f(147–148) f(148–149) Captopril

GLDIKQ VAGTW VAGTWY LAMA LDAQSAPLR LKPTPEGN LQKW IPA VFK VLDTDYK YL YLLF LLF LF CMENSA LVRT ALPMH ALPMHIR LPMHIR HIR HIRL IR RL C9H15NO3Se

Whey with fermentation then pepsin and trypsin Pepsin then trypsin and chymotrypsin Whey with fermentation then pepsin and trypsin Trypsin Thermolysin Whey with proteinase K Trypsin Pepsin, then trypsin and chymotrypsin Synthetic Synthetic Thermolysin Synthetic Pepsin, then trypsin and chymotrypsin Thermolysin Pepsin, then trypsin and chymotrypsin Trypsin/Synthetic Pepsin, then trypsin and chymotrypsin Synthetic Synthetic Synthetic Synthetic Synthetic drug

IC50 (µg ml–1)b 390 1054 1170 430d 616 >2309 19.9 42 404 807 35.9 95.3 31.2 97.2 515 1204 296 35.7 nd 404.8 620.1 199.9 700.9 0.0013

Reference Pihlanto-Leppälä et al. (1998) Pihlanto-Leppälä et al. (2000) Pihlanto-Leppälä et al. (1998) Pihlanto-Leppälä et al. (2000) Hernández-Ledesma et al. (2002) Abubakar et al. (1998) Pihlanto-Leppälä et al. (2000) Mullally et al. (1996) Hernández-Ledesma et al. (2002) Mullally et al. (1996) Pihlanto-Leppälä et al. (2000) Hernández-Ledesma et al. (2002) Pihlanto-Leppälä et al. (2000) Mullally et al. (1997b) Pihlanto-Leppälä et al. (2000) Mullally et al. (1997b) Mullally et al. (1996) Mullally et al. (1996)

a One letter amino acid code used; bConcentration of peptide needed to inhibit 50% of the ACE activity; cPeptides identified in caprine β-lactoglobulin; dValue obtained with synthetic peptide; eCondensed formula of Captopril, designated chemically as 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline; fStrong antihypertensive activity shown in SHR rats (–31 mm Hg); nd = not determined.

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Peptide fragment/ analogue

164

Table 8.2

Enzymatic production of bioactive peptides from milk and whey proteins

Fig. 8.2

165

Structure of Captopril (MW = 217.32), a potent synthetic drug applied for the treatment of hypertension.

8.2.2

Milk-derived ACE inhibitory peptides as an alternative to synthetic drugs in hypertension treatment Captopril, 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, Fig. 8.2, a mercaptoacyl amino acid, was the first non-peptide inhibitor potent enough to be clinically useful (Petrillo and Ondetti, 1982) and, to date, is the most widely used synthetic drug in the treatment of hypertension, together with some other synthetic drugs like enalapril, alecepril and lisinopril. An IC50 of 0.0013 µg ml–1 (0.006 µM) is reported for Captopril using hippuryl–histidyl–leucine as substrate (Table 8.2, Mullally et al., 1996), which makes it considerably more potent than any of the most potent milk-derived ACE inhibitory peptides reported so far. For instance, the αs1-caseinderived peptide f(25–27) and αs2-casein-derived peptide f(174–179) have IC50 values of 2.0 and 4.3 µM, respectively (FitzGerald et al., 2004), that is they are between 300–700 times less potent than the synthetic drug Captopril. Although several of these chemically produced drugs have a proven and more potent antihypertensive effect, they can have harmful side effects such as dry cough and alterations in serum lipid metabolism (FitzGerald and Meisel, 2000). The use of milk-derived antihypertensive peptides, on the other hand, does not cause all these side effects as they have their origin in a fundamental part of the diet. Milk-derived bioactive peptides could also be a more economical option for health care and even when they are not intended to replace synthetic drugs completely in hypertension treatment, they can be used as a complementary treatment for this condition in combination with lifestyle modifications, such as weight reduction, exercise and a controlled diet, which have been reported to be some of the most important tools for effective reduction of blood pressure (Hermansen, 2000).

8.3

Other bioactive peptides and their health benefits

A brief description of some other bioactivities found in casein and whey proteinderived peptides will be given below. Table 8.3 shows some examples of these peptides with some additional information about their protein precursors and origin.

166

Table 8.3

Other bioactivities found in casein and whey protein-derived peptides Fragment

Sequence

Name

αs1-CN

f(90–96) f(158–164)

RYLGGYLE YVPFPPF

α-CN exorphin Casoxin D

Loukas et al. (1983) Yoshikawa et al. (1994)

f(59–79) 4P QMEAES*IS*S*S*

Caseinophosphopeptide Trypsin

Mineral binding

f(66–74) 3P S*S*S*EEIVPN

Caseinophosphopeptide Jejunum (minipigs) Trypsin αs1-Immunocasokinin β-Casomorphin-11 Jejunum (minipigs) β-Casomorphin-7 Pepsin

Mineral binding

Meisel and Olieman (1998) Schlimme and Meisel (1995) Meisel and Frister (1988)

β-CN

Treatment or origin

Bioactivity

Pepsin Opioid Pepsin and Opioid (antagonist) chymotrypsin f(43–58) 2P DIGS*ES*TEDQAMEDIM Caseinophosphopeptide Trypsin Mineral binding

f(194–199) f(60–70)

TTMPLW YPFPGPIPNSL

f(60–66)

YPFPGPI

f(60–64) f(7–18) 3P

YPFPG NVPGEIVES*LS*S*

f(29–41) 1P KIEKFQS*EEQQQT f(1–25) 4P f(63–68)

RELEELNVPGEIVES*L S*S*S*EESITR PGPIPN

f(191–193)

LLY

β-Casomorphin-5 Caseinophosphopeptide Duodenum (human) Caseinophosphopeptide Stomach (human) Caseinophosphopeptide Trypsin Immunopeptide

Synthetic

Immunopeptide

Synthetic

Reference

Immunomodulatory Maruyama et al. (1987) Opioid Meisel (1986) Opioid, immunomodulatory Opioid Mineral binding

Brantl et al. (1981), Kayser and Meisel (1996) Meisel (1997) Chabance et al. (1998)

Mineral binding

Chabance et al. (1998)

Mineral binding, Hata et al. (1998, 1999) immunomodulatory Immunomodulatory Migliore-Samour et al. (1989) Immunomodulatory

Novel enzyme technology for food applications

Protein

YQQPVLGPVR

β-Casokinin

Synthetic

κ-CN

f(25–34) f(33–38) f(38–39)

YIPIQYVLSR SRYPSY(OCH3) YG

Casoxin C Casoxin 6 Unnamed

Trypsin Pepsin Synthetic

α-La

f(50–53)

YGLF

α-Lactorphin

Synthetic

f(50–51), f(18–19)

YG

Unnamed

Synthetic

f(18–20) f(102–105)

YGG YLLF

Immunopeptide β-Lactorphin

Synthetic Synthetic

YGFQNA

Serorphin

Pepsin

YLGSGY(OCH3) FKCRRWQWRMKKLG APSITCVRRAF

Lactoferroxin A Lactoferricin

Pepsin Pepsin

β-Lg

Serum f(399–404) albumin Lactoferrin f(318–323) f(17–41)

Immunomodulatory Kayser and Meisel (1996), Meisel and Schlimme (1994) Opioid (antagonist) Chiba et al. (1989) Opioid (antagonist) Chiba et al. (1989) Immunomodulatory Kayser and Meisel (1996), Mullally et al. (1996) Opioid Chiba and Yoshikawa (1986), Mullally et al. (1996) Immunomodulatory Kayser and Meisel (1996), Mullally et al. (1996) Immunomodulatory Kayser and Meisel (1996) Opioid Chiba and Yoshikawa (1986), Mullally et al. (1996) Opioid Tani et al. (1994) Opioid Yamamoto et al. (1994) Immunomodulatory Bellamy et al. (1992), Shinoda et al. (1996)

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f(193–202)

167

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8.3.1 Opioid peptides Typical opioid peptides are derived from enkephalins, endorphins and dynorphins, which have an affinity for an opiate receptor ligand and an opiate-like effect, inhibited by naloxone (Pihlanto-Leppälä, 2001). The receptors, found principally in the central nervous system and gastrointestinal tract, could be of different types: µ receptors participate in the control of intestinal motility and emotional behaviour, δ receptors control emotional behaviour and κ receptors are related to analgesia and satiety (FitzGerald and Meisel, 2003). Their structure always exhibits a definite N-terminal Y-G-G-F sequence (Clare and Swaisgood, 2000). Milk-derived opioid peptides, however, are known as atypical opioid peptides as their primary structure is different from that typical of opioid peptides, sharing a common sequence: Y-X1-F, Y-X1-X2-F or Y-X1-X2-Y. The N-terminal tyrosine residue is essential for their functionality; an aromatic amino acid, such as phenylalanine or tyrosine, is present in the second, third or fourth position in most of the cases (Clare and Swaisgood, 2000). They have been shown to exert opioid activities in both receptor and tissue culture (Meisel, 2001). Exogenous caseinderived opioid peptides are known as casomorphins and casoxins and whey protein-derived opioid peptides are called lactorphins. β-Casomorphins are fragments of β-casein and have been characterized as µ-selective ligands (Teschemacher et al., 1994), whereas α-casein-derived exorphins are δ-selective receptor ligands derived from αs1-casein (PihlantoLeppälä et al., 1994). β-Lactorphin and α-lactorphin have been found in β-lactoglobulin and α-lactalbumin sequences and are µ-opioid receptor agonists (Chiba and Yoshikawa, 1986). Opioid antagonists found in bovine κ-casein are known as casoxins; these are opioid µ-type receptor ligands, which were found to be more active after the Cterminal was methoxylated.

8.3.2 Antimicrobial peptides It is well known that milk has an inherent antimicrobial effect caused by immunoglobulins and other defence proteins like lactoferrin and lysozyme. However, the total antibacterial effect in milk cannot be explained as the sum of the individual contribution of each of these proteins. The most important contribution comes from the generation of antimicrobial peptide sequences from inactive precursor proteins (Clare and Swaisgood, 2000). Lactoferricin is an antimicrobial peptide originating from lactoferrin, an ironbinding glycoprotein found in milk and other mammal fluids. Lactoferricin displays antimicrobial effects against several Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi. Its antimicrobial activity seems to be correlated to its net positive charge, which causes a loss in cell membrane permeability that kills sensitive microorganisms (Bellamy et al., 1992). Lactoferricin has shown antibacterial activity against enterotoxigenic Escherichia coli and Listeria monocytogenes (Dionysius and Milne, 1998) and also against clinical isolates of E. coli O157:H7 (Shin et al., 1998).

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Casocidin-I is a cationic αs2-casein-derived peptide that inhibits the growth of E. coli and Staphylococcus carnosus (Zucht et al., 1995). Isracidin is an N-terminal segment of αs1-casein B that has been shown to protect mice against S. aureus and Candida albicans (Lahov and Regelson, 1996).

8.3.3 Antioxidant peptides The antioxidant properties of milk and milk-derived peptides have received less attention in comparison with other peptide-related bioactivities. Suetsuna et al. (2000) reported a casein-derived pectic hydrolysate, α1-CN f(144–149), which shows free radical scavenging activity and a potent super oxide anion activity. More recently, four β-casein-derived peptide sequences were found to possess antioxidant activity. These peptides inhibited enzymatic and non-enzymatic lipid peroxidation and were identified by electrospray ionisation mass spectrometry (ESI-MS) analysis as β-casein f(98–105), f(177–183), f(169–176) and f(170–176), which correspond mainly to the C-terminal moiety of this protein. In a recent study, Hernández-Ledesma et al. (2005) found that β-lactoglobulin and α-lactalbumin possess antioxidant properties. The β-lactoglobulin-derived peptide f(19-29) with the sequence Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-AspIle showed stronger antioxidant activity than BHA, a chemically synthesised food antioxidant. This activity was measured using the oxygen radical absorbance capacity (ORAC) method which measures the scavenging activity of a compound against peroxyl radicals.

8.3.4 Immunomodulatory peptides Most of the immunomodulatory peptides are hydrolysates derived from the major milk proteins (for a review, see Gill et al., 2000). Casein-derived immunopeptides from αs1-casein and β-casein, αs1-CN f(194–199) and β-CN f(63–68), f(191–193) and f(193–202), were shown to stimulate phagocytosis in sheep red blood cells by murine peritoneal macrophages in vitro and in human macrophages and to produce a protective effect against Klebsiella pneumoniae infection in mice after intravenous administration of peptides (Maruyama et al., 1987; Meisel and Schlimme, 1994; Migliore-Samour, et al., 1989). The C-terminal β-CN f(193–209) induced a significant proliferative response in rat lymphocytes (Coste et al., 1992). Kayser and Meisel (1996) showed that the residue Tyr-Gly [α-La f(18–19) and f(50–51) and κ-CN f(38–39)] as well as Tyr-Gly-Gly [α-La f(18–20)] could significantly enhance the proliferation of human peripheral blood lymphocytes in vitro. Recent studies have focused on caseinophosphopeptides with immunostimulatory activity. Hata et al. (1998) found that phosphopeptides from αs1-CN and β-CN had mitogenic activity and enhanced immunoglobulin production in mouse spleen cells. Lactoferricin B, obtained by hydrolysis of lactoferrin with pepsin, has been found to stimulate phagocytosis of human neutrophils (Miyauchi et al., 1998).

170

8.4

Novel enzyme technology for food applications

Production of bioactive peptides from milk and whey proteins

The production of bioactive peptides derived from caseins is more developed than that from whey proteins, as the former were the first bioactive peptides identified in milk and are produced naturally in fermented dairy products such as cheese and yoghurt. Caseins are an important source of a wide variety of potent bioactive peptides, such as those with antihypertensive activity and mineral binding properties. Therefore several processes have been developed for the enrichment and/or production of these peptides and some of them have resulted in commercialised products (see Section 8.5). This section will focus mainly on the enzymatic production of whey protein-derived bioactive peptides, as an increasing number of studies demonstrate their in vitro bioactivities in relation to a wide range of biological functions and a limited number of processes have been developed.

8.4.1 Proteolytic enzymes The production of bioactive peptides from milk and whey proteins has been carried out using a wide range of proteases from different sources. Digestive enzymes produced from animal tissues, such as porcine or bovine, are by far the most commonly used for the hydrolysis of whey proteins and production of bioactive peptides such as, trypsin, pepsin, chymotrypsin, elastase and kallikrein (Table 8.2). These are enzymes found in the gastrointestinal tract whose function is to break down dietary protein molecules into their component peptides and amino acids. Pepsin (aspartic endopeptidase, E.C. 3.4.23.1) is the principal acid protease in the stomach and possesses a broad specificity. It is synthesised in the gastric mucosa and secreted into the stomach as a zymogen called pepsinogen, which is stable in neutral and alkaline conditions and is converted into pepsin when it reaches the acidic conditions of the stomach (Tang, 1998). Pig pepsin is commercially available from Fluka, Sigma and Boehringer. Trypsin (serine endopeptidase, EC 3.4.21.4) is one of several digestive enzymes secreted into the intestine of animals, where a slightly alkaline environment (around pH 8, varying within species) with moderate amounts of CaCl2 (20 mM) promotes its maximal activity and stability. It is found in all animals, including fish, insects and mammals. In bovine pancreatic secretions, it represents approximately 15% of the digestive enzymes. Trypsin is synthesised by the pancreas as a preproenzyme and then stored as the proenzyme trypsinogen in secretory granules. Once activated, the enzyme is responsible for the activation of the proenzymes of all digestive enzymes, such as chymotrypsin, and contributes to the digestion of consumed protein (Halfon and Craik, 1998). This enzyme cleaves peptide bonds in protein molecules that have carboxyl groups donated by arginine or lysine, that is, amino acids with basic side chains. Cattle pancreatic trypsin is commercially available from Sigma, Boehringer, Manheim, Worthington and Fluka. However it is important to note that trypsin from commercial sources is generally contaminated with other pancreatic enzymes such as chymotrypsin.

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Chymotrypsin (serine endopeptidase, EC 3.4.21.1) is the second major proteinase component of the pancreatic juice. It is produced as a zymogen, chymotrypsinogen, and acts upon peptide bonds formed by the carboxyl groups of the amino acids tyrosine, phenalanine, tryptophan, methionine and leucine. Chymotrypsin hydrolyses, preferentially, peptide bonds adjacent to the carbonyl groups of aromatic amino acids (tyrosine, phenylalanine and tryptophan) together with leucine and, secondly, peptides having asparagine, glutamine, glycine, cysteine, histidine, isoleucine, lysine, serine, valine or threonine residues in the P1 position of substrates (third aminoacid from the amino end of protein). The secondary specificity of the enzyme is expressed to a minor degree and depends strongly on the P1' residue, so that there is a certain preference for hydrophobic aminoacids in the P2 and P1' position of the chymotryptic substrates (second and fourth amino acids, respectively, starting from the amino end of protein) (Gráf et al., 1998). Microbial proteinases have also been used for the production of bioactive peptides from whey and, although their use has been more limited, there is a vast variety of them in the market. Some sources are Aspergillus spp., Bacillus spp., Rhizomucor miehei, Cryphonectria parasitica, Penicillium citrinum and Rhizopus niveus (Law, 2002). The use of microbial enzymes can have certain advantages over the animal extracted ones. The first one is based on political geography and transportation costs as it is desirable for an enzyme user to have a reliable, predictable source of an enzyme that is pivotal for a manufacturing process. Microbial enzymes can be produced anywhere in the world and the enzyme yield is predictable from the fermentation parameters. It also avoids problems of diseases in the animal population that can cause problems of enzyme availability (Law, 2002). Proteinase K, thermolysin and subtilisin are examples of microbial proteinases. Proteinase K (a member of the subtilisin family of proteinases, E.C. 3.4.21.64) is an alkaline endolytic proteinase produced by Tritirachium album (Limber) fermentation. It cleaves peptide bonds at the carboxylic site of aliphatic, aromatic or hydrophobic amino acids, preferring the last two types. It is supplied by different companies such as Boehringer, Fluka and Sigma (Bond, 1996; Saenger, 1998). Thermolysin (E.C. 3.4.24.27) is a neutral thermostable metalloendopeptidase secreted by the Gram-positive thermophilic bacterium Bacillus thermoproteolyticus. It hydrolyses peptide bonds on the amino terminal site of hydrophobic amino acids such as leucine, phenylalanine, isoleucine and valine, although hydrolysis of bonds with methionine, histidine, tyrosine, asparagine, serine, threonine, alanine, glycine, lysine, glutamic and aspartic acid at P1' has been observed. A hydrophobic residue is preferred in the P1 position, alanine or phenylalanine is preferred to glycine in P2 and in P2', and the order of preference is Leu>Ala>Phe>Gly. Some of its suppliers are Boehringer, Sigma and Fluka (Beynon and Beaumont, 1998; Bond, 1996). Another example of a microbial enzyme that is used for production of bioactive peptides is subtilisin (serine endoprotease, E.C. 3.4.21.62). This enzyme was first isolated from Bacillus subtilis and the term now includes also those secreted by

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other Bacillus species. Subtilisin Carlsberg, an alkaline protease from B. licheniformis, has been used for production of whey-derived bioactive peptides and it is commercially available as Alcalase. Subtilisins are stable over the range of pH of 6–10 and they hydrolyse proteins with a broad specificity for peptide bonds. They have two subsites with strong preferences named S1 and S4; the former prefers large non-β-branched hydrophobic side chains and the S4 subsite strongly prefers hydrophobic side chains (Ballinger and Wells, 1998). In addition, commercial enzyme mixtures have also been used for the production of whey-derived bioactive peptides. The advantages of using these as opposed to the more pure enzymatic preparations can be that a wider range of peptides may be produced which could result in increased bioactivity at reduced cost. One example is the work being developed by the authors on the production of bioactive peptides from β-lactoglobulin using Protease N ‘Amano’. In this work, hydrolysates that contain a mixture of peptides with high bioactivities (IC50 ≈ 100 µg ml–1) are produced at relative short times (6–8 h) and mild conditions of temperature and pH. The specificities of some commercially available proteinase preparations have been described and related to the final characteristics of the hydrolysates produced from whey protein concentrates (WPC) (Smyth and FitzGerald, 1998; Mullally et al., 1994; Mullally et al., 1995). For instance, Mullally et al. (1994, 1995) described the proteolytic and peptidolytic activities of commercially available pancreatic preparations and related to the molecular characteristics of the hydrolysates produced dividing them in two groups. Group II proteases contain only trypsin and chymotrypsin activity, whereas group I proteases contain exopeptidase activity as well. A similar work is described by Smyth and FitzGerald (1998) for commercial protease preparations from fungal, microbial and mammalian origin. In a later work, Mullally et al. (1997a) report the ACE inhibition activity of hydrolysates generated from WPC and fractions enriched in α-lactalbumin and β-lactoglobulin using a range of purified and commercially available proteinase preparations of gastric and pancreatic origin. New commercial proteolytic preparations are appearing every day in the market, however, most of them are not well characterized, hindering their potential for application to the production of bioactive peptides. Knowledge of the specific activities available in each proteolytic preparation would be a very useful starting point in the design of processes encompassing production of functional peptides.

8.4.2 Enzymatic processes and enzyme reactors Several studies have suggested the need for rapid and cost-effective methods for the production and isolation of bioactive peptides from whey, which can also be used at a larger industrial scale (Groleau et al., 2002; Korhonen, 2002; Korhonen and Pihlanto, 2003; Perea and Ugalde, 1996). Since the mid-1990s, various processes have been developed for the production of bioactive peptides utilising various technologies, which will be described in this section together with their underpinning basic principles. However, most of these methods have some

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technological or economical limitations that have made them difficult to scale-up and thus hinder their application to industrial processes. Membrane bioreactors Membrane reactors have been widely studied for the continuous production of protein hydrolysates and peptides. They integrate enzymatic hydrolysis, product separation and catalyst recovery into a single operation. Hydrolysis has been carried out in batch or continuously using different combinations of membrane reactors and ultrafiltration membranes. Currently, most enzymatic processes used in industry are carried out in batch reactors in which the enzyme is in its soluble or ‘free’ form. Some examples of areas in which this type of reactor is used are agriculture, chemical processing, cosmetics, food and beverages production, paints and coatings, pharmaceutical and medical production, and so on. This is a widely practised method that has some advantages; however it has several limitations (Cheryan, 1998, Prazeres and Cabral, 1994):

• less efficiency than continuous processes because of the start up and shut down procedures

• batch to batch variations in the product • high capital costs for equipment owing to their low productivity • the need to inactivate or recover the enzyme at the end of each batch, which increases the costs

• long process times for reaction completion, mainly owing to substrate depletion and product inhibition. Immobilisation of enzymes is a way of overcoming the disadvantages of using the biocatalyst free in solution. It allows a continuous process, which gives the following advantages: better process control, higher productivity, more uniform products and the integration of a purification step in the process. However, problems related to losses in activity of 10–90% have been reported (Cheryan, 1998, Prazeres and Cabral, 1994). Membrane reactors use synthetic semipermeable membranes to create a selective barrier between the enzyme and the substrate or product in order to keep the catalyst within the reaction vessel or to separate the permeable solutes from the reaction mixture by the action of a driving force (chemical potential, pressure, electric field) (Cheryan, 1998, Prazeres and Cabral, 1994). The enzyme can be present free in solution or immobilised at the surface of the membrane or inside its pores. The reaction products should permeate through the membrane pores to attain their continuous removal from the reaction mixture; this will keep the equilibrium towards the products. Membrane bioreactors have several intrinsic advantages that make them a good alternative to the conventional enzymatic systems like batch, fixed or fluidised beds. Their main advantages are (Cheryan, 1998; Prazeres and Cabral, 1994; Rios et al., 2004):

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• continuous operation mode which contributes to a higher productivity and • • • •

might also contribute to the economic viability of the process retention of catalyst within the system and its reuse integration of unit operations (single-step reaction/separation) enrichment and concentration of products in process streams better control of product properties by enzyme (specificity) and/or membrane (selectivity) choice.

The efficiency of the overall system depends on several parameters, which include biochemical parameters (for example catalytic activity, reaction kinetics, concentration, immobilisation stability), geometric parameters (e.g. membrane configuration, pore size distribution, morphology) and hydrodynamics parameters (such as transmembrane pressure and flow velocity) (Charcosset, 2006). The selection of the membrane for the enzymatic membrane bioreactor is very important and the size of the enzyme, substrate(s) and product(s) as well as the chemical nature of the species and of the membrane itself should be considered; special attention should be given to this last factor as the membrane material can contribute to a loss of enzyme activity (enzyme poisoning) (Prazeres and Cabral, 1994; Rios et al., 2004). Ultrafiltration (UF) membranes with a nominal molecular weight cut off of 1–100 kDa are normally used for enzyme membrane bioreactors. For instance, Bordenave et al. (2000) used an UF membrane enzyme reactor to concentrate β-lactoglobulin (β-Lg) from goat whey in the retentate and recovered a mixture of α-lactalbumin (α-La) derived peptides in the permeate obtained by pepsin hydrolysis; the opioid and ACE inhibitory peptide α-lactorphin were among them. According to previous work (Sannier et al., 2000) larger molecular weight cut-off (MWCO) membranes are needed for improved results in the continuous process reaching a compromise between permeate flux, retention of βLg and permeation of α-La peptides. They suggested that for further fractionation of the peptides smaller MWCO membranes may be used. Bouhallab et al. (1993) used a stirred tank membrane reactor for the extraction of the immunomodulatory peptide β-CN f(193–209) from a bovine β-casein/chymosin hydrolysate. Their work showed that the feasibility of the process depends on the nature and area of the UF membrane used. As in every process, membrane reactors have also some disadvantages which are associated with a decrease in process performance during operation. These problems are generally related to loss in enzyme activity and/or mass transfer inefficiencies. An example of these problems is found in the work reported by Visser et al. (1989). They applied an enzyme recycle reactor for the continuous production and isolation of peptide fractions from β-casein (β-CN) degraded by plasmin, finding that this system can only be operated over limited periods of time owing to membrane fouling and loss of enzyme activity after longer conversion periods. Enzyme leakage through the membrane, enzyme poisoning and shear stress effects are the main reasons for losing part of the catalytic activity versus time during the operation of the system. The reaction temperature should be chosen

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carefully as it is considered to be one of the main parameters causing enzyme inactivation. In order to retain activity over a long period of time temperatures lower than the optimum should be used in membrane bioreactors (Cheryan 1998; Lewis, 1996). The loss of mass transfer efficiency is due mainly to two different phenomena: membrane concentration polarisation and fouling. Concentration polarisation occurs whenever a component is rejected by the membrane causing an increase in the concentration of that component at the membrane surface and a concentration gradient over the boundary layer. Therefore an increase in resistance is registered and, in the case of macromolecules, it could give rise to the formation of a gelled layer on the surface of the membrane (Lewis, 1996). Lower flux in polarisation-limited ultrafiltration systems is due to the hydrodynamic resistance of the boundary layer; initially the solute is transported convectively to the membrane causing a steep concentration gradient within the boundary layer which causes a back-transport of the solute into the bulk caused by diffusion. These phenomena reach a steady state and cause the solute to precipitate out and foul the membrane (Cheryan, 1998). Concentration polarisation is a reversible phenomenon that can be reduced by manipulating operational conditions (increased turbulence, cycle backflushing or pulsating flow) or by introducing a cleaning procedure between consecutive operations (Prazeres and Cabral, 1994; Rios et al., 2004). Fouling is a major operational problem that is characterized by an irreversible decline in flux over time which takes place when feed components are collected on the surface or adsorbed in the membrane pores. The nature and extent of membrane fouling is strongly influenced by the physicochemical nature of the membrane and the solute(s) (Cheryan, 1998). Membrane fouling leads to a reduced life of the membrane and affects its separation characteristics and the composition of the products. Hydrolytic membrane bioreactors have been used widely for the production of bioactive peptides from whey or whey protein isolates or concentrates and also from the main milk proteins (caseins, β-lactoglobulin, α-lactalbumin, lactoferrin). Righetti et al. (1997) proposed a multicompartment enzyme bioreactor operating under an electric field for the continuous production of peptides from milk proteins, like β-CN. However, none of the peptides produced with this system were tested for bioactivity. Perea and Ugalde (1996) utilised a membrane recycle reactor to hydrolyse whey proteins by alcalase, finding that this system offers higher conversion levels, productivity and enzyme yield, with respect to batch processes. However, the application of the system may be limited by short reactor stability and high cost of filtration units. Other works include the use of an immobilised enzyme reactor for the partial hydrolysis of a whey protein concentrate (WPI), where the degree of hydrolysis was controlled and maintained at less than 10% in order to minimise bitterness of hydrolysates with the subsequent production of a mixture of protein and peptides and with relatively low ACE inhibitory activity (Scholthauer et al., 2004). As it can be seen from this section, membrane bioreactors offer a broad range

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of possibilities for future industrial applications. They have many advantages compared with more conventional systems. However, a better understanding of the complex phenomena underlying the process is needed in order to enable optimum performance and the design of cost-effective integrated processes for the production of bioactive components.

Chromatographic bioreactors Ion-exchange membranes have also been used for the isolation of bioactive peptides. This approach has the advantage of simultaneous removal of the products during the reaction, which favours thermodynamic equilibrium towards the products and, as the separation is performed during the reaction step, no additional purification is required, reducing production costs (Podgornik and Tennikova, 2002). Ion-exchange membranes with four different reactive groups have been investigated: strongly acidic groups (sulphonic acid), strongly basic (quaternary ammonium), weakly acid (carboxylic acid) and weakly basic (diethylamine). They have been commercialised in the form of flat sheet systems by Pall (New York, USA), membrane stacks by Sartorius (Göttingen, Germany), radial flow cartridges (CUNO Europe, France) and hollow-fibre modules (Kinetic System Inc., St Louis, MO). One of the major limitations of membrane chromatography is non-uniform flow distribution across the membrane caused by the large diameter-to-length ratio of the modules (Charcosset, 2006). Recio and Visser (1999) used a cation-exchange membrane selectively to bind lactoferrin from cheese whey. The bound lactoferrin was hydrolysed in situ with pepsin and the resulting active peptide lactoferricin B was retained in the ion exchanger and eluted with a 50% yield. This is an interesting approach as it allows the production of peptides from a specific protein yet starting from a complex feedstock such as whey. However, it has some limitations for application at larger scales: long hydrolysis times and limited binding capacity of the adsorptive membranes used which limits the scalability of the process. Alternatively this system could be used for the production of new low molecular mass peptides at the laboratory scale. Chromatographic reactors have not been as widely applied as membrane bioreactors in the production of bioactive peptides. Membrane chromatography is a relatively new area that has not obtained the success and acceptance that was expected owing to the reticence of the potential industrial users to apply new technologies and also because of the difficulties that may be posed to scale-up. As a result, suppliers like Millipore do not provide membrane discs any more. In addition, membranes for chromatography are more attractive for preparative chromatography as initially developed by Sepracor Inc. (Brandt et al., 1988) and for this purpose, hollow fibres are preferable to flat sheets (Brandt et al., 1988). Once again a better understanding of physicochemical mechanisms involved in the ion-exchange process with the subsequent generation of good predictive models will facilitate their industrial implementation for a range of applications, including production of bioactive peptides.

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8.5

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Future trends

A small number of dairy products have already been commercialised such as Evolus and Flora pro-activ, which contain peptides with antihypertensive effects. However, there is a need for fundamental research that could substantiate these health claims. In addition these products contain a mixture of peptides together with other components which could hinder, reduce or probably enhance their bioactivity. Therefore, it might be advantageous to produce specific peptides from given milk proteins that could be used as food supplements or developed for pharmaceutical purposes. At present, one of the main challenges in this area is to develop a cost-effective process for the production of specific highly functional peptides from a given protein but starting from complex feedstock mixtures. This may be possible, for instance, by developing integrative processes in which separation of a specific protein from the complex feedstock, its enzymatic hydrolysis and separation of peptides takes place in a single reaction vessel and/or within the same process step. Also research needs to focus on new improved enzymes for production of specific peptides such as novel commercial proteolytic mixtures or more efficient genetically modified enzymes. The advantages of using these compared with pure enzymes is not only reduced cost, which is particularly important in the larger scale, but also the possibility of producing a wider range of peptides with different bioactivities which could result in synergistic effects that could provide the opportunity to formulate products with more than one bioactivity. On the other hand, the enzymatic production of bioactive peptides will have to meet some regulatory challenges. The advantage of dairy food and ingredients over novel foods is that they are considered traditional food with potentially easier regulatory approval processes and also the enzymes typically used are food grade. However, their regulatory approval will still be subjected to demonstration of their safety and efficacy.

8.6

Sources of further information and advice

Fox, P.F. and McSweeney P.L.H. (2003). Advanced Dairy Chemistry Volume 1: Proteins, 3rd edition, Kluwer Academic/Plenum Publishers, New York. Antimicrobial peptide database, http://aps.unmc.edu/AP/main.php Protein Data Bank, RCSB PDB: Structure Explorer, http://www.rcsb.org/pdb/cgi/explore.cgi

8.7

References

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Meisel H. (1997). ‘Biochemical properties of bioactive peptides derived from milk proteins: potential nutraceuticals for food and pharmaceutical applications’. Livestock Production Science, 50(1–2), 125–138. Meisel H. (2001). ‘Bioactive peptides from milk proteins: a perspective for consumers and producers’. Australian Journal of Dairy Technology, 56(2), 83–92. Meisel H. and Frister H. (1988). ‘Chemical characterization of a caseinophosphopeptide isolated from in vivo digests of a casein diet’. Biological Chemistry Hoppe-Seyler, 369(12), 1275–1279. Meisel H. and Olieman C. (1998). ‘Estimation of calcium-binding constants of casein phosphopeptides by capillary zone electrophoresis’. Analytica Chimica Acta, 372(1–2), 291–297. Meisel H. and Schlimme E. (1994). ‘Inhibitors of angiotensin-converting enzyme derived from bovine casein (casokinins)’, in Brantl V. and Teschemacher H. (eds) β-Casomorphins and Related Peptides: Recent Developments, VCH, Weinheim, 27–33. Migliore-Samour D., Floc’h F. and Jollès P. (1989). ‘Biologically active casein peptides implicated in immunomodulation’. Journal of Dairy Research, 56(3), 357–362. Miyauchi H., Hashimoto S., Nakajima M., Shinoda I., Fukuwatari Y. and Hayasawa H. (1998). ‘Bovine lactoferrin stimulates the phagocytic activity of human neutrophils: identification of its active domain’. Cellular Immunology, 187(1), 34–37. Mullally M.M., O’Callaghan D.M., FitzGerald R.J., Donnelly W.J. and Dalton J.P. (1994). ‘Proteolytic and peptidolytic activities in commercial pancreatic protease preparations and their relationship to some whey protein hydrolysate characteristics’. Journal of Agricultural and Food Chemistry, 42(12), 2973–2981. Mullally M.M., O’Callaghan D.M., FitzGerald R.J., Donnelly W.J. and Dalton J.P. (1995). ‘Zymogen activation in pancreatic endoproteolytic preparations and influence on some whey protein hydrolysate characteristics’. Journal of Food Science, 60(2), 227–233. Mullally M.M., Meisel H. and FitzGerald R.J. (1996). ‘Synthetic peptides corresponding to α-lactalbumin and β-lactoglobulin sequences with angiotensin-I converting enzyme inhibitory activity’. Biological Chemistry Hoppe-Seyler, 377(4), 259–260. Mullally M.M., Meisel H. and FitzGerald R.J. (1997a). ‘Angiotensin-I converting enzyme inhibitory activities of gastric and pancreatic proteinase digests of whey proteins’. International Dairy Journal, 7(5), 299–303. Mullally M.M., Meisel H. and Fitzgerald R.J. (1997b). ‘Identification of a novel angiotensinI converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine β-lactoglobulin’. FEBS Letters, 402(2–3), 99–101. Perea A. and Ugalde U. (1996). ‘Continuous hydrolysis of whey proteins in a membrane recycle reactor’. Enzyme and Microbial Technology, 18(1), 29–34. Petrillo E.W. and Ondetti M.A. (1982). ‘Angiotensin converting enzyme inhibitors: Medicinal chemistry and biological actions’. Medicinal Research Reviews, 2(1), 1–41. Pihlanto-Leppälä A. (2001). ‘Bioactive peptides derived from bovine whey proteins: opioid and ACE-inhibitory peptides’. Trends in Food Science and Technology, 11(9–10), 347– 356. Pihlanto-Leppälä A., Antila P., Mäntsälä P. and Hellman J. (1994). ‘Opioid peptides produced by in vitro proteolysis of bovine caseins’. International Dairy Journal, 4, 291– 301. Pihlanto-Leppälä A., Rokka T. and Korhonen H. (1998). ‘Angiotensin-I converting enzyme inhibitory peptides derived from bovine milk proteins’. International Dairy Journal, 8(4), 325–331. Pihlanto-Leppälä A., Koskinen P., Piilola K., Tupasela T. and Korhonen H. (2000). ‘Angiotensin-I converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides’. Journal of Dairy Research, 67(1), 53–64. Podgornik A. and Tennikova T.B. (2002). ‘Chromatographic reactors based in biological activity’, in Freitag R. (ed.) Modern Advances in Chromatography, Springer-Verlag, Berlin, 167–206.

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Prazeres D.M.F. and Cabral J.M.S. (1994). ‘Enzymatic membrane bioreactors and their applications’. Enzyme and Microbial Technology, 16(9), 738–750. Recio I. and Visser S. (1999). ‘Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin – in situ enzymatic hydrolysis on an ionexchange membrane’. Journal of Chromatography A, 831(2), 191–201. Righetti P.G., Nembri F., Bossi A. and Mortarino M. (1997). ‘Continuous enzymatic hydrolysis of β-casein and isoelectric collection of some of the biologically active peptides in an electric field’. Biotechnology Progress, 13(3), 258–264. Rios G.M., Belleville M.P., Paolucci D. and Sanchez J. (2004). ‘Progress in enzymatic membrane reactors – a review’. Journal of Membrane Science, 242(1–2), 189–196. Saenger W. (1998). ‘106. Proteinase K’, in Barret A.J., Rawlings N.D. and Woessner J.F. (eds) Handbook of Proteolytic Enzymes, Academic Press, London, 322–325. Sannier F., Bordenave S. and Piot J.M. (2000). ‘Purification of goat β-lactoglobulin from whey by an ultrafiltration membrane enzymic reactor’. Journal of Dairy Research, 67(1), 43–51. Schlimme E. and Meisel H. (1995). ‘Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects’. Nahrung, 39(1), 1–20. Scholthauer R.C., Schollum L.M., Reid J.R., Harvey S.A., Carr A.J. and Fanshawe R.L. (2004). Bioactive Whey Protein Hydrolysate. USA Patent 20040086958, May 6. Shin K., Yamauchi K., Teraguchi S., Hayasawa H., Tomita M., Otsuka Y. and Yamazaki S. (1998). ‘Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7’. Letters in Applied Microbiology, 26(6), 407–411. Shinoda I., Takase M., Fukuwatari Y., Shimamura S., Koller M. and Konig W. (1996). ‘Effects of lactoferrin and lactoferricin on the release of interleukin 8 from human polymorphonuclear leukocytes’. Bioscience, Biotechnology and Biochemistry, 60(3), 521–523. Smyth M. and FitzGerald R.J. (1998). ‘Relationship between some characteristics of WPC hydrolysates and the enzyme complement in commercially available proteinase preparations’. International Dairy Journal, 8(9), 819–827. Spyroulias G.A., Galanis A.S., Pairas G., Manessi-Zoupa E. and Cordopatis P. (2004). ‘Structural features of angiotensin-I converting enzyme catalytic sites: Conformational studies in solution, homology models and comparison with other zinc metallopeptidases’. Current Topics in Medicinal Chemistry, 4(4), 403–429. Sturrock E.D., Natesh R., van Rooyen J.M. and Acharya K.R. (2004). ‘Structure of angiotensin-I converting enzyme’. Cellular and Molecular Life Sciences, 61(21), 2677– 2686. Suetsuna K., Ukeda H. and Ochi H. (2000). ‘Isolation and characterization of free radical scavenging activities peptides derived from casein’. Journal of Nutritional Biochemistry, 11(3), 128–131. Tang J. (1998). ‘272. Pepsin A’, in Barret A.J., Rawlings N.D. and Woessner J.F. (eds), Handbook of Proteolytic Enzymes, Academic Press, London, 805–814. Tani F., Shiota H., Chiba H. and Yoshikawa M. (1994). ‘Serorphin, an opioid peptide derived from bovine serum albumin’, in Brantl V. and Teschemacher H. (eds), β−Casomorphins and Related Peptides: Recent Developments, VCH, Weinheim, 49–53. Teschemacher H., Koch G. and Brantl V. (1994). ‘Milk protein derived atypical opioid peptides and related compounds with opioid antagonist activity’, in Brantl V. and Teschemacher H. (eds), β−Casomorphins and Related Peptides: Recent Developments, VCH, Weinheim, 3–17. Vermeirssen V., Van Camp J. and Verstraete W. (2004). ‘Bioavailability of angiotensin-I converting enzyme inhibitory peptides’. British Journal of Nutrition, 92(3), 357–366. Visser S., Noorman H.J., Slangen C.J. and Rollema H.S. (1989). ‘Action of plasmin on bovine β-casein in a membrane reactor’. Journal of Dairy Research, 56(3), 323–333. Yamamoto N., Akino A. and Takano T. (1994). ‘Antihypertensive effect of the peptides

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derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790’. Journal of Dairy Science, 77(4), 917–922. Yoshikawa M., Tani F., Shiota H., Usui H., Kurahashi K. and Chiba H. (1994). ‘Casoxin D, an opioid antagonist/ileum-contracting/vasorelaxing peptide derived from human αs1casein’, in Brantl V. and Teschemacher H. (eds), β−Casomorphins and Related Peptides: Recent Developments, VCH, Weinheim, 43–48. Zucht H.D., Raida M., Adermann K., Mägert H.J. and Forssmann W.G. (1995). ‘CasocidinI: a casein-αs2 derived peptide exhibits antibacterial activity’. FEBS Letters, 372(2–3), 185–188.

9 Production of flavours, flavour enhancers and other protein-based speciality products Stuart West, Biocatalysts Ltd, UK

9.1

Introduction

Proteins are complex macromolecules and although they have the repeating unit of the peptide bond they are extremely heterogeneous in their structure. They are composed of 20 amino acids which gives rise to 400 combinations of amino acid pairs around the repeating peptide bond. So according to the nature of the complexity of proteins and the often highly specific catalytic nature of enzymes, proteases often only break down a small part of the protein structure. In addition all enzymes are proteins and any enzyme that was able to degrade all proteins totally would rapidly hydrolyse itself. The 20 amino acids found in proteins form a diverse group of molecules including those which have side chains that are positively charged, negatively charged, hydrophilic, hydrophobic, sulphur containing and hydroxyl group containing. This becomes important when flavour production is considered later. In addition amino acids and peptides will react with sugars via the Maillard reaction and this is a key reaction for producing a whole class of reaction savoury flavours. Many proteins have a globular structure whereby the polypeptide chain folds with the hydrophobic amino acids ending up in the interior and the hydrophilic amino acids on the exterior. Hence, as a protein is digested, the structures encountered by the enzyme will change. Proteins which have molecular weights in excess of 10 000 Daltons are too

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Table 9.1

Flavour characteristics of some amino acids

Taste

Amino acid

Intense bitter Bitter Sweet

Trytophan, phenylalanine, arginine, iso-leucine Leucine, histidine, valine, ornithine, methionine, lysine Hydroxyproline, praline, alanine, lysine, valine, threonine, serine, glycine, glutamic acid Aspartic acid, glutamic acid, asparagine, histidine, serine

Sour

Source: Nagodawithana, 1995a.

large to be able to be tasted. There are some exceptions to this. The plant protein thaumatin (which is one of several sweet proteins, Kant, 2005) has a sweet liquorice type flavour which makes it useful for several applications within the food industry. Several other proteins, for example haemoglobin which has a slight metallic taste, have a flavour that is due to the iron-containing haem group and not the protein itself. Often industrial food proteins will have characteristic flavours but these flavours are due to molecules associated with the proteins and not the proteins themselves. Most amino acids have mild but characteristic flavours (Table 9.1; Nagodawithana, 1995a). When we consider protein breakdown and flavour production, the key molecules are peptides that have a molecular weight roughly in the range 200 Daltons (molecular weight region of dipeptides) to 2000 Daltons. So there are two main routes for producing flavours from proteins. The proteins can be hydrolysed into peptides and this will produce flavours such as those found in soy sauce or cheeses like brie and camembert (other cheese types will also have peptide flavours but these are in combination with fat breakdown flavours, for example free fatty acids and methyl ketones (Gatfield, 1988; Gripon, 1993). Alternatively, the protein can be more extensively hydrolysed and then reacted with sugars via the Maillard reaction to form strong savoury flavours (Nagodawithana, 1995b). For example, a chicken flavour can be made by reacting the sulphurcontaining amino acid cysteine with sugars such as glucose and xylose.

9.1.1 Protease classification Enzymes that catalyse the breakdown of proteins come in the International Union of Biochemists (IUB) group EC 3.4.x.x. They can then be subdivided into those that are endo-acting, that is hydrolyse the peptide chain in the middle or exo-acting, that is those which will cleave amino acids (or small peptides) from the end of the chain. Exo-acting enzymes are generally called peptidases and they are further classified into amino- and carboxy-peptidases depending upon which end of the peptide bond they attack. The term used to describe the extent of protein hydrolysis is degree of hydrolysis or DH. A DH of zero is an intact protein and a DH of 100 would be a fully hydrolysed soup of amino acids. To achieve a high DH (>20) a combination of both endo- and exo-acting proteases are usually required. Proteases can also be classified by the nature of their catalytic site and their bond specificity. Groups 3.4.11 to 3.4.19 are exopeptidases, for example group 3.4.11 is the

Production of flavours and flavour enhancers Table 9.2

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IUB classification of proteases

IUB group number

Protease type (group at catalytic centre)

Commercial examples

3.4.21

Serine

3.4.22

Cysteine

3.4.23 3.4.24 3.4.25

Aspartic MetalloThreonine

Trypsin (pancreatic protease); subtilisin (alkaline Bacillus protease) Papain (papaya protease), bromelain (pineapple protease) Chymosin, Mucor rennin, pepsin Neutral Bacillus protease No commercial examples

Table 9.3

Bonds hydrolysed in the insulin β-chain

Enzyme

No. of bonds vigorously broken

Chymosin Microbial coagulants Trypsin Ficin Pepsin Neutral bacterial

2 2 2 4 5 6

Alkaline bacterial (subtilisin) Acid fungal Papain

7 9 9

Main bonds attacked

Glu-Ala; Leu-Val Glu-Ala; Leu-Val Lys-Ala; Arg-Gly Glu-Ala; Tyr-Leu; Phe-Tyr Leu-Val; Phe-Tyr His-Leu; Ser-His; Ala-Leu; Gly-Phe; Arg-Gly Gln-Leu; Ser-His; Leu-Tyr His-Leu; Gly-Phe; Phe-Phe Asn-Gln; Glu-Ala; Leu-Val; Phe-Tyr

pH optimum range 3.5–6.5 3.5–6.5 6–9 5–7.5 1.8–3 6.5–7.5 7.5–9.5 2.5–4 5–7

amino-peptidase group. Groups 3.4.21 to 3.4.25 are the endopeptidases or proteases. These are characterized by their amino acid or prosethetic group being the catalytic site. Commercial examples are given in Table 9.2. As mentioned in the introduction no protease can fully hydrolyse a protein. Proteases have specificities against certain amino acids forming the peptide bond. This will range from a highly specific protease which can only cleave at one amino acid site (for example clostripain which only hydrolyses bonds next to arginine), to those which act at a few sites, ‘broad spectrum’ proteases which will cleave at up to around 10 different amino acids. Table 9.3 (West, 1996) gives several common proteases and the extent to which they can hydrolyse the oxidised β-chain of insulin. Most commercially available proteases are medium to broad specificity.

9.1.2 Industrial proteases A protease-containing extract of pancreas was the first industrial use of enzymes. In 1913, Otto Rohm patented a pre-soaking detergent product containing pancreas extract. This was not an ideal product as the pH of the pre-soak was above the pH

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optimum for the pancreatic proteases, but it ultimately led to the whole market of biological soap powders. Today, soap powder (detergent) proteases make up the largest part of the industrial enzyme market. However, they are produced by fermentation from bacteria and have pH optima totally in line with the soap powder products for maximum efficacy (Barfoed, 1983). These detergent Bacillus-derived proteases are also made as food grade enzymes, for example Alcalase™ from Novozymes and Protex™ from Genencor (Danisco). They are the most cost effective proteases available and are widely used within the food industry. They are broad spectrum, as can be seen from Table 9.3, which is good if protein breakdown with no consideration of flavour is the only goal. Whereas, they are usually far from ideal if either a bland tasting hydrolysate or a meaty (savoury) hydrolysate is the commercial goal, as they usually produce excessive bitterness and a variety of other off-flavours (Kilara, 1985).

9.2

Production and usage of monosodium glutamate (MSG)

Amino acids are made by fermentation in huge amounts (Hacking, 1986). A large proportion of these go into the animal feed industry (Hepner, 2007). Some, such as lysine, an essential amino acid, are used as nutritional supplements in foods. However, it is monosodium glutamate (MSG) that plays the largest role in food and in particular flavouring applications. MSG is also used by the pharmaceutical industry, for example for the treatment of gastric ulcers. Glutamic acid is present in most proteins and is often in a high percentage, on extensive hydrolysis the glutamic acid becomes free and can contribute flavour. Hence it is present in most naturally hydrolysed food proteins, for example cheese, soy sauce and yeast extract. Originally MSG was extracted from natural materials and hydrolysed proteins but from the late 1950s it has been made by fermentation (Kusumoto, 2001). An outline of the production process is given in Fig. 9.1. This process is highly efficient, producing ~100 g of MSG per litre of fermentation broth. MSG was discovered in 1907 by Kikunae Ikeda at the Tokyo Imperial University. He identified it as being a major flavour in products like soy sauce and he coined the flavour term umami which is now accepted as the fifth taste, as it is the Table 9.4

Natural levels of MSG in certain foods

Food Eggs Meat (depending on type) Peas Parmesan cheese Soy sauce (depending on type) Tomatoes Yeast extract

MSG concentration (mg/100g) 20–30 20–50 200 1200 700–1100 140 1400–2000

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187

Fed-batch aerobic fermentation using GM-bacteria with glucose, ammonia, minerals and growth factors as fermentation substrates

Remove cells from fermenter broth by centrifugation and microfiltration

Remove ionic impurities with ion exchange resin and chromatography

First crystallisation step production of crude crystals

Decolorisation and depyrogenation

Second crystallisation and drying

Fig. 9.1 Outline process for producing MSG by fermentation.

savoury flavour associated with many foods (Dewis, 2004). MSG stimulates the T1R1/T1R3 receptor amongst others (Li, 2002). MSG is naturally present in many foods as the free amino acid (as opposed to being part of a protein) and Table 9.4 gives some examples. MSG is used widely in a range of meaty/savoury tasting foods including canned soups, stock cubes and concentrates, gravies, seasoning mixes, meat sauces, meat flavours for savoury snacks, and so on. MSG is a controversial food additive as although it is found in a wide range of totally natural foods there have been many claims against it including that many people have an allergic response to it or that it induces severe conditions (Heath and Reineccius, 1986). Most studies have failed to find any conclusive problems and it is still regarded as GRAS in the USA. There is a possibility that a small percentage of the population experience a mild and transitory reaction under certain circumstances when MSG is consumed in foods that contain high levels. Claimed symptoms include skin flushing, headache and nausea (Nagodawithana, 1995c).

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Fig. 9.2

9.3

Chondroitin sulphate A.

Chondroitin sulphate

Chondroitin sulphate is used in health food supplements where it is effective as an agent to aid repair and maintenance of joint tissues (Lipiello et al., 2000; Ronca and Conte, 1993), particularly in older individuals. It is a glycosaminoglycan produced by cartilage cells called ‘chondrocytes’ and it is essential for cartilage growth, nutrition and repair. Chondroitin sulphate is composed of repeating disaccharide units; one of the monosaccharides is always either N-acetylglucosamine or N-acetylgalactosamine, and the other is, in most cases, uronic acid (see Fig. 9.2). One of the hydroxyls of the amino sugar is esterified with sulphate which gives it a very high density negative charge. This then favours an electrochemical attraction to water, thus giving cartilage its elasticity and fluidity (Morreale et al., 1996). Chondroitin sulphate is attached to extracellular proteins (aggrecan) in which chains of chondroitin sulphate and keratan sulphate (also a glycosaminoglycan) are attached to the central portion of the core protein by glycosidic bonds between sugar residues and the hydroxyl groups of serine residues in the protein. There are 100–150 chondroitin sulphate chains per aggrecan monomer and individual monomers interact with hyaluronic acid to form an aggregate with a very high molecular weight (Hardingham et al., 1994). The production of chondroitin sulphate by chondrocytes declines with age and is also disrupted by stress or injury (Lauder at al., 2001). Clinical studies have shown that supplementing the diet with chondroitin sulphate boosts the level in the cartilage matrix and in turn the chondrocytes respond to this increase by renewing their production (Lipiello et al., 2000). The chondroitin sulphate used in health food supplements is extracted from cartilage tissue, for example bovine trachea. The extraction is facilitated by the use of protease enzymes, as shown in Fig. 9.3, which solubilise the solid matrix of cartilage so releasing the chondroitin sulphate which is then extracted by further processing (Biocatalysts, 2006). Chondroitin sulphate can be extracted from a wide variety of sources such as bovine trachea, bovine shoulder blade, shark cartilage and so on. (Jin-Ho et al., 2004; Hoffman and Mashburn, 1967; Luo et al., 2002). Bovine trachea is the most common source containing 3–4% chondroitin sulphate. Shoulder blade is a richer source containing >6% chondroitin sulphate. As well as the type of cartilage

Production of flavours and flavour enhancers

189

Intact or chopped cartilage tissues

Enzyme hydrolysis, 16–24 h, pH 5–7, 1–1.5 volumes solute, 55–70º C, P144P (500 TU) 2–3 g, P648L 7–10 ml per kg cartilage

Cooling to 40° C and separation of fat and solid wastes

Removal of soluble proteins and peptides from aqueous solution

Precipitation of chondroitin sulphate from aqueous solution

Drying and grinding

Yield of chondroitin sulphate (mg/g)

Fig. 9.3 Enzymatic production of chondroitin sulphate. 70 60 50 40 30 20 10 0 Whole shoulder blade

Fig. 9.4

Finely chopped shoulder blade

Finely chopped trachea

Effect of substrate type on yield of chondroitin sulphate.

tissues used, the age of the animal and processing conditions will also affect the final yield of chondroitin sulphate obtained, as shown in Fig. 9.4 (Biocatalysts, 2006). 9.3.1 Recommended conditions for enzyme digestion The digest is carried out in water or acetate buffer at around pH 5.5 for 16 to 20 h

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Novel enzyme technology for food applications

at 65 °C with agitation using papain or a bacterial protease such as Promod 648L at a dose rate of 5–10 ml per kg of cartilage (Biocatalysts, 2006). In most cases, this should result in complete solubilisation of the animal tissues. The yield and purity of the final product will vary considerably according to the follow on processes selected for the removal of free proteins and precipitation of chondroitin sulphate. After separation of the fat and solid wastes, filtration can be carried out to remove small particulates before proceeding to the next stage (Khare, 2004). Soluble proteins and peptides can be removed by precipitation using salts, ethanol or alternative suitable processes, for example selective filtration. The precipitation of chondroitin sulphate can be accomplished by the addition of 4 volumes of cold ethanol to 1 volume of liquid filtered digest. The solid chondroitin sulphate is then separated, washed, dried and ground to yield the final product.

9.4

Production of aspartame

Aspartame is a dipeptide (containing aspartic acid and phenylalanine, Fig. 9.5) synthetic sweetener over 200 times sweeter than sucrose and is the market leading product in the high intensity sweetener market of over US$1bn (www.nutrasweet. com). This is an unusual property for a peptide which, if they have taste, are usually cheesy, savoury or bitter, as is the isomeric form β-aspartame. It was discovered by accident in 1969. Aspartic acid contains two carboxylic groups and early chemical methods of synthesis produced large quantities of β-aspartame as a by-product which is actually bitter and not sweet. Aspartame was one of the first commercial successes for the use of enzymes as biocatalysts (Ajinimoto, 1985). In 1976, the Tosoh Corporation of Japan discovered that the proteolytic enzyme thermolysin would catalyse the amide bond formation between phenylalanine methyl ester and Z-aspartic acid without the Phenylalanine

Aspartate

O

Methanol O

HO

CH3

NH O

O

NH2 L -aspartyl- L -phenylalanine

Fig. 9.5

methyl ester

Structure of aspartame.

Production of flavours and flavour enhancers

191

need to protect the β-carboxylic acid. In addition the reaction catalysed is also stereospecific, only catalysing the reaction between the L-amino acids. The reaction is reversible and the reaction equilibrium favours the hydrolysis direction. However, with excess phenylalanine methyl ester, the dipeptide precipitates out as it is formed and hence drives the reaction in the direction of aspartame synthesis (West, 1996).

9.5

Enzymes for vanilla extraction

Enzymes currently play little role in the overall extracted plant flavour market. Plants contribute a vast array of flavouring substances to the food industry but most of these are in forms that cannot or do not require enzymes to assist in their production. A review of flavour producing plants is given by Hodge (1975). Herbs and spices are just dried leaves or ground plants/seeds. Essential oils are solvent extracted and hence there is no scope for enzymes to contribute. Enzymes can play a role where an aqueous or aqueous/alcoholic extraction is done and they can help in either breaking down the botanical tissue to make the extraction easier or by releasing bound flavours into their fully active form (Reyne et al., 1992). One of the most important flavourings is vanilla. Both synthetic and natural forms are produced. Traditionally natural vanilla is extracted from the fully grown fruit of a variety of orchid. Madagascar is currently the most important production region for vanilla pods. Owing to the high price of natural vanilla, several fermentation/biocatalytic processes have been patented to make natural vanilla cheaper and to facilitate more controllable ways of making it. Naturally extracted vanilla is not just composed of the molecule vanillin and any synthesised product whether by chemistry, fermentation or biocatalysis will also taste different to the naturally extracted product (Riley and Kleyn, 1989; Cheetham, 1993). The ‘green’ beans as found on the plant do not have the characteristic vanilla flavour and the vanilla is present in the bean in a bound form, where it is chemically linked to a glucose molecule and has no flavour. So after the bean has been picked, the flavour has to be developed before it can be extracted; this is done by a curing process (Riley and Kleyn, 1989). The curing process can take between 5 weeks and 5 months. First the beans are ‘killed’ by heat (for example 20 s in boiling water or 48 h in an oven) or by freezing. Then they are wrapped in blankets, heated in the sun and allowed to sweat followed by drying and conditioning. During this process, enzymes naturally present in the beans (glycosidases, proteases and oxidases) ferment the beans, which shrink by up to 400% and turn their characteristic brown colour. The best grades of beans develop a visible white coating of vanillin. The glycosidases convert the bound vanilla into the free flavoursome form as shown in Fig. 9.6. However, not all the vanilla is converted and this leaves scope for a further enzyme treatment of the beans to release the extra bound vanilla. The ideal type of enzyme to assist in vanilla extraction is one that contains high levels of cell wall degrading enzymes, for example cellulose and pectinase and a high level of β-glucosidase. This enables

192

Novel enzyme technology for food applications O

C

H

OMe OH

Fig. 9.6

Chemical structure of vanillin.

the whole plant structure to be opened up and any remaining bound vanilla converted into free flavoursome vanilla. An outline process for the enzymatic-assisted digestion of vanilla beans is shown in Fig. 9.7 (Biocatalysts, 2005). A weak alcoholic solution is used because if the alcohol is any greater than 15% the enzyme will be precipitated and Cured vanilla pods

finely chop

Ethanol (15%) + water (can use 0–15 % ethanol)

mix to form slurry

adjust pH to 4.0–4.5

digest at 40-60 o C for 3–16 h

Depol 40 l (0.75–2.0% dry matter)

adjust pH to 7.0–7.5

95% ethanol (to 75 % final concentration)

alcohol extraction

separate solids in a decanter or separator Concentrated vanilla extract

Fig. 9.7 Process for enzyme-assisted vanilla extraction.

Production of flavours and flavour enhancers

193

deactivated. To limit the amount of alcohol used later a minimum of liquid should be used at this point, just enough to cover the beans and make a thick slurry. A mixed carbohydrase/glycosidase is used which both digests the beans and releases any bound vanilla. At the end of the reaction the system is neutralised and the vanillin fully solubilised by increasing the alcohol level to 75% w/w. Although this process will give more yield, the flavour of the final extract will not be exactly the same, as vanilla extract is not just a solution of vanillin, and the enzyme process will yield different concentrations of the other flavours that are contained within the vanilla extract.

9.6

Enzyme modified cheese as a flavour ingredient

Cheese is the original enzyme processed food, dating back to around 8000 years ago. Both the structure (production of curds by chymosin) and flavours (by proteolytic enzymes produced by the starter cultures) are produced enzymatically (Dalgliesh, 1993; Fox, 1993). It is possible to produce cheese by fermentation or addition of acid alone but this produces cottage or cream cheese type products. The more common rennet curd cheeses such as cheddar are made by a combination of enzyme actions. Chymosin curd is produced by the action of animal rennet or some other fungal acid proteases on the κ-casein where the phe(105)–met(106) bond is broken and which results in cleavage of the hydrophilic micelle stabilising peptide segment. For cheese gel or curds to form, the temperature has to be above 20 °C and calcium must be present (Fox, 1984). Our main concern here is the production of cheese flavours and although the chymosin (rennet) hydrolyses some other casein bonds it does not produce a significant cheese flavour. This is produced by proteases and peptidases produced by the starter culture bacteria (mainly Lactobacillus spp.) or in the case of a cheese like brie, by surface bacteria such as Brevibacterium spp. (Reps, 1993). It is the enzymes produced by the bacteria and moulds that are added to cheese that produce the vast array of different varieties of cheese with different cheese flavours. However in this chapter the focus is on the production of cheese flavours and not the production of flavours within cheese for which there are very many good books and reviews (see Section 9.10, Sources of further information and advice). Before moving on to cheese flavour production we need to look at processed cheese production as this is one of the main markets where cheese flavours are used. Whereas real cheese has been made for thousands of years, processed cheese has been made for just under 100 years. The original process was developed in Switzerland by Gerber and Stetzler of the Gerber Company in 1911. Processed cheese is basically made by heating ordinary cheese with emulsifiers such as citrates and phosphates. It melts and takes on a smooth free flowing texture but most importantly it is now stable and can be stored for many years without deteriorating. Work has been done to try to make processed cheese without making real cheese first and a review of this is given by Pal (2002). In the mid-1940s work started on adding enzymes to the processed cheese to try

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Novel enzyme technology for food applications

Fig. 9.8

Schematic process for making enzyme modified cheese.

to help it develop a more consistent flavour, but it was not until the mid-1970s that enzyme modified cheese (EMC) was developed and permitted for use in processed cheese (FDA, 2003). EMC is an intense cheese flavour, sold either as a paste or a powder, which has many different food applications. EMC and processed cheese is an area where there is little in the science literature but many patents, although many of these have now expired. One example for EMC manufacture is a Kraft patent (Han et al., 2001). The simplest cheese flavour is dried cheese; this has a flavour intensity two to five times that of ordinary cheese. Dried cheese finds applications in savoury snacks and cheese flavoured items but it is an expensive way to make cheese flavours as strong flavoured cheeses have been stored for many months or years and this makes them quite expensive. EMC is made from rapidly matured real

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Fig. 9.9 Cooker used for EMC production. Photograph courtesy of Teagasc, Dairy products Research Centre, Cork, Ireland.

cheese using enzymes. The process essentially mimics what happens during real cheese production by adding extra enzymes to the cheese and then incubating at around 50 °C. A cheese flavour that is 10–20 times stronger than real cheese can be made in as little as 24–48 h instead of months or years. A typical process for making EMC is shown in Fig. 9.8 and a typical cooker used for making EMC is shown in Fig. 9.9 A cheese emulsion is made first (similar to making processed cheese); this is pasteurised and then cooled to a suitable temperature for the enzymes to work. For fungal enzymes that are used in EMC manufacture, this is around 45–50 °C. A combination of enzymes is used containing lipase, protease and peptidases. Once the reaction is complete, which can be monitored by measuring the fatty acid release, the cheese is heated to inactivate the enzyme totally. This is very important in order to prevent the enzyme continuing to work when it is added to the processed cheese. The enzyme can be inactivated by heating for a long time at 70 °C+, for example half an hour, or for a very short time at 90 °C+. This high degree of heat is required as the lipase is stabilised by the presence of fat and the reduced level of water. The EMC is then concentrated to a paste (for use in processed cheese and cheese sauces) or dried to a powder for use in products such as savoury flavourings. Most EMC is made in the USA where there is a much bigger demand for processed cheese and where well over half of all cheese made is consumed as ingredients by the food industry (and in particular the fast food industry), whereas

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Table 9.5

Cheeses and their different flavours

Cheese

Type

Flavours

Brie

Soft – surface bacteria ripened

Cheddar

Hard, flavour produced by starter cultures Internal blue mould ripened made with homogenised milk Made with additional enzymes – PGEa Internal blue mould ripened

Protein owing to extensive protein breakdown by surface bacteria Protein notes with some lipid breakdown flavours Strong blue owing to high level of lipolysis Piquant owing to short chain free fatty acids Strong methyl ketone flavour (fatty acid breakdown products) Creamy and smooth texture but with bitter protein notes

Danish Blue Provolone Roquefort Serra da Estralab

Made with thistle rennet (water extract of cardoon flowers)

Pre-gastric esterase, an animal derived (calf, lamb, sheep) lipase which exhibits a strong preference for releasing short chain fatty acids. b Famous Portuguese cheese made from plant not animal/microbial rennet.

in Europe it is the other way round with about three-quarters of cheese sold as retail and mainly eaten as a food product in its own right. The emphasis so far has been on the production of cheese flavours by proteases and peptidases but this is only part of the picture. Table 9.5 shows different types of cheeses and the main types of flavour they contain. This shows that with the exception of brie and Serra da Estrela, the other cheeses all contain flavours that are derived from fat breakdown. Hence EMCs are typically made by using a combination of lipase, peptidase and protease. Many of these enzymes are derived from cultures that are used in cheese manufacture such as Penicillium and Mucor spp. Animal-derived pre-gastric esterases (PGEs) are used to produce Italian EMC. Many cheese flavours are just EMC but more complex flavours can be made by adding fermentation derived top notes. Many cheese flavours are about balance, having the right degree of bitterness; too little bitterness and the cheese flavour will be bland, too much and it will be unpalatable. The cheese also needs the right balance between short, medium and long chain fatty acids. Too high a level of short chain fatty acids and the flavour will be too sharp and piquant; too high a level of long chain fatty acids and the product will taste soapy. So whilst there are many different proteases, peptidases and lipases on the market, few can be used to create a successful cheese flavour. Alkaline proteases and papain produce too much bitterness and many of the commercial lipases have too high a level of long chain fatty acids and produce a soapy flavour. Examples of the free fatty acid profiles produced by the hydrolysis of butter oil are given in Fig. 9.10. Generating the right type of protein notes is more difficult than getting the fatty acid profile right. In general bacterial endo-proteases are of no use and will produce off-flavours and bitterness that cannot be eliminated by the use of

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Fig. 9.10

197

Typical hydrolysis profile of butter oil (a) by Lipomod® 187P; (b) by pregastric esterase (PGE); (c) by Lipomod® 338.

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debittering peptidases. Most commonly used are fungal proteases and either fungal or bacterial (from Lactobacillus spp.) peptidases (West and Pawlett, 1996). (Note that some of the peptidases mentioned in this reference were produced by Imperial Biotechnology which no longer exists as an independent company. Some of these peptidase products are still available from Danisco).

9.6.1 Applications of EMC EMC is used in a wide range of products such as processed cheese, cheese sauces, pizza toppings, cheese flavourings for savoury snacks, macaroni cheese, cheese dips and so on. All of these products can be made using real cheese instead of EMC, so what advantages does EMC give the food processor over using real cheese? Real cheese is a living product that constantly changes (ages) with time. Hence to obtain the right flavour addition the same type of cheese of exactly the same age would have to be used every time. Highly flavoured real cheese is also the oldest and hence the most expensive. When cheese alone is used in processed cheese manufacture it serves two purposes: it generates the flavour and the texture and this makes exactly the right type of cheese even more difficult to source. By using EMC to generate the flavour of the product, cheese can be selected to contribute the right texture and this can often be done with a young cheese (which is much cheaper than aged cheese). The advantages of using EMC for cheese flavour and product manufacture are:

• • • • •

reduces cost by eliminating expensive mature cheese reduces cost by allowing a higher percentage of immature cheese to be used enables the end product to be made more consistently makes the ingredient buying process easier allows cheese off-cuts to be recycled back into the main product.

Processed cheese still has to be made to compositional standards to conform to processed cheese regulations (FDA, 2003). So in the USA at least 51% of the ingredients must come from cheese and this excludes any EMC used. In addition the moisture content must not exceed 44% and the fat content must be greater than 23%. These regulations limit the amount of cheese that can be substituted. EMC also has the potential to be used for the accelerated ripening of traditional cheese. Hannon et al. (2006) showed that by adding 1% EMC to cheese curd they could produce a cheese after 4 months aging that had a flavour profile equivalent to that of a traditionally 6 month ripened cheese.

9.7

Enzymes used in savoury flavouring

Two very common savoury flavourings are soy sauce and yeast extract. They are both enzyme-produced peptide-based savoury flavours (Schultz, 2005). Soy sauce is produced by fermentation (and sometimes additional enzymes) and yeast extract is produced by autolysis (with additional enzymes). Soy sauce as a fermentation

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product will not be discussed further here; for more information see Nagodawithana (1995d). Another soy-based savoury flavour which is chemically produced is acid hydrolysed vegetable protein (HVP). This is low-cost intense savoury flavour produced by acid hydrolysis of soy flour with hydrochloric acid (6N, 180 °C, 6 atmospheres). As well as the protein being hydrolysed, the soy carbohydrates are hydrolysed to sugars and these react with the amino acids via the Maillard reaction to produce an intense savoury flavour. The chemistry of the Maillard reaction is well described by Ames (1990). Acid HVP has declined in popularity as it is not natural and in the late 1980s was found to contain the carcinogens MCP and DCP (mono- and dichloropropanol, respectively). Acid HVP (from soy) is a key ingredient in non-brewed or synthetic soy sauce, whereas traditional brewed soy sauce is produced by enzyme hydrolysis during a fermentation process with Aspergillus moulds (koji fermentation). Much work has been done to produce a natural enzymatically hydrolysed e-HVP (with enzymes and not fermentation) with limited success, as stated earlier, extensive enzyme hydrolysis of proteins is difficult and expensive (in comparison with hydrochloric acid) to achieve. To achieve a savoury-tasting protein hydrolysate from soy protein a degree of hydrolysis (DH) >30 needs to be achieved. This requires more than one protease. In addition if a low cost alkaline bacterial protease is used the resulting hydrolysates is more bitter than savoury. There are two different routes to achieve extensive enzyme hydrolysis of proteins. These are the all-in-one system and the Cascade system (Godfrey, 1990). The all-in-one system involves adding several different proteases and exo-peptidases all in one go at the start of the reaction. The Cascade system involves a sequential hydrolysis with one enzyme being added after another. The advantages and disadvantages of the two systems are given in Table 9.6.

9.8

Enzymes used in yeast extract manufacture

Yeast extract was one of the food and drinks industries first commercial successes at moving a waste product up the value chain. The main by-product of beer brewing is waste yeast and although some is recycled for the next brew, most is surplus to requirements. The main outlet, as with many food waste products, was for animal feed. Brewer’s yeast (Saccharomyces cerevisiae mainly) is a rich source of the B vitamins and hence of high commercial value. Late in the 19th century waste yeast was converted into a strong flavoured highly nutritious savoury spread. This process still continues today but in addition yeast extract is also made from specially grown Baker’s yeast and other yeast strains such as Candida utilis (called Torula) and Kluyveromyces fragilis (Kelly, 1983). An additional step might be required with some Brewer’s yeast to remove the hop isohumolones which would make the yeast extract very bitter if left. These can be removed by washing the intact yeast at pH 9. Yeast extract has three main market outlets, these are as a consumer savoury spread, for example Marmite®, as a bulk savoury flavouring used by the food industry and as a fermentation substrate.

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Table 9.6

Protein hydrolysis advantages and disadvantages Cascade system

All-in-one system

Advantages

Disadvantages

Advantages

Disadvantages

Few or no pH changes required

Requires several operator interventions and hence possible process mistakes

Very simple

Only one hydrolysate route can be achieved and hence only one product (at any DH value) Different pH optima of the various proteases cannot be taken advantage of Different temperature optima of the various proteases cannot be taken advantage of Different proteases will digest each other reducing their effectiveness

Can take advantage of proteases with different pH and temperature optima Limited digestion of the different proteases by each other One protease produces the substrate peptides for the next protease A whole range of different hydrolysates can be produced Easier to control flavour generation and keep bitterness to a minimum

Operator error unlikely

As a flavour product, yeast extract is very complex. The basic process is yeast autolysis (Nagodawithana, 1992, the yeast’s own enzymes digest the yeast) with supplementary enzymes (of which the main one used is papain). As well as protein breakdown flavours, there are also nucleic acid breakdown products (nucleotides which are also potent flavour compounds) and sugars from carbohydrate breakdown which will, at a later stage, react with the amino acids via the Maillard reaction to form further savoury flavours. The basic process is outlined in Fig. 9.11 (Kelly, 1983). Several types of enzyme can be used to assist in the manufacture of yeast extract. Two of the types, namely the protease and carbohydrase, are there to increase the yield. The remaining enzymes are used to create differently tasting yeast extracts. These are summarised in Table 9.7

9.9

Future trends

The readily available use of GM technology in the development and production of

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Fig. 9.11

Table 9.7

201

Outline procedure for the manufacture of yeast extract.

Enzymes used in yeast extract manufacture

Enzyme type (Source)

Reaction

Purpose of addition

Papain (Papaya)

Broad specificity protease

Will further digest the yeast protein and increase the yield of extract to about 70%

Peptidase (Aspergillus)

Releases amino acids from peptides

Can improve the flavour of the extract

Glutaminase (Bacillus)

Converts glutamine to glutamic acid

Improves flavour of extract

Ribonuclease Converts nucleic acid to nucleotides (Penicillium)

Produces nucleotide flavour components

Deaminase (Aspergillus) Glucanase (Bacillus)

Inosinic acid is a strong flavour ingredient Increases yield of solubles and gives extra sugars for Maillard reaction

Converts adenylic acid to inosinic acid Breaks down glucan (from cell wall)

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enzymes has made the development of new enzymes cheaper and faster than ever. Previously every new enzyme identified from a new microbial source had to go through its own fermentation and purification development process. Invariably the yield from any wild-type organism was never economically viable and a whole expensive process of strain improvement had to be gone through. Whereas the first GM enzyme (detergent protease by Genencor) cost over US$10m to develop, the costs now are substantially less than US$1m. Having a cheap development route for enzymes for the flavour industry is important as often the enzyme cost is a small percentage of the overall process of making a flavour. For example, if a flavour has a US$50m market and costs US$25m to make and the enzyme contribution to the cost is 4% then the enzyme will have a market value of US$1m. This is not large enough to generate a decent return on the cost of development, particularly if regulatory approval is required in many countries around the world. As the cost of enzyme development has come down, the balance of costs between scientific development and regulatory approval has changed. When enzyme development costs were high, the cost of regulatory approval, whilst still being expensive, was a minor part of the total cost. It is now more on a par with the cost of enzyme development. However, within the EU, where currently there are no pan-European enzyme regulations, enzymes used in the manufacture of flavours and food additives will not need to be approved for food use as the flavour or additive has to be. The new pending EU enzyme regulations only apply to enzymes added to food and not used to make flavours or food ingredients. Thus these new regulations will greatly reduce the total development package for enzymes for flavour manufacture. Whilst there are concerns in some countries about eating GMO foods, this is different from eating foods containing enzymes produced by GMMs (genetically modified microorganisms). Enzymes made using new GM technology are purer and cheaper to make than with the old wild-type microorganism technology and less energy is consumed in their manufacture. The GMMs are totally contained and none of the modified DNA ends up in the enzyme let alone the finished food. Enzymes made in this way have been available in Nature for thousands of years; the only thing that has changed in the way they are made. The same cannot be said for enzymes created by directed evolution (genetic shuffling); here enzymes are made with a novel amino acid sequence possibly never seen in Nature before, so we are truly dealing with totally novel enzymes and these should be treated cautiously. This technology, although currently suitable for industrial enzymes, is not tried and tested as a way of making enzymes safe for the food industry.

9.10 Sources of further information and advice The following websites are worth exploring: www.biocatalysts.com www.genencor.com www.amano-enzyme.co.jp www.novozymes.com

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Recommended further reading: Cheese: Chemistry, Physics and Microbiology (Volumes 1 and 2), edited by P. F. Fox, Chapman and Hall, London, 1993. Chemistry and Technology of Flavours and Fragrances, edited by D. Rowe, Blackwell Publishing, 2004. Enzymes in Food Processing (2nd edition), G. A. Tucker and L. F. J. Woods, Springer, 1995, Enzymes in Industry, edited by W. Aehle, Wiley VCH, 2004. Enzymic Hydrolysis of Food Proteins, J. Adler-Nissen, Elsevier Applied Science Publishers, London, 1986. Industrial Enzymology (Edition 1), edited by T. Godfrey and J. Reichelt, MacMillan Publishers, London, 1983. Industrial Enzymology (Edition 2), edited by T. Godfrey and S. West, MacMillan Publishers, London, 1996. Process Cheese, Vincent L. Zehren and D. D. Nusbaum, Cheese Reporter Publishing, Madison, 1992.

9.11 References Ajinomoto Coop. Inc. (1985), Japan Patent 60-62998. Ames, J. M. (1990), Trends in Food Science and Technology, 1, 150–4. Barfoed, H. C. (1983), in T. Godfrey and J. Reichel (eds), Industrial Enzymology, Chap 4.7, The Nature Press, London. Biocatalysts Limited, (2005), Technical Bulletin 110, The Use of Enzymes in Vanilla Extraction, Biocatalysts, Cardiff. Biocatalysts Limited, (2006), Technical Bulletin 106, The Use of Enzymes in the Extraction of Chondroitin Sulphate, Biocatalysts, Cardiff. Cheetham, P. S. J. (1993), TIBTECH, 11, 478 – 482. Dalgliesh, D. G. (1993), in P. F. Fox (ed.), Cheese: Chemistry, Physics and Microbiology, Volume 1, Chap 3, Chapman and Hall, London. Dewis, M. L., (2004), in D. Rowe (ed.), Molecules of Taste and Sensation in Chemistry and Technology of Flavours and Fragrances, Blackwell Publishing, Oxford. FDA (2003), Title (CFR) 21, part 133. Fox, P. F. (1984), in B. J. F. Hudson (ed.), Developments in Food Proteins, Chap 3, Elsevier Applied Science Publishers, London. Fox, P. F. (1993), in P. F. Fox (ed.), Cheese: Chemistry, Physics and Microbiology, Volume 1, Chap 10, Chapman and Hall, London. Gatfield, I. L. (1988), Food Technology, October, 110–122, 169. Godfrey, T. (1990), European Food and Drink Review, Autumn, 5–8. Gripon, J. C. (1993), in P. F. Fox (ed.), Cheese: Chemistry, Physics and Microbiology, Volume 1, Chap 4, Chapman and Hall, London. Hacking, A. J. (1986), in Economic Aspects of Biotechnology, Chap 5, Cambridge University Press, Cambridge. Han, X-Q, Silver, R. S. and Brown P. H. (2001), US Patent No. 6,251,445. Hannon, J. A., Kilcawley, K. N. , Wilkinson, M. G., Delahunty, C. M. and Beresford, T. P. (2006), J. Dairy Science, 89, 3749–3762. Hardingham, T. E., Fosang, A. J. and Dudhia, J. (1994), Eur. J. Clin. Chem. Clin. Biochem., 32, 249–257. Heath, H. B. and Reineccius, G. (1986), in Flavour Chemistry and Technology, Chap 9, MacMillan Publishers, Westport. Hepner, L. (2007), Feed Amino Acids Market Research Report, Leo Hepner and Associates, London. Hodge, W. H. (1975), Int. Flavours Food Additives, 6, 244–245.

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Hoffman, P. and Mashburn, T.A. (1967), The Journal of Biological Chemistry. 242, 3805– 3809. Jin-Ho Jo. et al. (2004), Food Science Biotechnology, 13, 622–626. Kant, R. (2005), Nutrition J, 4, 5–8. Kelly, M. (1983), in T. Godfrey and J. Reichelt (eds), Industrial Enzymology (Edition 1) Chapter 4.24, MacMillan Publishers, London. Khare, A. B. (2004), International Patent WO 2004/044009 A1 Kilara, A. (1985), Process Biochemistry, October, 149–157. Kusumoto, I. (2001), Journal of Nutrition, 131, 2552–2555. Lauder, R. M., Huckerby, T. N., Brown, G. M., Bayliss, M. T. and Nieduszynski, I. A. (2001), Biochem. J., 358, 523–528. Li, X., Staszewski, L., Xu, H., Durick, K., Zoller, M. and Adler, E. (2002), Proc Natl Acad Sci USA, 99(7), 4692–4696, April. Lippiello, L., Woodward, J., Karpman, R. and Hammad, T. A. (2000), Clinical Orthopedics and Related Research, 381, 229–240. Luo, X. M., Fosmire, G. J. and Leach, R. M. (2002), Poultry Science, 81, 1086–1089. Morreale, P., Manopulo, R., Galati, M., Boccanera, L., Saponati, G. and Bocchi, L. (1996), The Journal of Rheumatology, 23, 1385–1391. Nagodawithana, T. W. (1992), Food Technology, November, 138–144. Nagodawithana, T. W. (1995a), Savoury Flavours, Chapter 6, p 249, Esteekay Associates, Milwaukee. Nagodawithana, T. W. (1995b), Savoury Flavours, Chapter 5, p 211, Esteekay Associates, Milwaukee. Nagodawithana, T. W. (1995c), Savoury Flavours, Chapter 1, p 11, Esteekay Associates, Milwaukee. Nagodawithana, T. W. (1995d), Savoury Flavours, Chapter 6, p 226, Esteekay Associates, Milwaukee. Pal, M. A. (2002), Int. J. Food Sci. Technol., 37, 229–237. Reps, A. (1993), in P. F. Fox (ed.), Cheese: Chemistry, Physics and Microbiology, Volume 2, Chap 5, Chapman and Hall, London. Reyne, V., Salles, C. and Crouzet, J. (1992). ‘Formation of aroma by hydrolysis of glycosidically bound components’, in G. Charalambous (ed.), Food Science and Human Nutrition, Elsevier Science Publishers, Amsterdam. Riley, K. A. and Kleyn, D. H. (1989), Food Technology, October, 64–77. West, S. I. (1996), Industrial Enzymology, MacMillan Press Ltd, London, Chapter 2.8, p 161, Ronca, G. and Conte, A. (1993) Int J. Clin Pharm. Res, 13, 27–34. Schultz, M. (2005), Food Product Design, April. West, S. I. and Pawlett, D. (1996), Industrial Enzymology, MacMillan Press Ltd, London, Chapter 2.12, p 220.

10 Applications of cold-adapted proteases in the food industry A. Guðmundsdóttir and J. Bjarnason, University of Iceland, Iceland

10.1 Introduction Enzyme technology has been an integral part of food processing and improvement of food quality for a long time and advances in enzyme technology will be important for the production of better and safer foods. Furthermore, utilization of by-products from the fishing and agricultural industries for added value largely depends on a thorough understanding of enzyme reactions. Relatively few enzymes are used in a wide variety of food processes such as cheese making, brewing, for the production of corn syrup and speciality fats, and to facilitate juice extraction from fruits, in addition to their use as antimicrobial preservatives (Table 10.1). Notably, the most common food processing enzymes are hydrolases including the proteases, glucosidases and lipases. Proteolytic cold-adapted ‘superactive’ enzymes from marine sources offer advantages for the food industry although their purification and structure determination are often problematic (Bjarnason et al., 1993; Smalås, 2000). This is mainly due to their suceptibility to thermal inactivation and autolytic degradation beyond other enzymes. As proteases are commonly used in industry and medicine (Bickerstaff, 1987), it is important to understand the relationship between their structure and function (Aghajari et al., 2003). Such knowledge may, for example, be used for improvement of the enzymes through the use of gene technology for better functioning of the enzymes in food systems (Wong, 1995). The cold-adapted Atlantic cod (Gadus morhua) serine proteases will be discussed

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Table 10.1 Important industrial food enzymes and their applications (Wong, 1995) Enzyme Proteases Chymosin Papain

Applications and functions Cheese making – curding of milk by specific proteolysis of κ-casein In meats – as a tenderizer Brewing – to prevent chill haze formation by digesting proteins reacting with tannic substances that may result in insoluble colloid particles

Microbial proteases Processing of raw animal and plant proteins. Production of fish meals, meat extracts, texturized proteins and meat extenders Atlantic cod Cryotin Seafood flavourants (pastes) Glucosidases α-amylase

Glucoamylase

β-amylase β-glucanase Lipases Lipase

Other enzymes Pectinase

Production of corn syrup (liquefaction) – conversion of starch to dextrins Dough making – additive to flour low in α-amylase to ensure a continous supply of fermentable sugar for yeast growth and gas production Brewing – solubilization of barley and other cereal carbohydrates Production of corn syrup (saccharification) – conversion of dextrins to glucose Brewing of light (low carbohydrate) beer – conversion of residual dextrins to fermentable sugar Production of high maltose syrup Brewing – breakdown of β-glucans in malt to aid filtration of wort after mashing Enhancement of cheese ripening – shorter time of flavour development and ripening time Production of specialty fats – improved qualities Enzyme modified cheese and butter from cheese curd and butterfat

Fruit juice production – treatment of fruit pulp to facilitate juice extraction and for clarification and filtration of juice Lactase An additive – in dairy products for lactose intolerant individuals Breaktown of lactose in whey products for manufacturing polylactide Acetolactate In wine making – reducing maturation time by converting acetolactate decarboxylase to acetoin Lysozyme Antimicrobial preservative Glucose oxidase In egg products – conversion of glucose to gluconic acid to prevent Maillard browning in heat dehydrated egg products In food packaging – removal of O2 to potentially protect against oxidative deteriation Xylose (glucose) Production of high fructose corn syrup – isomerization of glucose to isomerase fructose Cellulases Ethanol or single cell protein production – conversion of cellulose wastes to fermentable feedstock

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in relation to their use for food production. Some examples of applications of those and other enzymes will also be addressed. Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted trypsins (Ásgeirsson et al., 1989). Overexpression of the recombinant form of cod trypsin I in microorganisms is explained, as well as the advantages of using site-directed mutagenesis to increase its stability towards autolysis and thermal inactivation (Jónsdóttir et al., 2004; Pálsdóttir and Guðmundsdóttir, 2004). The Atlantic cod is an economically important fish species in Iceland and elsewhere in the northern hemisphere. The pyloric caeca, serving the role of a digestive organ in the Atlantic cod, is a by-product from the fishing industry found in abundance in Iceland. As should be expected, it is rich in digestive enzymes such as serine proteases. Serine proteases from the Atlantic cod such as trypsin (Ásgeirsson et al., 1989), chymotrypsin (Ásgeirsson and Bjarnason, 1991), elastase (Ásgeirsson and Bjarnason, 1993) and serine collagenase (Kristjánsson et al., 1995) have typical characteristics of cold-adapted enzymes. The serine proteases play important roles in a number of biological functions including digestion (Neurath, 1984) where trypsin has a dual role in that it cleaves ingested proteins and activates the precursor forms of several other digestive proteases including chymotrypsin. The Atlantic cod is known to produce numerous trypsin isozymes (Ásgeirsson et al., 1989; Guðmundsdóttir et al., 1993; Helgadóttir, 2002). Several of these have been isolated from their native source, with trypsin I being predominant. It also has the highest catalytic efficiency and is by far the best characterized of the trypsin isozymes (Ásgeirsson et al., 1989; Jónsdóttir et al., 2004). The cDNAs of two trypsin isozymes (I and X) (Guðmundsdóttir et al., 1993) in addition to a novel trypsin, termed trypsin Y (Spilliaert and Guðmundsdóttir, 1999), have been isolated from a cod pyloric caeca cDNA library. Characterization of the recombinant trypsin Y polypeptide demonstrated that it is very different from classical trypsins such as trypsin I (Pálsdóttir and Guðmundsdóttir, 2004) and it may be the digestive enzyme produced under cold-shock conditions (Roach, 2002). In general, trypsins from the Atlantic cod and other fish adapted to cold environments differ somewhat from their mammalian analogues in that they have higher catalytic efficiencies, especially at low temperatures (Ásgeirsson et al., 1989; Schrøder Leiros et al., 2000; Gerday et al., 2000). These enzymes are also more sensitive to inactivation by heat, low pH and autolysis than their mesophilic analogues (Simpson et al., 1989; Ásgeirsson et al., 1989). In addition, native proteins are readily hydrolysed by the cold-adapted fish proteases. These traits and the fact that the cold-adapted enzymes do indeed function properly at low temperatures have stimulated an interest in their commercial use as they are generally better suited for enzymatic processes than their mesophilic counterparts (Bjarnason et al., 1993; Bjarnason, 2000a,b; Bjarnason and Benediktsson, 2001; Shahidi and Janak Kamil, 2001). Data presented on the expression of Atlantic cod trypsin I (Jónsdóttir et al., 2004) and trypsin Y (Pálsdóttir and Guðmundsdóttir, 2004) are, to our best knowledge, the first reports on the expression of psychrophilic or cold-adapted proteolytic enzymes

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from fish in an active form. The reason for the lack of other publications in this area may be difficulties related to the sensitivity of cold-adapted proteases to autolytic degradation, thermal inactivation as well as molecular aggregation (Ásgeirsson et al., 1989; Ásgeirsson and Bjarnason, 1991; Kristjánsson et al., 1995; Helgadóttir, 2002). The fact that the problems with the expression of cod trypsins I and Y have been largely overcome opens up a new era for future studies and applications of these enzymes, in particular in the pharmaceutical field. Site-directed mutagenesis of their cDNAs may be used to produce new and improved recombinant enzyme variants (Narinx et al., 1997; Benjamin et al., 2001; Gerike et al., 2001). Atlantic cod trypsin I has already proven its usefulness in industrial (Bjarnason et al., 1993; Bjarnason and Benediktsson, 2001) and medical (Bjarnason, 2000a) applications as will be discussed later in this review. Other cold-adapted proteolytic enzymes have been applied as processing aids in the food and feed industries as thoroughly described in a review by Shahidi and Janak Kamil (2001).

10.2 Use of proteolytic enzymes in food processing Hydrolytic enzymes, especially proteases, have many uses and potential applications in industry, medicine and research. Among these applications are detergent production, leather processing, chemical modifications, food processing and drug development (Bjarnason, 2000b). One of the oldest and most successful examples of protease use in the food industry is that of rennet, an enzyme found in the stomach of calves that causes milk to curdle during the production of cheese. Rennet mainly consists of chymosin with a small amount of pepsin (Fox, 1988). Papain from the leaves and the unripe fruit of papaya (Carica papaya) has also been used as meat tenderizer for a long time (Glazer and Smith, 1971). Proteases can be used at various pH values and they may be either highly specific in their choice of cleavable peptide links or quite non-specific. Previously, enzymes were mainly isolated from plant as well as animal sources and relatively few expensive enzymes were available to the food processor. Developments in enzyme technology have led to applications in the food industry of a wide range of new proteases from many sources, mostly microbial. Proteolysis of inexpensive materials such as soya proteins can increase the range and value of their usage, as indeed occurs naturally in the production of soy sauce. Partial hydrolysis of soya proteins greatly increases their whipping qualities and further hydrolysis improves their emulsifying capacity (Aoki et al., 1980). Soya protein hydrolysates may also be added to cured meats. Proteases are used to recover proteins from parts of fish and land animals that would otherwise go to waste after slaughter. About 5% of the meat can be removed mechanically from animal bones. The meat slurry produced can be used in canned meats and soups or other foods. Also, large quantities of blood are available but, it is not generally acceptable in foodstuffs because of its colour. Blood proteins are of high nutritional quality and can be de-haemed using the enzyme subtilisin, but excessive degradation is avoided to prevent the formation of bitter peptides (Wong, 1995).

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Dough may be prepared more quickly if its gluten is partially hydrolysed. A heat-labile fungal protease is used so that it is inactivated early in the subsequent baking. Weak-gluten flour is required for biscuits in order to be able to spread the dough thinly and retain decorative impressions. In the past this has been obtained from European domestic wheat but this is being replaced by high-gluten varieties of wheat. The gluten in the flour derived from these must be extensively degraded if such flour is to be used efficiently to make biscuits or to prevent shrinkage of commercial pie pastry.

10.3 Application of cold-adapted serine proteases in food processing Enzymes isolated from cold water marine organisms may prove to be especially useful for food processing purposes. The cold-active or psychrophilic enzymes are frequently more active at low temperatures than their mammalian or bacterial counterparts, a characteristic which could be beneficial in many industrial processes and medical applications. Atlantic cod serine proteases, such as trypsin and chymotrypsin have already proven their usefulness in various industrial applications (Bjarnason et al., 1993; Bjarnason and Benediktsson, 2001). The high catalytic efficiency of Atlantic cod trypsin I is especially useful in the processing of fresh foods where protein digestion at low temperatures is required (Bjarnason et al., 1993; Bjarnason and Benediktsson, 2001). Food processing at low temperatures minimizes undesirable chemical reactions as well as bacterial contamination which may indeed be elevated at higher temperatures. In cases where the enzymatic activity needs to be controlled, the cold-adapted enzymes are easily inactivated by relatively low heat. Also, cold-adapted proteolytic enzymes are in most cases more economical as their high catalytic efficiencies facilitate the use of lower amounts of enzymes than are required using analogous mesophilic enzymes. A mixture of proteases is already being used in a patented process to produce seafood flavours, bases and stocks for the food industry (Bjarnason and Benediktsson, 2001). These products, called NorthTaste, are already available on the international market and consist of natural digests of seafood such as lobster, shrimp, crab and cod, containing no additives. Other uses of cold-adapted proteolytic fish enzymes in the food and feed industries have been reviewed by Shahidi and Janak Kamil (2001). More recently it was shown that three cold-adapted serine protease preparations, cod chymotrypsin, krill trypsin and cryotin, a protease mixture from cod, are effective against Pseudomonas aeruginosa biofilms in the absence of milk (Augustin and Ali Vehmas, 2004).

10.3.1 Cryotin from Atlantic cod A mixture of proteolytic digestive enzymes, called cryotin, prepared by neutral extraction from the pyloric caeca of Atlantic cod (Gadus morhua) has many unique

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Table 10.2 Definition of different cryotins from Atlantic cod (Gadus morhua) Samples

Definition

Crude cryotin Cryotin A Cryotin B Cryotin C Cryotin D Cryotin E Cryotin F Cryotin G Cryotin H

Trypsin, chymotrypsin, elastase and serine collagenase Chymotrypsin, elastase and serine collagenase Concentrated purifed elatase (CM column) Purified chymotrypsin Mixture of cryotin B (elastase) and cryotin C (chymotrypsin) Concentrated cryotin A Cryotin A + crude cryotin ( ratio 9:1) Purified elastase (CM column) Purified trypsin

CM = carboxyl methyl

characteristics (Table 10.2). Cryotin has been shown to contain trypsin, chymotrypsin, elastase and, perhaps most importantly, collagenolytic enzymes, as well as other proteolytic and peptidolytic activities. However, it is practically devoid of lipase, amylase and nuclease activities (Ásgeirsson et al., 1989; Ásgeirsson and Bjarnason, 1991; Ásgeirsson and Bjarnason, 1993; Kristjánsson et al.,1995). The proteinases in the mixture, studied so far, are more active at low temperatures than their mammalian counterparts. They are also thermolabile as well as acid sensitive. Crude cryotin, is prepared by neutral aqueous extraction from the Atlantic cod byproducts, in particular digestive organs such as the pyloric caeca. In one study, a novel cryotin A protease mixture, derived from crude cryotin, and several purified protease preparations, called cryotins B to H derived from cryotin A and crude cryotin, were developed to test their use in cheese ripening, leather processing and skinning of squid. These cryotin preparations (Table 10.2) contained various amounts and proportions of the proteases trypsin, chymotrypsin, elastase and serine collagenase in differing degrees of purity. Cryotin A contains chymotrypsin, elastase and serine collagenase, cryotin B and G contain purified elastase in differing concentrations, cryotin C contains purified chymotrypsin and cryotin H contains purified trypsin, all from Atlantic cod. Stable formulations of the cryotins have been developed for storage, transport and use. Purification processes for various cryotin derivatives have been developed, as well as formulations for the cryotins for the purpose of stabilizing the proteinase activities of trypsin, chymotrypsin and elastase in the different cryotins (Bjarnason, 2004; Rodriguez et al., 2004). The cold-active proteases, in the various cryotin formulations, have many other potential uses in industry, medicine and research, especially in food processing applications that require hydrolysis at low temperatures, inactivation under mild conditions or native collagen digestion. Cryotin has, for example, proven promising in various fish processing applications such as skinning of fish, removal of membranes and ripening of herring. Cryotin also has potential as a digestive aid, both for humans and animals. It is now being tested as an adjunct in microdiets for fish larvae and in the preparation of fish feed. Cryotin is presently used in a patented process to prepare high quality all-natural flavourings for food processing and innovative cooking (Bjarnason and Benediktsson, 2001).

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Various additional food processing applications are being considered, such as in the chill-proofing of beer, biscuit manufacture, tenderizing of meats, preparation of minimally treated fruit and vegetable beverages and hydrolysis of various food proteins, such as gelatin, vegetable proteins and collagens.

10.4 Modifying marine proteases for industrial use The thermal stability of proteins is determined by factors such as structural stability as well as stability or resistance to chemical degradation processes. In the case of proteases, autolysis also plays a major role in their stability (Abraham and Breuil, 1995). Autolysis is known to be one of the regulatory mechanisms for the protease activity of serine proteases (Halfon and Craik, 1998). The ‘autolysis loop’ in trypsins, composed of residues 144–154, is a relatively flexible structure considered to be a primary autolysis target (Smalås et al., 1994). The autolysis loop of the cold-adapted salmon trypsin has a different structure to that of its mesophilic analogues (Smalås et al., 1990). Atlantic cod trypsin I is cleaved by autolysis, predominantly at residue Lys154 in its ‘autolysis loop’ (Helgadóttir, 2002) with a secondary cleavage site at residue Lys107. A Lys154Gln mutation has been introduced into the cod trypsin I cDNA sequence. Preliminary data demonstrate that this mutation has a stabilizing effect on the recombinant trypsin I molecule (Jónsdóttir et al., 2004). Our previous research involved increasing the thermal stability of a multifunctional proteolytic enzyme from Antarctic krill (Euphausia superba), called euphaulysin, through point mutations of its cDNA sequence (Benjamin et al., 2001). Euphaulysin has a high sequence identity to a protease previously isolated from fiddler crab (Uca pugilator), called crab collagenase I (Eisen et al., 1973). These two enzymes have been classified as brachyurins, a subclass of serine proteases, owing to their broad substrate specificity and their ability to cleave collagen (Guðmundsdóttir, 2002). Loop D, extending from residues 143–153 in crab collagenase I (Perona and Craik., 1997) and euphaulysin, is analogous to the ‘autolysis loop’ of trypsins. A molecular model of euphaulysin, based on the known three-dimensional structure of crab collagenase I, was used to guide the design of amino acid substitutions in the molecule which could increase the thermal stability of the enzyme by decreasing its susceptibility towards autolysis (Benjamin et al., 2001). The model revealed that two residues of loop D, Lys143 and Phe149, might be a target for autolysis since euphaulysin has a high affinity towards Phe, Lys and Arg residues (Kristjánsdóttir, 1999). The Phe149 residue appeared to be more exposed on the surface of the molecule than the Lys143 residue and euphaulysin is the only type I brachyurin known to contain a Phe residue at position 149. This difference may in part be responsible for the decreased stability of the coldadapted euphaulysin relative to crab collagenase I. Euphaulysin had previously been shown to have a low affinity for Asp and Glu residues (Kristjánsdóttir, 1999). Thus, two mutations were incorporated into the cDNA encoding the

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precursor form of recombinant euphaulysin, resulting in Phe149Glu and Phe149Asp substitutions in the enzyme. The precursor forms of the two mutants were expressed in a Pichia pastoris expression system and fully activated by cod trypsin as previously described (Kristjánsdóttir and Guðmundsdóttir, 2000) for the wild-type recombinant euphaulysin. Interestingly, the amounts of the mutated recombinant euphaulysin forms recovered in the expression experiments were approximately two times greater than that of the wild-type recombinant euphaulysin under the same growth conditions. This indicates that the Phe149Asp and Phe149Glu mutants are more stable towards autolysis during expression than the wild-type form of the enzyme. The melting temperature, Tm, of the Phe149Glu mutant (48 °C) was approximately 5 °C higher than that of the wild-type recombinant euphaulysin (43 °C). For comparison, the Tm of the Phe149Asp mutant was around 2.5 °C higher than that of the wild-type recombinant enzyme or the native enzyme (Benjamin et al., 2001).

10.5 Future trends The tremendous knowledge gathered on enzyme structures and function in recent years has opened up a new era for food processing applications with enzymes. A new phase of enzyme technology may revolutionize the ways foods are prepared and processed in the near future. It is becoming easier to obtain relatively large numbers of naturally rare proteins, crystallography requires smaller amounts of purified proteins and smaller crystals are easier to work with than ever before. The database of known protein structures is expanding rapidly leading these to be more meaningful questions that need to be answered at the atomic level of resolution. An understanding of the principles of protein structure is important in molecular biology, drug development and food processing. The high catalytic efficiency of cold-adapted serine proteases is especially useful in the processing of fresh foods where protein digestion at low temperatures is required. Food processing at low temperatures minimizes undesirable chemical reactions as well as bacterial contamination that may indeed be elevated at higher temperatures. In cases where the enzymatic activity needs to be controlled, the cold-adapted enzymes are easily inactivated by relatively low heat. Also, cold-adapted proteolytic enzymes are in most cases more economical as their high catalytic efficiencies facilitate the use of smaller amounts of enzymes than are required using analogous mesophilic enzymes.

10.6 References Abraham, L.D. and Breuil, C. (1995). ‘Factors affecting autolysis of a subtilisin-like serine proteinase secreted by Ophiostoma piceae and identification of the cleavage site.’ Biochim Biophys Acta, 1245, 76–84. Aghajari, N., Van Petegem, F., Villeret, V., Chessa, J.P., Gerday, C., Haser, R. and Van Beeumen. J. (2003). ‘Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases’. Proteins, 50(4), 636–647.

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Aoki, H., Taneyana, O. and Inami, M. (1980). ‘Emulsifying properties of soy protein: characteristics of 7S and 11S proteins’. J Food Sci, 45, 534–540. Ásgeirsson, B. and Bjarnason, J.B. (1991). ‘Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin’. Comp Biochem Physiol B, 99(2), 327–335. Ásgeirsson, B. and Bjarnason, J.B. (1993). ‘Properties of elastase from Atlantic cod, a cold adapted proteinase’. Biochim Biophys Acta, 1164(1), 91–100. Ásgeirsson, B., Fox, J. and Bjarnason, J.B. (1989). ‘Purification and characterization of trypsin from the poikilotherm Gadus morhua’. Eur J Biochem, 180(1), 85–94. Augustin, M. and Ali Vehmas, T. (2004). ‘Assessment of enzymatic cleaning agents and disinfectants against bacterial biofilms’. J Pharm Pharmaceut Sci, 7(1), 55–64. Benjamin, D.C., Kristjánsdóttir, S. and Guðmundsdóttir, Á. (2001). ‘Increasing the thermal stability of euphauserase: a cold active and multifunctional serine protease from Antarctic krill’. Eur J Biochem, 268(1), 127–131. Bickerstaff, G.F. (1987). Enzymes in Industry and Medicine, Edward Arnold, Victoria, Australia. Bjarnason, J.B. (2004). Pharmaceutical and Cosmetic Composition Comprising Cod Serine Proteases and their Pharmaceutical and Cosmetic Use, European Patent 1 202 743 B1, filed 15 June 2000; issued: 6 October 2004, Bulletin 2004/41, European Patent Office. Priority: 18.06.1999, IS 508699. Bjarnason, J.B. (2000a). Fish Serine Proteases and their Pharmaceutical and Cosmetic Use, PCT Patent, WO 00/78332 A2 (28 December 2000). Bjarnason, J.B. (2000b). ‘Biotechnological applications of fish offal in Iceland’, in Proceedings from a Nordic Conference: Verdiskaping av marine biprodukter etter År 2000. Held in Stjördal, Norway on January 24–25. Bjarnason, J.B. and Benediktsson, B. (2001). Protein Hydrolysates Produced with the Use of Marine Proteases. PCT Patent, WO 01/28353 A2 (26 April 2001). Bjarnason, J.B., Asgeirsson, B., Kristjansson, M.M., Guðmundsdottir, Á, Fox, J.W., Chlebowski J.F. and Craik, C.S. (1993). ‘Characteristics, protein engineering and applications of psychrophilic marine proteinases from Atlantic cod’. in W.J.J. van den Tweel, A.Harder and R.M. Buitelaar (eds), Stability and Stabilization of Enzymes, Proceedings of an International Symposium held in Maastricht, The Netherlands. Elsevier Science B.V, The Netherlands, 205–214. Eisen, A.Z., Henderson, K.O., Jeffrey, J.J. and Bradshaw, R.A. (1973). ‘A collagenolytic protease from the hepatopancreas of the fiddler crab, Uca pugilator. Purification and properties’. Biochemistry, 12(9), 1814–1822. Fox, P.F. (1988). ‘Rennets and their action in cheese manufacture and ripening’. Biotechnol Appl Biochem, 10, 522–535. Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J.P., Claverie, P., Collins, T., D’Amico, S., Dumont, J., Garsoux, G., Georlette, D., Hoyoux, A., Lonhienne, T., Meuwis, M.A. and Feller, G. (2000). ‘Cold-adapted enzymes: from fundamentals to biotechnology’. Trends Biotechnol, 18(3), 103–107. Gerike, U., Danson, M.J. and Hough, D.W. (2001). ‘Cold-active citrate synthase: mutagenesis of active-site residues’. Protein Eng, 1489, 655–661. Glazer, A.N. and Smith, E.L. (1971). ‘Papain and other plant sulfhydryl proteolytic enzymes’. The Enzymes, 3, 501–547. Guðmundsdóttir, Á. (2002). ‘Cold-Adapted and Mesophilic Brachyurins’. Biol Chem, 383(7–8), 1125–1131. Guðmundsdóttir, Á., Guðmundsdóttir, E., Óskarsson, S., Bjarnason, J.B., Eakin, A.K. and Craik, C.S. (1993). ‘Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen’. Eur J Biochem, 217(3), 1091–1097. Halfon, S. and Craik, C.S. (1998). ‘Family S1 of trypsin (clan SA)’, in Barrett, A.J., Rawlings, N.D. and Woessner, J.F. (eds), Handbook of Proteolytic Enzymes, Academic Press, San Diego CA, 5–12.

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Helgadóttir, L. (2002). Trypsin I from Atlantic cod: Purification, Specificity, Stability and Autolysis. MSc Thesis, Department of Chemistry, Faculty of Sciences, University of Iceland, Reykjavík, Iceland. Jónsdóttir, G., Bjarnason, J.B. and Guðmundsdóttir, Á. (2004). ‘Recombinant cold-adapted trypsin I from Atlantic cod – expression, purification and identification’. Protein Expression Purification, 33(1), 110–122. Kristjánsdóttir, K.S. (1999). Isolation and Characterization of a Broad Specificity Collagenolytic Protease from Antarctic Krill. MSc Thesis, Department of Chemistry, Faculty of Sciences, University of Iceland, Reykjavík, Iceland. Kristjánsdóttir, S. and Guðmundsdóttir, Á. (2000). ‘Propeptide dependent activation of the Antarctic krill euphauserase precursor produced in yeast’. Eur J Biochem, 267(9), 2632– 2639. Kristjánsson, M.M., Guðmundsdóttir, S., Fox, J.W. and Bjarnason, J.B. (1995). ‘Characterization of a collagenolytic serine proteinase from Atlantic cod (Gadus morhua)’. Comp Biochem Physiol B Biochem Mol Biol, 110(4), 707–717. Narinx, E., Baise, E. and Gerday, C. (1997). ‘Subtilisin from psychrophilic Antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold’. Protein Eng, 10(11), 1271–1279. Neurath, H. (1984). ‘Evolution of proteolytic Enzymes’. Science 224, 350–357. Pálsdóttir, H.M. and Guðmundsdóttir, Á. (2004). ‘Recombinant trypsin Y from Atlantic cod – properties for commercial use’. J Aquat Food Prod Technol, 13(2), 85–100. Perona, J.J. and Craik, C.S. (1997). ‘Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold’. J Biol Chem, 272(48), 29987–29990. Roach, J.C. (2002). ‘A clade of trypsins found in cold-adapted fish’. Proteins, 47, 31–44. Rodriguez, I., Ferreiro, S., Cochard, S., Roghe, R., Bjarnason, J.B. and Benediktsson, B. (2004). Use of New Enzymatic Processes in the Food Industry (EU CRAFT research project, ENZYPRO, QLK1-CT-2002-70871). Actas dle III Congreso Espanol de Ingeniería de Alimentos, Pamplona. Schrøder Leiros, H.K., Willassen, N.P. and Smalås, A.O. (2000). ‘Structural comparison of psychrophilic and mesophilic trypsins. Elucidating the molecular basis of cold adaptation’. Eur J Biochem, 267(4), 1039–1049. Shahidi, F. and Janak Kamil Y.V.A. (2001). ‘Enzymes from fish and aquatic invertebrates and their application in the food industry’. Trends Food Sci Technol, 12(12), 435–464. Simpson, B.K., Simpson, M.V. and Haard, N.F. (1989). ‘On the mechanism of enzyme action: digestive proteases from selected marine organisms’. Biotechnol Appl Biochem, 11, 226–234. Smalås, A.O., Hordvik, A., Hansen, L.K., Hough, E. and Jynge, K. (1990). ‘Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the North Atlantic Salmon (Salmon salar)’. J Mol Biol, 214(2), 355–358. Smalås, A.O., Heimstad, E.S., Hordvik, A., Willassen, N.P. and Male, R. (1994). ‘Cold adaptation of enzymes: structural comparison between salmon and bovine trypsins’. Proteins, 20(2), 149–166. Smalås, A.O., Schrøder Leiros, H.K., Os, V. and Willassen, N.P. (2000). ‘Cold-adapted enzymes’. Biotechnol Annu Rev, 6, 1–57. Spilliaert, R. and Guðmundsdóttir, Á. (1999). ‘Atlantic cod trypsin Y – member of a novel trypsin group’. Marine Biotechnol, 1(6), 598–607. Wong, D.W.S. (1995). Food Enzymes – Structure and Mechanism, Chapman and Hall, USA.

11 Health-functional carbohydrates: properties and enzymatic manufacture Simon Hughes and Robert A. Rastall, University of Reading, UK

11.1 Introduction For the past 40 years there has been an increased understanding of the sources, processing, uses and physiological effects of dietary fibre. However, it is only since about the mid-1990s that certain dietary fibres have been awarded functional food status, and have been regarded as health promoting ingredients in staple foods like bread and breakfast cereals, as well as in foods designed for the treatment of particular physiological or medical conditions, such as celiac sensitivity, ulcerative colitis, gastric infections and weight control. When dietary fibre is added to food it should provide a health benefit without sacrificing the food’s qualities, for example taste, appearance, and texture characteristics. In this chapter the potential for using enzyme technology to realize and extend the health benefits of dietary fibre will be discussed.

11.2 Dietary fibre The carbohydrates of interest in this chapter come under the heading of dietary fibre and their functional attributes are primarily exerted upon the colon, whereby consequent systemic effects may ensue. The currently accepted definition for dietary fibre is ‘any dietary component that reaches the colon without being absorbed in a healthy human gut’. Dietary fibre is characterized in three ways:

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Fig. 11.1 Schematic division of plant carbohydrates (McLean Baird and Ornstein, 1981). Of the plant carbohydrates, dietary fibre primarily includes all non-starch polysaccharides (NSPs) and resistant starch.

(i) whether or not it is digested by the colonic microbiota, (ii) its origin and (iii) its chemical characteristics (Ha et al., 2000). Indeed, the largest contribution to dietary fibre is from plant cell wall material (the division of plant carbohydrates is illustrated in Fig. 11.1). Physically entrapped materials (starch, sugars, protein, minerals) also reach the colon undigested and come under the heading ‘dietary fibre’. The indigestible plant cell wall material is of interest to the food industry as it is relatively easy to isolate and modify by industrial processing, it is cheap, and reaches the colon intact, where it may exert a health benefit (that is, primarily through microbial degradation). Dietary fibre resists digestion and absorption in the small intestine and enters the large intestine where it undergoes varying degrees of fermentation by the colonic microbiota. The main consequences of fermentation by the gut microbiota are an increased faecal bulk through both bacterial proliferation and the mechanical water holding-capacity of cellular structures, combined with short-chain fatty acid (SCFA) and gas production (Fig. 11.2) (Cummings, 1984). Dietary fibre has been shown to improve health and prevent certain diseases; beneficial physiological effects include laxation, blood cholesterol reduction and blood glucose attenuation (Marlett et al., 2002). Continued exposure of the gut microbiota to adequate quantities of dietary fibre improves the levels and activity of selected beneficial bacterial populations over time. This has been shown to provoke steady and continued prevention of the onset of diseases like colorectal cancer (CRC) and inflammatory bowel disease (IBD) (Bingham, 1990; Hill, 1997). The features considered to be responsible for the health effects of dietary fibre

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Non-starch polysaccharides

Large intestines

Highly fermentable fibre

Microbial

SCFA

Poorly fermentable fibre

Gas

Water holding

Physical properties intact

growth

Increased digesta bulk

Increased transit time

Fig. 11.2

Increased stool bulk

Precursors to attaining increased transit time and stool bulk by non-starch polysaccharides in the large intestines.

include: water solubility, fermentability and viscosity (Davidson and Maki, 1999). Increased solubility improves the availability of fibre for microbial fermentation. Polysaccharides that increase the viscosity of the gut contents increase the waterholding capacity, which in turn increases the transit time of ingested material through the colon and increases faecal mass (Tomlin and Read, 1988).

11.3 Prebiotics ‘A prebiotic is a non-digestible food ingredient that affects the host by selectively targeting the growth and/or activity of one or a limited number of bacteria in the colon and, thus, has the potential to improve host health’ (Gibson and Roberfroid, 1995). Although prebiotics can occur naturally, there is growing interest in manufacturing them with additional functional attributes, such as affording a baking benefit (for example adjusting viscosity, fat substitute). Although prebiotics by definition can be dietary fibres as they have to reach the colon intact, selective fermentation by ‘beneficial’ colonic bacteria such as bifidobacteria and lactobacilli must also be achieved. The prebiotics with the most potential are the non-digestible oligosaccharides (NDO) such as fructo-oligosacharides (FOS) (Côté et al., 2003) and galacto-

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Distal colon

SCFA accumulation/absorption and selective microbial growth

Fast fermenting prebiotics Slow fermenting prebiotics

Accumulation/absorption of SCFA and selective microbial growth throughout large intestine

Fig. 11.3

Depiction of fermentation persistence, comparing fast and slow fermenting prebiotics, and the region of their effect.

oligosaccharides (TOS) (Hylla et al., 1998); their fermentation has been shown to selectively to promote Bifidobacterium spp. and Lactobacillus spp., which are the favoured target bacterial groups for health promotion in the colon (Gibson, 1998). These species occupy both the lumen and the mucosa of the gut, and preferentially digest soluble over insoluble food matter. Prebiotics, such as FOS, TOS and inulin, are readily fermented in the proximal colon, where the microflora are adapted to a more saccharolytic metabolism (Macfarlane et al., 1992); therefore any beneficial effects rarely persist to the distal colon (Fig. 11.3). Colonic diseases (principally ulcerative colitis and tumours) predominantly originate in the distal colon (Bufill, 1990, Roediger, 1980), consequently there is much interest in finding prebiotics that can persist to more distal regions of the colon and induce health-promoting effects, like those related to saccharolytic fermentation and prebiotic benefits. One approach to achieving the goal of prebiotic persistence is molecular weight regulation by enzymatic modification of polysaccharides (Olano-Martin et al., 2000; Wichienchot et al., 2006). This difference in persistence was demonstrated in a study comparing the effect of two dietary fibre sources on SCFA profiles and cell proliferation indices along the length of the large intestine (Lupton and Kurtz, 1993). It was hypothesized that the less fermentable fibre would have a slower yet more persistent effect in the colon. A rat model was used and supplementation of highly fermentable pectin caused increased propionate concentrations in proximal regions compared with rats fed control diets with no fibre. However when wheat bran, which is less fermentable, was introduced to the rat diets, butyrate levels increased throughout the colon, which also correlated with a greater reduction in pH and to lower cell proliferation indices. As such, dietary fibre type can influence the resulting SCFA profile and have an impact on the large intestinal epithelium in different regions of the colon.

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Table 11.1 Physicochemical characteristics of chicory and artichoke inulin

Average degree of polymerization Dry matter (%) Inulin/oligofructose content (% on d.m.) pH (10% w/w) Sweetness (vs. sucrose = 100%) Solubility in water at 25 °C (g l–1)

Standard chicory inulin

High performance chicory inulin

Artichoke inulin

12 95 92 5–7 10% 120

25 95 99.5 5–7 None 25

46 95 99.5 5–7 None 5

11.4 Inulin Inulin belongs to a class of carbohydrate compounds called the fructans and has a chain length of between 2 and 60+ fructose units with β, 2-1 links and with a glucose terminal end linked by an α, 1-2 bond, as in sucrose (Gibson and Roberfroid, 1995). Around 36 000 plants contain inulin, many of which are commonly found in human diets (Davidson and Maki, 1999), in particular plants in the Compositae family, including artichoke, chicory and dahlias (Lopez-Molina et al., 2005). Inulin functions as a storage polysaccharide in plants and, as such, inherits a polydispersity that fluctuates with the seasons, with lower molecular weight and more soluble fructosans predominating in the winter months and higher molecular weight and more insoluble fructosans prevailing during the summer months (Phelps, 1965). Variations in the physical and chemical characteristics of different inulins are shown in Table 11.1. When sheared in water or milk the higher molecular weight inulin forms microcrystals that, when eaten, provide a fat-like sensation in the mouth, yet retain a neutral flavour. Inulin has been successfully used to replace fat in many foods such as spreads, baked goods and frozen products (Lopez-Molina et al., 2005). Inulin’s prebiotic efficacy has also been well documented (Gibson et al., 1995; Yazawa et al., 1978; Yazawa and Tamura, 1982; Roberfroid et al., 1998) and its bifidogenic effect encompasses a range of related health benefits, including innate immune system stimulation, anti-mutagenic effects on colonocytes (Jenkins et al., 1999; Roberfroid, 1999), vitamin B production (Deguchi et al., 1985), inhibition of the growth of clostridia (Gibson et al., 1995; Hopkins and Macfarlane, 2003) and other pathogens (Shiba et al., 2003; Servin, 2004; van Nuenen et al., 2003), and inulin may also help reduce the hypercholesterolemic effects of some foods (Davidson and Maki, 1999). Because inulin resists digestion in the stomach and small intestine (Knudsen and Hessov, 1995; Andersson et al., 1999) and is fermented by the colonic microbiota, it could be used as a protective envelope for drugs that need to evade degradation by upper gastrointestinal tract (GIT) enzymes and acids which would otherwise prevent the effective delivery of drugs to target regions of the distal GIT. On arrival in the large intestine the protective inulin coat would be degraded by

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Fig. 11.4 Proposed pathway for the synthesis of inulin type fructans from sucrose by sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT) in Helianthus tuberosus (Jerusalem artichoke); ‘G’, glucose; ‘F’, fructose (Edelman and Jefford, 1968).

colonic bacteria, thus enabling the release and direct effect of therapeutic drugs on the site of malady (Chourasia and Jain, 2002). Diseases like CRC, Crohn’s and ulcerative colitis (UC) would benefit from direct drug treatment in this way.

11.4.1 Inulin synthesis in plants In Jerusalem artichokes, the proposed pathway for inulin-type fructan synthesis involves the enzymes sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT) (Edelman and Jefford, 1968). Starting with two sucrose molecules, a trisaccharide of 1-ketose, with β, 2-1 linkages, and one glucose molecule are liberated from the catalytic action of 1-SST. These subsequently become transferred as fructose from one fructan chain to another, which is catalysed by 1-FFT (Fig. 11.4). The resulting fructans have diverse chain lengths. This pathway was confirmed by isolating and purifying 1-SST and 1-FFT from Chicorium intybus (chicory) and synthesizing in vitro inulin-type fructans from physiologically relevant sucrose concentrations (van den Ende and van Laere, 1996); this strongly qualifies it as an in vivo pathway.

11.4.2 Novel production of inulin Inulin can also be extracted directly from its host plant, the artichoke. Inulin contributes 3% to the fresh weight of an artichoke; however, only the flower portion is eaten, leaving a considerable quantity of waste plant matter, containing a valuable amount of inulin. In Spain (which hosts a strong artichoke industry), a team endeavoured to extract inulin by milling the waste material from globe artichokes (Cynara scolymus L.) to produce an aqueous solution that underwent ultrafiltration, precipitation and phase-splitting by centrifugation, with no addition of any organic solvents, thereby keeping it an environmentally sound process (Lopez-Molina et al., 2005). The average degree of polymerization (DP) of the product was 46, which is larger than chicory- and dahlia-derived inulin, thus

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providing a useful higher molecular weight fraction of inulin for the food industry. It was also successfully degraded using exo- and endo-inulinases from Aspergillus niger to produce fructose and FOS, both of which are desirable baking ingredients. The FOS and higher molecular weight fraction of inulin both exerted bifidogenic effects, as shown by Lopez-Molina et al. (2005). Chicory and dahlias are typically used as sources of inulin; however, developing these new extraction processes from waste material could present environmentally and economically friendly opportunities for producing the prized carbohydrate, inulin.

11.4.3 Isolation of high-chain-length inulin Standard extraction, purification and isolation techniques of inulin from plants generally reduces the DP of the product by 1–1.5 (compared with native inulin) (Moerman et al., 2004). This change in DP affects inulin sweetness, water-binding capacity, digestibility and prebiotic activity, which are all important factors in the food industry. An attempt was made to increase the DP of inulin using ultrafiltration, whereby membranes with a nominal molecular weight cut-off (NMWCO) were used to filter out inulin that fell short of a molecular weight threshold (Moerman et al., 2004). A membrane with NMWCO of 2 kDa enabled removal of all monosaccharides and inulino-oligosaccharides <10 DP from chicory inulin. When the NMWCO was increased to 3 kDa, mid-range inulino-oligosaccharides were also removed (10 < DP > 20). A membrane with NMWCO of 5 kDa isolated just 7% of the starting inulin material, with DP 15–16. In an industrial situation this yield would represent an uneconomical and time consuming process. However, when using higher MW starting material originating from native dahlia inulin (unlike the processed inulin used above) and a membrane with a NMWCO of 5 kDa, the resulting average DP was higher at 29–30, yet the yield was again low at just 10%. This process was energy intensive and the membranes were unreliable, costly and fragile; therefore other methods of isolation were tested. Another method for increasing the yield of high DP inulin was by crystallization of aqueous solutions. In a solution with a concentration of 10% inulin (w/v), and following 6 days of storage, the resulting precipitate had a higher DP (n=42) than when a lower (5%, w/v) inulin concentration was used (n=40) (Moerman et al., 2004). This process is again expensive, time consuming and large quantities of high-chain-length inulin remained in the supernatant; as such it was an unattractive process for industrial use. To improve this technique, solvents of varying concentrations were added as supplements to the crystallizations. Acetone was the most effective as a carbohydrate solvent and appeared to have an optimum effect at around 50–60% (v/v) where yield was high and appreciable quantities of high DP inulin was produced. The inulin was then isolated by centrifugation or in a pressure filter. There were, however, losses of high DP inulin caused by dissolution of the aqueous solution. Again, this was a complex, laborious and expensive process, and the use of large quantities of solvents would pose a threat to the environment. The methods investigated by Moerman et al. (2004) are a useful resource for future research into facilitating the cost and time efficient isolation of high DP inulin.

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Another quite different approach tapped into the use of transgenic plants designed specifically to synthesize high chain-length inulin. The two enzymes, 1-SST and 1-FFT, from Jerusalem artichokes (Edelman and Jefford, 1968) were used to catalyse the production of inulin. Starting with two sucrose molecules, a trisaccharide of 1-ketose and one glucose molecule were liberated from the catalytic action of 1-SST, these subsequently become transferred as fructose from one fructan chain to another, catalysed by 1-FFT (see Section 11.4.1). The result was diverse chain lengths. H. tuberosus (Jerusalem artichoke) and C. intybus (chicory) produced inulin chain lengths of up to DP 60, where as Cynara scolymus (globe artichoke) synthesized inulin chains up to DP 200. Transgenic petunia plants, when incorporated with the genes required to produce 1-SST and 1-FFT from H. tuberus, were able to produce inulin with a DP of up to 25 (van der Meer et al., 1998). More recently, transgenic Solano tuberosum (potato) were implanted with 1-SST and 1-FFT from C. scolymus and produced inulin with the same high chain length as C. scolymus (DP 200) (Hellwege et al., 2000). These transgenic potatoes concurrently reduced starch production suggesting that the overall carbohydrate sink remained the same and that inulin production merely replaced starch production, rather than becoming an additional sink. Further work is investigating a line of potato tubers that have the same 1-SST and 1-FFT genes, but are unable to produce starch, so that inulin is the sole carbohydrate sink, ensuring that energy and resources are invested into inulin synthesis and storage and not expended on starch. This is an economically important discovery as chain length is of considerable importance in the industrial manufacture of inulin, and given the large biomass of potato tubers, a high quantity of inulin may potentially be produced.

11.5 Transgalacto-oligosaccharides Transgalacto-oligosaccharides (TOS) are a mixture of oligosaccharides produced from lactose by transgalactosylation. They are linear oligomers consisting of varying proportions of D-glucose and D-galactose, primarily in 1-4 and 1-6 linkages (Alles et al., 1999). They resist digestion by β-galactosidases in the small intestine and enter the colon where, like other dietary fibres, they are fermented by the colonic microbiota. TOS can be synthesized by a number of different microorganism-derived galactosidases, each with varying specificities for the different glycosidic linkages within TOS. As such, many potential TOS products exist that can each be expected to have different effects on the colonic microbiota. β-galactosidases were extracted from different probiotic strains of bifidobacteria and used to synthesize TOS in the presence of lactose (Rabiu et al., 2001). Product linkage ratios varied distinctly between the enzymes used. The starting probiotic strains were then grown in pure culture on their homologous TOS product to evaluate how well they grew. Bifidobacterium angulatum, Bifidobacterium infantis, and Bifidobacterium pseudolongum displayed the highest growth rates of all and gave greater growth than Oligomate 55, a commercial TOS preparation. The TOS product of the

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β-galactosidase from B. angulatum was added as a supplement to a mixed culture containing faecal-derived bacteria and it showed a more selective fermentation than Oligomate 55, because although they had similar bifidogenic effects, the B. angulatum TOS did not stimulate the growth of Bacteroides and Lactobacillus spp., whereas Oligomate 55 did. Further investigations into the precise strains that may benefit from different TOS products and the chemical and physical properties of TOS that are paramount in affecting its prebiotic affect are required. A human trial demonstrated that the fermentation of TOS was selective for bifidobacteria (Ito et al., 1993). The study also showed additional health benefits, including a reduction in faecal nitroreductase activity and decreased levels of indole and isovaleric acid. In another human study (Bouhnik et al., 1997) volunteers were administered 10g/d of TOS for 21 days; significant increases in faecal bifidobacteria were noted without affecting other faecal characteristics such as pH, faecal water characteristics and stool weight. The bifidogenic effect of TOS also benefits the infant gut. A low-molecularweight TOS (in combination with high-molecular-weight FOS) was introduced into an infant formula which was fed to infants for a 28-day period following which a significant rise in faecal bifidobacteria was observed compared with a control diet with no TOS and FOS (Moro et al., 2005). In another human trial (Alles et al., 1999), 40 patients were fed 0, 7.5 and 15 g/d of TOS and breath and faecal samples were analysed. TOS fermentation was complete for both dose rates (according to faecal analysis of TOS) and breath hydrogen increased significantly in the highest dose of TOS. TOS had no significant effect on faecal sample analysis, which included bowel habit, stool size, microflora profile, concentration of SCFA and bile acid, and pH. TOS also has anti-adhesive properties, whereby it can inhibit pathogens from binding to human colonic cells. The hypothesis is that anti-adhesive oligosaccharides mimic the structural nature of the binding sites on the surface of host gastrointestinal epithelial cells; thus pathogens bind to these decoy oligosaccharides and are prevented from binding to the gut wall, where they would otherwise induce infection. In vitro purified TOS reduced the adhesion of enteropathogenic Escherichia coli (EPEC) to the human cell lines HEp-2 and Caco-2 cells by 65% and 70%, respectively, and reduced the number of bacteria per microcolony from an average of 14 to 4 (Shoaf et al., 2006). In the same study, TOS was added as a supplement to human cell lines that already had adhered EPEC; there was no anti-adhesive effect, indicating that under this situation antiadhesion works before the pathogen binding to the host gut wall, as opposed to after the host–pathogen interaction.

11.6 Gluco-oligosaccharides Gluco-oligosaccharides (GOS) are composed of α, 1-6 and α, 1-2 linked β-Dglucose subunits. The α, 1-2 linkages are indigestible to human gastric enzymes, making GOS an important candidate prebiotic (Hylla et al., 1998).

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Methods for producing oligosaccharides generally have been restricted to acid and enzymic hydrolysis of polysaccharides, plant source extraction or synthesis by transglycosylation reactions (Chung and Day, 2002). However, Leuconostoc fermentation is an alternative method for producing GOS. A dextransucrase derived from Leuconostoc mesenteroides NRRL B-1299 can catalyse the production of the high molecular weight polysaccharide dextran. However, in the presence of an efficient acceptor, maltose, a mixture of homologous α, 1–6 and α, 1–2 GOS were produced, each with a maltosyl residue at the reducing end (DolsLafargue et al., 2001). The products varied greatly depending on the strain used to catalyse the reaction (Dols-Lafargue et al., 2001). This reaction was investigated further, whereby an increase in temperature from 25 to 40 °C and an increase in pH from 5.4 to 6.7 lead to significant increases in yield of the favoured product α, 1– 2 glucooligosaccharide, with maltosyl residues at the reducing end; this was due to a concurrent decrease in the synthesis of the less desirable products: dextran and leucrose. Further fine tuning of the reaction revealed that using a sucrose:maltose ratio of 4 and a total sugar concentration of 45% w/v, enabled 88% yield of GOS, which also further reduced the side reactions and yield of dextran and leucrose. Moreover, the product was 56.7% α, 1-2 glucooligosaccharide, which was the most desirable type of glucooligosaccharide product. Similar GOS products, from L. mesenteroides B-742, were tested for their prebiotic efficacy. They were readily fermented by Bifidobacterium and Lactobacillus spp., but not by Salmonella sp. or E. coli, suggesting a prebiotic effect (Chung and Day, 2002). In another study (Djouzi et al., 1995), the ability of Bacteroides thetaiotaomicron, Bifidobacterium breve and Clostridium butyricum, all clinically significant species of the human gut microbiota, to break down GOS was tested in vitro and in vivo. In pH-regulated fermenters, α, 1-2 GOS was more resistant to degradation than α, 1-6 GOS. In vitro and in vivo, B. thetaiotaomicron was the most effective at degrading the α, 1-2 linkages, whereas C. butyricum was the least effective and its growth was inhibited in vitro. The effects of TOS and GOS were compared in germ-free rats, inoculated with human faecal microbiota (gnotobiotic, Djouzi and Andrieux, 1997). Although this approach has its limitations owing to physiological differences between humans and rats, it shifts in vivo studies away from the inter-individual variations that commonly plague human studies and provides rats with the major bacterial population, enzymatic activity and metabolic profile of the donor human. GOS increased bifidobacteria by 2 log values and increased CH4 excretion. However, TOS did not affect the microflora profile, but did increase both CH4 and H2 excretion significantly. GOS fermentation produced a higher proportion of propionate and TOS produced more butyrate. Structural differences between these oligosaccharides were hypothezised as being the reason for differences in the effects on the microflora.

11.7 Alternansucrase–maltose acceptor oligosaccharides Dextransucrases from L. mesenteroides can catalyse the synthesis of a range of

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14 12

12 h 24 h

10 8 PI 6 4 2 0 No FOS DP3 DP4 DP5 DP5.7 DP6.7 Carbohydrate Carbohydrate degree of polymerization (DP)

DP7.4

Fig. 11.5 Prebiotic index (PI) of six different alternansucrase–maltose acceptor oligosaccharides, FOS and a no treatment control, in 37 °C batch cultures at 12 and 24 h (Sanz et al., 2005).

oligosaccharides from a reaction between the glucosyl group of sucrose and low MW sugars. The products are either oligosaccharides containing one, two, three or more D-pyranosyl units or a high MW glucan. An investigation (Côté and Robyt, 1982) using an alternansucrase extracted from L. mesenteroides NRRL B-1355 produced an alternan polysaccharide with alternating α, 1-6 and α, 1-3 linked D-glucosyl products. Despite the reaction taking place in the presence of a number of different low MW acceptor sugars, it was maltose that was the most effective in the production of more oligosaccharide relative to polysaccharide material. Despite both α, 1-6 and α, 1-3 linkages being formed, the enzyme could only produce the α, 1-3 linkages when the non-reducing end of the acceptor glucose was linked by a α, 1-6 linkage to another glucose residue. In work by Sanz et al. (2005), alternansucrase-maltose acceptor oligosaccharides were separated into fractions with distinct DPs; the influence of each DP fraction on gut-derived pure cultures of bacteria and on the whole colonic microflora in vitro was investigated. The acceptor reaction conditions were carried out according to Côté et al. (2003) and produced three pure products with oligosaccharides of DP 3, 4 or 5, and three mixed products where the DP averaged 5.7, 6.7 or 7.4 in each product (as determined by a thin layer chromatography densitogram after separation on a Bio-Gel P2 fine mesh column). The prebiotic index (PI), which evaluates the change in bifidobacteria and lactobacilli (generally accepted as health promoting groups) versus the change in bacteroides and clostridia (generally accepted as detrimental to health) as a proportion of the overall change in total bacterial counts, was used as a quantitative tool to compare the prebiotic effect of the alternansucrase-maltose acceptor oligosaccharide fractions (Palframan et al., 2003). Bifidobacteria grew well on all of the fractions apart from DP 7.4 and when considering the prebiotic

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index (PI) (Fig. 11.5) all fractions showed selectivity for lactobacilli and bifidobacteria groups similar to FOS (positive prebiotic control) apart from DP 7.4, as it did not stimulate bifidobacteria growth. DP 6.7 contained mostly hexasaccharides and DP 7.4 contained mostly octasaccharides, indicating a DP cut-off where carbohydrates become less available for fermentation by LAB.

11.8 Resistant starch Starch is the major storage polysaccharide in plants and forms a substantial part of our diet. It is a polysaccharide that has two forms. Amylose is a linear polymer which has mainly α-1,4 linked glucose subunits and has a DP of up to 6000. Amylopectin has an α, 1-4 linked backbone of glucose subunits that have α, 1-6 bonded branches every 24–30 subunits, molecules have an average DP of 2 million (Sajilata et al., 2006). There are three categories of starch according to enzymic degradation (Berry, 1986). Rapidly degradable starch (Alexander and Christine, 2004) is degraded into glucose molecules within 20 min of enzyme digestion; it is common in starchy foods such as bread and potato. Slowly digestible starch (SDS) is also degraded to completion, but takes up to 100 min. Both RDS and SDS are expected to undergo complete degradation in the small intestine. Lastly, there is resistant starch (RS), which was first classified by Englyst et al. (1992); it resists degradation by exhaustive treatment with pullulanase and α-amylase in vitro. Physiologically RS is defined as the fraction of starch that resists degradation in the small intestine and undergoes fermentation by the large intestine microbiota. RS can be further sub-divided: RS1 is starch entrapped within a food matrix, which thus prevents access of amylase to the starch; RS2 is starch with a granular structure resistant to digestion; RS3 is retrograded starch formed by food processing (Englyst et al., 1992). RS3 is deemed to be the least digestible/fermentable of the RSs. RS has a bland flavour and white appearance, yet in the food industry it has a number of useful qualities including: water-holding capacity, viscosity increase, swelling, gel formation and small particle size (causing less interference with texture) (Faraj et al., 2004). RS can in fact be used to replace flour to a certain extent before the texture and rheology of the product becomes affected. Most sources of dietary fibre have a high capacity to bind water and form bulky foods; conversely, RS improves mouthfeel, texture and appearance, in addition to providing a low calorie source of dietary fibre (Sajilata et al., 2006). The ratio of amylose:amylopectin is a key determinant in the amount of RS present; in a meal containing normal corn flour, with 20% amylose, there were 3 g/100 g (dry weight) of RS, but a high amylose corn flour (70%) had 20 g/100 g of RS. The surface area:volume ratio is also hypothesized to be important to the enzymatic degradation of starches by amylases (Sajilata et al., 2006), whereby a small surface area:volume ratio makes enzymatic hydrolysis harder, as in potato starch and high amylose maize starch which both have a high RS content. Heating alone is one method of increasing the RS content of flours and starches. Heating of starch to 120 °C for 20 min causes gelatinization; to optimize RS yield

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the starch gels can be left to cool during retrogradation and frozen at –60 °C overnight and finally thawed before milling (Christou et al., 1999). Each step in this process is suitable to scaling-up for industrial purposes. Alternatively, pyrodextrinization is a method used in the paper industry to modify starch; it involves heating dry and occasionally acidified starch to produce pyrodextrinized starch. It was proposed that pyrodextrin conversion could increase the amount of RS from starch in the food industry to produce highly soluble RS with non-starch linkages, particularly as the conversion already occurs on an industrial scale. A study (Alexander and Christine, 2004) comparing the effect of native starch and starch following pyrodextrinization on SCFA production, in a simple in vitro model of the gut was conducted to investigate if the increased RS content could adjust the response of faecal-derived microbiota. Potato, lentil and cocoyam pyrodextrinized starch were produced by heating at 140 °C for 3 h in the presence of hydrochloric acid at 1.82 g kg–1 starch. Both native and pyrodextrin starches were pretreated with pepsin and pancreatic enzymes to mimic the digestion of starches in the upper gut before reaching the large intestine. The resulting pretreated substrates were fermented anaerobically by faecal microbiota for 24 h and SCFA profiles were assessed. Compared with 10 mM per gram of pyrodextrinized starch, 6.8 mM SCFA were produced per gram of native starch. A doubling in the concentration of propionate and a 25% reduction in acetate were thought to have contributed to this change in total SCFA content and may have represented a preference of the propionate-producing microflora for more soluble and/or nonstarch linked pyrodextrinized starch. A murine study corroborated this finding for the enhanced production of propionate from RS (Cheng and Lai, 2000), whereby a dose–response study of the fermentation of resistant rice starch and corn starch was conducted. This also compared the effect of starch granule structure on microbiota fermentation, where resistant rice starch was an aggregation of smaller granules and the corn starch consisted of larger single granules. The cornstarch diet produced no propionate, but at a dose of >30%, resistant rice starch propionate increased significantly. This result was correlated to changes in cholesterol and triglyceride concentrations. At a dose of over 45% of rice starch in the diet, the rats’ total serum cholesterol was significantly reduced compared with corn starch diets and at 63% hepatic and total cholesterol concentrations were significantly reduced. This relationship between propionate production and cholesterol levels has been demonstrated previously in rat (Chen et al., 1984) and human (Muir et al., 1998) studies. Fermentation of starch has been shown to favour butyrate production (Weaver et al., 1992) which is thought to be beneficial for human epithelial cells in the large bowel (Gamet et al., 1992). In addition, propionate has also been shown to inhibit invasive human colon cancer (in vitro) (Emenaker et al., 2001) and to reduce the growth and differentiation of the cancer cell line: HT29 (Gamet et al., 1992). Propionate has been implicated in the stimulation of leptin production by mouse adipocytes, which play a central role in controlling metabolic rate and feeding behaviour (Xiong et al., 2004). RS appears to be readily fermentable by the human gut microbiota (Topping et al.,

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1997); as such its benefits may only be reaped in the proximal regions of the colon (see Section 11.3. for implications of this). To increase the persistence of RS psyllium was combined with high-amylose starch which was fed to rats (Morita et al., 1999). Total SCFA and butyrate concentrations increased significantly along the length of the rat large intestine, compared with a diet containing just high-amylose starch. In addition, faecal butyrate concentration correlated positively with faecal starch content, showing that a slower and more persistent fermentation was occuring. Bifidobacterium lactis LaftiTM B94 was demonstrated to hydrolyse a highamylose maize starch, Hi-MaizeTM, indicating a potential synbiotic combination; whereas other closely related probiotic strains of bifidobacteria could not do so (Crittenden et al., 2001). In addition, B. lactis LaftiTM B94 could ferment other well known prebiotics, such as inulin, TOS, FOS, XOS and soybean oligosaccharides.

11.9 Arabinoxylan Cereal products boasting ‘whole grain’ benefits can attribute many of their effects to their non-starch polysaccharide (NSP) content. Arabinoxylan (AX) is the primary NSP in the endosperm of wheat kernels, accounting for about 60–70% of the cell wall polysaccharides which in turn account for about 9.5% of the wholemeal and 3% of white flour (Southgate, 1976). AX has a long 1-4-linked backbone of β-D -xylopyranose residues with a varying degree of α-L arabinofuranosyl residue substitutions at the C-2 and C-3 positions; occasionally double substitutions occur (C2,3) (Fig. 11.5). Previous work (Lu et al., 2000) showed that AX supplementation to the diet of rats resulted in reduced epithelial cell proliferation indices, the impact being greater than with whole wheat bran, guar gum and a control with no fibre added. In addition, AX supplementation stimulated the greatest increases in faecal bulk, as reported by Eastwood et al. (1986) and McIntosh et al. (2001). Work by Adam et al. (2001) showed that a highly viscous and AX-rich wheat flour could increase steroid content (bile acids and sterols) in excreted faeces by 78% above that excreted by rats fed a control diet of purified wheat starch. AX has even been demonstrated to increase natural killer cell activity, increase IL-2 and INF-γ production and reduce the symptoms of atopic dermatitis in mice (Ogawa et al., 2005). The benefits of AX, with regards to cancer, cholesterol levels, feacal bulking and inflammatory responses are becoming more apparent, so the production of particularly active AX fractions is receiving interest. A recent study by Hughes et al. (2007) investigated the relationship between the MW of AX and its prebiotic effect. Wheat-flour derived AX fractions, with average MW of 354, 278 and 66 KDa, were added as supplements to miniaturescale batch cultures that were inoculated with faeces from three healthy donors. The prebiotic index (PI) was used as a quantitative tool to compare the prebiotic effect of the three AX fractions (Palframan et al., 2003). It was concluded that with decreased MW of AX there was an increase in PI, suggesting that lower MW polysaccharides have more prebiotic efficacy.

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Fig. 11.6 (a) Structure of part of a hypothetical arabinoxylan molecule; (b) simplified version of the same arabinoxylan molecule: (X)=β-Dxylopyranose residue, (A)=α-L-arabinofuranosyl residue substitution.

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11.10 Oligosaccharides from non-starch polysaccharides NSP (Arrigoni et al., 2002) are an interesting and very abundant fibre source and also present a potential source for production oligosaccharides. There are a large number of NSP by-products including cereal bran, fruit pomace, sugar beet pulp, potato fibre and press cake of oleaginous seeds (van Laere et al., 2000). Extracting and isolating NSPs and treating them with certain enzymes can produce a variety of potentially useful oligosaccharides. Such novel oligosaccharides were produced from NSPs in a study (van Laere et al., 2000) investigating their impact on, and preferential degradation by, individual bacterial strains normally resident in the human colon. It should be noted that the oligosaccharides discussed often frequent the large intestine anyway because they are produced from the enzymatic degradation of NSP by the microflora. Bacteria are present in the colon that have evolved to degrade these oligosaccharide by-products. (Arabino)galacto-oligosaccharide (ATOS) was produced from dehulled soy beans (van Laere et al., 2000). These were initially defatted, deproteinated, destarched and treated with NaOH, thus producing an arabinogalactan polysaccharide-rich extract (AGP) containing predominantly arabinose (38%) and galactose (52%) residues. AGP was subsequently treated with purified endogalactanase, cloned from Aspergillus aculeatus, and remaining monomers and polymers were removed by gel filtration to produce the oligosaccharide product profile listed in Table 11.2. The prevalent ATOS present were β, 1-4 linked. When fermented in vitro by various strains of bacteria the medium pH fell in nearly all vessels and all strains either partially or fully degraded the ATOS product (except three Chlostridium strains and E. coli). However, no selectivity for just bifidobacteria or lactobacilli spp was noted, because the less desirable and potentially pathogenic strains also degraded ATOS. Arabinanoligosaccharides (AOS) were produced from sugar beet pulp-derived linear α, 1-5 arabinan (van Laere et al., 2000) which was incubated with endoarabinanase from A. aculeatus. A column fractionation technique was used to isolate oligomers in the range DP 2-6 all with linear α, 1-5 arabinan which were subsequently used as supplements to pure culture experiments. Following degradation the oligosaccharides were analysed to gauge which chain lengths were preferentially degraded by each strain of bacteria. Oligosaccharides with DP 2-6 were all fermented by Bifidobacterium longum and Clostridium clostridiiforme. Table 11.2 Manufacture and products summary of (arabino)galacto-oligosaccharides (ATOS) AGP

endo-galactanase

ATOS mixture

▲

DP

2

3

4

5

6

7

8

9

DP distribution of ATOS by %

22

26

16

12

9

9

3

3

AGP: arabinogalactan enriched polysaccharide fraction, DP: degree of polymerization

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Table 11.3 Arabinoxylano-oligosaccharide (AXOS) structure, derived from wheat flour AXOS (DP) 5.5

5.1

Structure of oligosaccharide β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4)  α-A-(1-2) β-X-(1-4) - β-X-(1-4) - β-X-(1-4) α-A-(1-3)

6.1

α-A-(1-2)

β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) α-A-(1-2)

8.1

α-A-(1-3)

β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) α-A-(1-2)

α-A-(1-3) α-A-(1-3)

9.1

β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) α-A-(1-2)

α-A-(1-2) α-A-(1-3)

10.1

α-A-(1-3)

β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) - β-X-(1-4) α-A-(1-2) α-A-(1-3)

α-A-(1-2) α-A-(1-3)

(X) = β-D-xylopyranose residue, (A) = α-L-arabinofuranosyl residue substitution.

Arabinotriose was degraded by E. coli and Bifidobacterium adolescentis and arabinotetraose was fermented by Clostridium butyricum, indicating individual species chain length preferences. Arabinoxylo-oligosaccharides (AXOS) were produced from a wheat flourderived arabinoxylan-enriched fraction (van Laere et al., 2000) which was degraded by an endo-xylanase from Aspergillus tubigensis. The products were fractionated and analysed by HPAEC and are summarised in Table 11.3. The branched AXOS were fermented fully by B. adolescentis and Bacteroides vulgatus and partially by Bacteroides ovatus and B. longum. Previous work supports the fermentation of AXOS by a range of Bifidobacterium spp. in pure culture experiments (Hopkins et al., 2003; Jaskari et al., 1998; Yamada et al., 1993) and this investigation (van

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Laere et al., 2000) indicated that B. adolescentis produced three different types of enzymes required for the degradation of AXOS. All the oligosaccharide fractions mentioned were successfully fermented in vitro by a number of different strains of bacteria; in particular, Bacteroides strains were able to degrade all substrates to some extent, illustrating that the bacteroides group hosts a diverse variety of glycosidases and glycanases that degrade an assortment of substrates. Previous work has also demonstrated that bacteroides are a metabolically versatile group with respect to their ability to utilize many types of plant polysaccharides as substrates (Salyers et al., 1981; Salyers et al., 1977; Hopkins et al., 2003). Although this was a useful study for comparing the fermentation of different oligosaccharides by particular strains of bacteria the growth rates were not measured and predictions from this in vitro study on the effect of the substrates in mixed cultures and in vivo would be speculative, particularly regarding their prebiotic activity.

11.11 Pectins Pectins are complex polysaccharides that have polydisperse, polymolecular and heterogenic structure and composition (Gulfi et al., 2005). Two main regions exist. The ‘smooth’ region is composed of homopolymeric partially methylated α-D-1,4galacturonic acid which forms an extended curved chain with a lot of flexibility. The ‘hairy’ region is composed of a rhamnogalacturonan (RG) backbone comprising either arabinan, galactan or arabinogalactan side chains (1–20 residues long, known as RG I) or a side chain with a variety of different monosaccharides (known as RG II), for example apiose, xylose, fucose (Vidal et al., 2000). Pectins are important for health; benefits include reducing serum cholesterol following oral intake (Anderson et al., 1994), reduced glucose absorption (Jenkins et al., 1977), delayed gastric emptying (Schwartz et al., 1982) and reduced transit time (Spiller et al., 1980). The effects of pectin are thought to be related to its gel forming and water-holding capacity (Roberfroid, 1993). Because of their gel-forming ability at low concentrations, pectins are widely used as natural food additives to improve the viscosity of liquid foods and at the same time act to replace fat because they mimic the organoleptic properties of fat. A structure–function study was carried out comparing the effect of methoxyl content of pectins on the cholesterol and fat content in the blood and liver of rats (Ahrens et al., 1986). A dose-dependent hypocholesterolemic effect was noted whereby pectins with high methoxyl content were most effective at a low dose rate. However, this effect was not mimicked in humans (Judd and Truswell, 1982) where high and low methoxyl content had a similar effect on blood and faecal lipids. A study investigated the influence of pectins and partially hydrolysed pectins on fermentation by the gut microflora (Gulfi et al., 2005). The degree of methylation was the only factor that influenced fermentation, whereby the more highly

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esterified pectins induced a faster and more extensive degradation; pectin source (apple and citrus) and viscosity did not affect fermentation. Partially hydrolysed pectin fractions had similar rates of fermentation and resulting SCFA production shared profiles related to their corresponding starting materials (that is, nonhydrolysed pectin). This means that larger amounts of hydrolysed pectin can be added to food to increase the health benefit for the gut, but without having the unwanted gelling effect of non-hydrolysed pectin. Pectic-oligosaccharides are also particularly bioactive foodstuffs. They were shown to have applications as liver lipid accumulation repressors in rats; additional uses include: antifungal phytoalexin-elicitors in plants (Bishop et al., 1984) and as inducers of flowering and antibacterial agents (Iwasaki et al., 1998). The continuous production of pectic-oligosaccharides using an enzyme endo-polygalacturonase was investigated using an ultrafiltration stirred cell which acted as a membrane reactor to control pectic-oligosaccharide MW and characteristics (Mountzouris et al., 1999). Two pectin substrates were used for the investigation: high methylated (60–66% degree of esterification; HMP) and low methylated (8% degree of esterification; LMP) pectin, and factors likely to influence the reactor performance, such as substrate concentration (1–5%), enzyme concentration (90–2700 U l–1), and residence time (40–120 min) were recorded. Conversions of pectin to pectic-oligosaccharide ranged from 67–99.7% (under the range of conditions mentioned) and the average MW distribution of the products obtained was similar for both products used, with 3.5 kDa when high methylated pectin was used and 3.8 kDa when low methylated pectin was used. Thus, the method for pecticoligosaccharide production displayed good rates of conversion for both HMP and LMP, and similar end-products were produced, elucidating this as an efficient and appropriate technique. The success of the method in the laboratory warrants progression to a pilot scale investigated, perhaps using cross-flow membrane reactor systems. The efficacy of these pectic-oligosaccharides and pectins as prebiotics was investigated to ascertain whether oligosaccharides were more readily fermented than their parent polysaccharide material and whether the degree of esterification was influential. The substrates used were HMP and LMP and pecticoligosaccharides from HMP (POS I) and pectic-oligosaccharides from LMP (POS II). Controlled pH batch mixed faecal cultures were set to pH 6.8 and 37 °C to simulate the gut environment, in vitro, and the PI (see Section 11.7) was calculated to compare the prebiotic effect of the four substrates at 8, 24 and 48 h (Table 11.4). In general, the HMP fraction resulted in slower bacterial growth rates than the LMP fraction, indicating that esterification inhibited fermentation (Olano-Martin et al., 2002) which is in agreement with previous work (Dongowski and Lorenz, 1998). The size of the pectin had an impact upon the growth rates of bacteria, whereby a higher PI was seen for the pectic-oligosaccharides than for their parent pectin; in addition the selectivity of the fermentation for bifidobacteria persisted for longer with pectic-oligosaccharides than for the pectins. In the pure culture experiments that complimented this study (Olano-Martin et al., 2002), there was an increase in all Bacteroides and Clostridia species tested on HMP and LMP,

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Table 11.4 Prebiotic index (PI) of two pectin (with high or low degree of methylation) and two pectic-oligosaccharide fractions (with high or low degree of methylation) following 8, 24 and 48 h of anaerobic fermentation, at 37 °C and pH 6.8 Treatment

HMP LMP POS I POS II

PI 8h

24 h

48 h

0.016 0.066 0.046 0.082

–0.11 0.033 0.071 0.092

0.004 0.012 0.081 0.120

whereas some of them failed to grow on the pectic-oligosaccharides. Most bifidobacteria were able to utilize the oligosaccharides better than the polysaccharides. This suggested that the pectic-oligosaccharides were able to support the growth of bifidobacteria while inhibiting groups of bacteria hosting potentially pathogenic species of Bacteroides and Clostridia, to a greater extent than the pectin parent material.

11.12 Oligodextran Dextrans are large polysaccharides predominantly composed of α, 1-6 glycosidic linked glucose subunits. Oligodextran is a collective term for carbohydrate mixtures derived from dextran that consist of an isomalto-oligosaccharide (IMO) part and a relatively low molecular weight (<70 kDa) dextran polysaccharide part. Oligodextrans have a considerably larger DP than IMO and, as such, would be theoretically less digestible than commercially available IMO. Oligodextran can be produced by the controlled enzymic hydrolysis of L. mesenteroides B512 F dextran using an endo-dextranase in an ultrafiltration stirred cell membrane reactor, with a NMWCO of 10 kDa. This method for the production of novel oligodextrans was developed to enable effective control over the molecular size of the oligodextran products (Mountzouris et al., 1999). The specific enzyme used was endo-dextranase (1,6-(α)-D-glucan-6-glucan hydrolase; EC 3.2.1.11) from Penicillium lilacinum and three different fractions of oligodextran were produced under varying substrate concentration (20–50 g dm3), enzyme concentration (6250–62500 U dm3) and residence time (60–120 min) in the reactor; the DP of each fraction is summarized in Table 11.5, and the MW distribution is given in Fig. 11.7. These products were hypothesized to be less digestible because a high proportion of the fractions had a DP ≥ 3 which would be less susceptible to digestion by membrane bound disaccharidases in the small intestine and they would escape digestion by luminal enzymes owing to their α, 1-6 glycosidic linkages (Olano-Martin et al., 2000). Thus, oligodextrans would hypothetically arrive in the large intestine intact as a source of dietary fibre and be a potential

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Table 11.5 Composition (g kg–1) of different oligodextran products from controlled enzymic hydrolysis of L. mesenteroides B512 F dextran using an endo-dextranase in an ultrafiltration stirred cell membrane reactor (Mountzouris et al., 1999) Oligodextran I (g kg–1) DP 1 DP 2 DP 3 DP 4 DP 5 DP 6 DP 7 DP 8 DP 9 DP 10 DP 11+

Oligodextran II (g kg–1)

62.2 185.3 269.6 41.1 11.4 14.3 20.0 22.2 18.4 12.9 342.4

Oligodextran III (g kg–1)

75.2 50.7 98.2 27.9 12.4 12.9 13.5 14.7 14.5 15.0 665.0

151.3 0.0 58.2 11.6 6.6 8.3 8.8 8.3 7.6 7.6 731.7

prebiotic. FOS, dextran and three oligodextran fractions with different MW distributions (Fig. 11.7) were added as supplements to closed batch cultures and underwent 48 h fermentation at 37 °C at a starting pH of 7, although the pH was not maintained. Samples were analysed for SCFA content and bacterial changes were assessed by FISH. The three oligodextran fractions varied in weight: oligodextran I < oligodextran II < oligodextran III. The lowest MW fraction, oligodextran I, had a similar bifidogenic effect upon the microflora as FOS (a proven prebiotic) in the early stages of the fermentation in comparison with oligodextran II, oligodextran 60

0.18 kDa 1.15 kDa

Refractive Index (mV)

50

11.6 kDa 19.5 kDa

40

42 kDa 30 74 kDa 20 10 0

0

20

40

60

80

100

120

140

160

180

Elution volume (ml)

Fig. 11.7 Molecular mass distributions of oligodextran I (▲), oligodextran II (¡) and oligodextran III (u) used in the batch culture fermentations. Arrows indicate the elution volumes of the standards used to calibrate the chromatographic column (Sephacryl S-200 HR). Data adapted from Mountzouris et al. (1999).

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Prebiotic Index

1 0 –1 –2 –3 –4 1

2

3

1

Dextran

2

3

Oligodextran Vessel/Treatment

Fig. 11.8 Prebiotic index in each of three vessels following the fermentation of dextran, oligodextran and maltodextran in a validated three-stage continuous culture system (OlanoMartin et al., 2000).

III and dextran. Carbohydrate utilization was related to the molecular mass of the oligodextran fraction, where the low MW oligodextran I was utilized to a greater extent than dextran. This difference was thought to result from differences in the DP of the fractions. A more representative three-stage continuous culture fermentation system was used in the same study to investigate carbohydrate persistence and effect throughout the system. It incorporated three vessels (V1, V2, V3), where V1 represented the proximal colon, V2 represented the transverse colon and V3 represented the distal colon; a media reservoir, containing media closely representing the small intestinal effluent enriched with the test carbohydrate, was continuously fed to V1. V1 flowed into V2, V2 into V3, and V3 into a waste bottle. The pH increased through the system: V1 = 5.5, V2 = 6.2 and V3 = 6.8. It was validated against sudden death victims for fermentation events in situ (Macfarlane et al., 1998). Two fractions were added as supplements to the system at roughly 5g/day: oligodextran IV (a low MW fraction, with a similar MW profile to oligodextran I) and dextran (the parent material). (Figure 11.8 shows the PI in each vessel after supplementation of dextran, oligodextran IV and maltodextran.) Bifidobacteria and lactobacilli populations were at least 1 log value higher in all vessels resulting from oligodextran IV supplementation, compared to dextran, and when the microflora values were converted into PI values the lower MW fraction has a considerably greater selectivity for bifidobacteria and lactobacilli than dextran. However, this selectivity was restricted to VI, and although bifidobacteria and lactobacilli remained high

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in V2 and V3, the bacteroides and clostridia groups also increased substantially. Dextran had no impact on bifidobacteria and lactobacilli numbers and bacteroides and clostridia both increased in V2 and V3. The reduction in pH is known to inhibit the growth of clostridia (Gibson and Roberfroid, 1995) which may account for their increase in V2 where the pH was regulated at 6.2 and substrate availability was still overflowing from V1 acting as a carbon source for them. Butyrate formation occurred in V2 and V3 from both oligodextran and dextran which may be related to clostridia proliferation which is known for its production of butyrate. This work demonstrated that by decreasing dextran MW the prebiotic activity was increased.

11.13 Conclusion Developments in enzyme technology to isolate, modify and refine carbohydrate products will enable us to design enhanced forms of functional carbohydrates to increase their potential health benefits. Dietary fibre and, increasingly, prebiotics are very important for human wellbeing. There is a wealth of evidence elucidating the benefits that may be mediated via dietary fibre intake. However, data to date is mainly focused on physiological effects such as gastrointestinal transit time, faecal bulk and colonic/faecal pH. Little is known about the faecal bacteria fermentation profiles of different fibres. SCFAs are an indicator of colonic fermentation but as up to 95% is rapidly absorbed in vivo in the colon the data obtained from human studies lack reliability. Advances in molecular methodologies based on 16S rRNA to analyse changes in the microflora can allow for an accurate evaluation of the effect of dietary intervention studies which, combined with SCFA data, can give a more complete picture on the efficacy of fibre intake.

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‘Effect of purified cellulose, pectin, and a low-residue diet on fecal volatile fatty acids, transit time, and fecal weight in humans’. American Journal of Clinical Nutrition, 33, 754–759. Tomlin, J. and Read, N. W. (1988). ‘The relation between bacterial degradation of viscous polysaccharides and stool output in human beings’. British Journal of Nutrition, 60, 467– 475. Topping, D. L., Gooden, J. M., Brown, I. L., Biebrick, D. A., McGrath, L., Trimble, R. P., Choct, M. and Illman, R. J. (1997). ‘A high amylose (amylomaize) starch raises proximal large bowel starch and increases colon length in pigs’. Journal of Nutrition, 127, 615–622. Van Den Ende, W. and Van Laere, A. (1996). ‘De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)’. Planta, 200, 335–342. Van Der Meer, I. M., Koops, A. J., Hakkert, J. C. and Van Tunen, A. J. (1998). ‘Cloning of the fructan biosynthesis pathway of Jerusalem artichoke’. The Plant Journal, 15, 489– 500. Van Laere, K., Hartemink, R., Bosveld, M., Schols, H. and Voragen, A. (2000). ‘Fermentation of plant cell wall derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria’. Journal of Agricultural and Food Chemistry, 48, 1644–1652. Van Nuenen, M., Meyer, P. and Venema, K. (2003). ‘The effect of various inulins and Clostridium difficile on the metabolic activity of the human colonic microbiota in vitro’. Microbial Ecology in Health and Disease, 15, 137–144. Vidal, S., Doco, T., Williams, P., Pellerin, P., York, W. S., O’Neill, M. A., Glushka, J., Darvill, A. G. and Albersheim, P. (2000). ‘Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain’. Carbohydrate Research, 326, 277–294. Weaver, G., Krause, J., Miller, T. and Wolin, M. (1992). ‘Cornstarch fermentation by the colonic microbial community yields more butyrate than does cabbage fermentation; cornstarch fermentation rates correlate negatively with methanogenesis’. American Journal of Clinical Nutrition, 47, 61–66. Wichienchot, S., Prasertsan, P., Hongpattarakere, T., Gibson, G. and Rastall, R. (2006). ‘In vitro fermentation of mixed linkage glucooligosaccharides produced by gluconobacter oxydans NCIMB 4943 by the human colonic microflora’. Current Issues in Intestinal Microbiology, 7, 7–12. Xiong, Y., Miyamoto, N., Shibata, K., Valasek, M. A., Motoike, T., Kedzierski, R.M. and Yanagisawa, M. (2004). ‘Short chain fatty acids stimulate leptin production in adipocytes through the G protein coupled receptor GPR41’. Proceedings of the National Academy of Sciences USA, 101, 1045–1054. Yamada, H., Itoh, K., Morishita, Y. and Taniguchi, H. (1993). ‘Advances in cereal chemistry and technology in Japan. Structure and properties of oligosaccharides from wheat bran’. Cereal Foods World, 38, 490–492. Yazawa, K. and Tamura, Z. (1982). ‘Search for sugar sources for selective increase of bifidobacteria’. Bifidobacteria Microflora, 1, 39–44. Yazawa, K., Imai, K. and Tamura, Z. (1978). ‘Oligosaccharides and polysaccharides specifically utilizable by bifidobacteria’. Chemical and Pharmaceutical Bulletin, 26, 3306–3311.

12 Flavorings and other value-added products from sucrose* Gregory L. Côté, United States Department of Agriculture, USA

12.1 Introduction Farmers in the United States produce approximately eight million tonnes of sugar from cane and beets every year. In a typical year, stocks remaining unsold represent nearly 10% of this amount. Worldwide, farmers produce approximately 140 million tonnes, with an equivalent percentage remaining in stock at year end. Sugar prices typically hover around US$0.22–0.27 per kilogram in the USA. Various types of subsidies and price support systems are used by many sugarproducing countries to keep sugar producers in business. In addition to significant production surpluses and low prices, sugar producers face increasing competition from artificial, high-intensity sweeteners and from corn sweeteners such as fructose or high-fructose corn syrup. It has been claimed that cane and beet sugar, or sucrose, represents the largest production of any pure, single organic compound in the world. This represents a huge potential supply of relatively inexpensive, pure, food-grade raw material. Thus, any process for converting sucrose into more valuable products will be beneficial for sugar farmers, food processors, consumers and taxpayers alike. Companies and government agencies in several countries are sponsoring research into ways to convert sucrose to higher-value products. This chapter will describe the use of biotechnology to produce value-added products *Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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Fig. 12.1

Sucrose.

from sucrose. In particular, it will focus on the use of enzymes to convert sucrose into oligosaccharides or polysaccharides suitable for food use.

12.2 Di- and oligosaccharides from sucrose Structurally, sucrose is a disaccharide of the simple hexose monosaccharides D-fructose and D-glucose (Fig. 12.1). Its systematic chemical name is α-D-glucopyranosyl-β-D-fructofuranoside. It occurs in most higher plants, where it is used as the main transport sugar, similar to the role glucose plays in humans and other animals. Because of its ubiquitous role in nature, many microbial systems have evolved to metabolize sucrose. Although most of these systems simply hydrolyse sucrose into its two component sugars, some are capable of converting sucrose into unusual products.

12.2.1 Sucrose linkage isomers and their reduced polyols Sucrose represents just one of the ways in which fructose and glucose can be chemically linked. Of the various possible isomers, a few have been found in nature or produced enzymatically and exhibit potentially useful properties. Isomaltulose (palatinose) Isomaltulose was first described in 1954, when US Department of Agriculture (USDA) scientists isolated it from dextran production broths during the enzymatic conversion of sucrose to dextran (Stodola et al., 1954). Subsequently, scientists at Sudzucker AG in Germany discovered a more efficient method for production of the disaccharide, which they named palatinose (Weidenhagen and Lorenz, 1957). Isomaltulose is now produced using the latter method, in which a microbial isomerase converts the α1↔β2 linkage of sucrose to an α(1→6) linkage, forming α-D-glucopyranosyl-(1→6)-D-fructose (Fig. 12.2). The name isomaltulose derives from the fact that it is the ketose analogue of isomaltose, which is α-D-glucopyranosyl-(1→6)-D-glucose. Note that, unlike sucrose, isomaltulose is a reducing sugar. Isomaltulose is much more resistant to acid hydrolysis relative to sucrose.

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Fig. 12.2 Isomaltulose (palatinose).

The enzyme responsible for conversion of sucrose to isomaltulose is a sucrose isomerase known as isomaltulose synthase (E.C. 5.4.99.11). It is produced by a number of bacteria, the most often mentioned being Erwinia rhapontici, Protaminobacter rubrum and various Klebsiella species. The enzyme is an α-Dglucopyranosyltransferase and occurs mainly in the periplasmic space of the cells. The properties of the enzyme and the use of immobilized cells and immobilized enzyme for the production of isomaltulose have been described (Cheetham, 1984; Cheetham, 1987; Nakajima and Nishio, 1993). According to a recent review (Lina et al., 2002), isomaltulose is slowly absorbed and hydrolysed in the small intestine, and is completely metabolized. The rate of metabolism is slower than that of sucrose or maltose, so the glycemic index of isomaltulose is significantly lower. Isomaltulose is approximately half as sweet as sucrose and exhibits no cariogenicity. Owing to its absorption in the upper gastrointestinal (GI) tract and subsequent metabolism, it cannot be considered a prebiotic, but shows great potential as a non-cariogenic, low-glycemic index sweetener. Isomaltulose can be chemically reduced, yielding a mixture of α-D-glucopyranosyl-(1→6)-D-mannitol and α-D-glucopyranosyl-(1→6)-D-glucitol. This mixture is commercially produced as isomalt or Palatinit (Irwin, 1990; Schiweck and Munir, 1992). Like isomaltulose, it is sweet, non-cariogenic and exhibits a low glycemic index in humans. Since one of the two monosaccharide units has been reduced to a sugar alcohol, its metabolism differs from that of its parent sugar and it yields only about one-half of the total calories of isomaltulose or sucrose. Being a sugar alcohol, it does not undergo the same browning reactions as isomaltulose. Both isomaltulose and isomalt are produced on a commercial scale in Germany by Palatinit GmbH, a subsidiary of Sudzucker AG. Mitsui Sugar Co. of Japan also produces isomaltulose, as does Cargill Corp. under the name XTend Isomaltulose. The companies market the compounds as natural, non-cariogenic low-glycemic index sweeteners which can be substituted for sucrose in many applications. Trehalulose Chemically related to isomaltulose, trehalulose is the ketose analogue of trehalose. Its structure is α-D-glucopyranosyl-(1→1)-D-fructose. It is synthesized as a

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coproduct by the same isomerase enzyme that synthesizes isomaltulose, but in generally smaller yields. Whereas production of isomaltulose is kinetically favored, trehalulose is thermodynamically favored, so it tends to accumulate only after long reaction times (Cheetham, 1984). Protaminobacter rubrum is used primarily for production of isomaltulose (Nakajima and Nishio, 1993), but a system using Pseudomonas mesoacidophila has been described which synthesizes mainly trehalulose from sucrose (Kitahata, 2001). Since trehalulose is not as widely available as isomaltulose, food applications do not yet exist, although it has been suggested as a non-cariogenic sweetener, similar to isomaltulose (Kitahata, 2001; Hamada, 2002). Leucrose Leucrose was first described by the same USDA researchers who initially isolated isomaltulose and derives its name from the source organism, Leuconostoc mesenteroides (Stodola et al., 1952). Structurally, leucrose is α-D-glucopyranosyl(1→5)-D-fructopyranose (Fig. 12.3). It is formed by the dextransucrase-catalysed transfer of glucosyl units to fructose acceptors, forming an α(1→5) linkage. The nominal reaction of dextransucrase is to incorporate the α-D-glucopyranosyl units from sucrose into dextran, an α(1→6)-linked D-glucan. As the levels of liberated fructose accumulate in the reaction mixture, some glucosyl units are transferred to fructose in what are known as acceptor reactions (Koepsell et al., 1953). Acceptor reactions with fructose give rise to leucrose as the major product and isomaltulose as a minor product in dextransucrase reactions. Leucrose bears many similarities to isomaltulose. It is about one-half as sweet as sucrose, is non-cariogenic, resistant to acid hydrolysis and releases glucose into the bloodstream only slowly (Schwengers, 1991). Its susceptibility to hydrolysis by various carbohydrases has been reported (Iizuka et al., 1990). It is made industrially using immobilized dextransucrase (Reh et al., 1996; Buchholz et al., 1998) and Pfieifer and Langen of Germany is the major producer.

12.2.2 Honey oligosaccharides For many years it was believed that honey consisted solely of sucrose and its hydrolyzates, glucose and fructose, but it was eventually shown that honey also contains numerous other di- and oligosaccharides. A typical honey sample contains approximately 15–20% water, 30–40% each of glucose and fructose, and

Fig. 12.3

Leucrose.

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anywhere from about 1 to 15% of di- and higher oligosaccharides (Siddiqui, 1970; Doner, 1977). Besides sucrose, which is usually the prevalent disaccharide, many other saccharides have also been found in honey. These are believed to arise from the action of glycosidase enzymes, presumably originating with the bees. In highly concentrated solutions of sugars, which certainly includes honey, glycosidases catalyse transglycosylations as well as the reverse reaction of hydrolysis, namely, condensation. Thus, α-D-glucopyranosidases and β-D-fructofuranosidases present in honey result not only in the hydrolysis of the sucrose in nectar, but also in the formation of a variety of glucose and fructose-containing saccharides. Some of the more commonly encountered saccharides in honey include the glucose disaccharides maltose (α1→4 linked), kojibiose (α1→2 linked), isomaltose (α1→6 linked) and α,α-trehalose (α1↔α1 linked). The sucrose linkage isomers isomaltulose, leucrose, maltulose (α-D-Glcp-1→4 D-Fru) and turanose (α-D-Glcp-1→3 D-Fru) are also found at significant levels. Glycosylated sucroses found in honey include erlose, theanderose and various kestoses, which will be discussed in a later section. Searches of the Internet as well as the popular and scientific literature turn up countless references to the purported health benefits of honey. These are often attributed to the prebiotic effects of the various oligosaccharides present, but definitive proof is scarce. One recent study does indicate that honey oligosaccharides exhibit some prebiotic activity in vitro (Sanz et al., 2005a) but this is of very questionable relevance at normal levels of consumption.

12.2.3 Glycosyl sucroses Melezitose Melezitose is an unusual trisaccharide whose structure is α-D-glucopyranosyl(1→3)-β-D-fructofuranosyl(2↔1)-α-D-glucopyranoside (Hehre, 1953; Fig. 12.4). Note that this structure contains both turanose and sucrose substructures. Often found in honey, the concentration of melezitose sometimes reaches levels high enough to cause it to crystallize in the comb and the term ‘melezitose honey’ refers to this undesirable phenomenon. Melezitose also occurs in the plant exudates

Fig. 12.4 Melezitose.

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known as honeydew and various types of so-called ‘manna’ from trees (Hudson, 1946). In the latter instance, it was long observed that certain trees would periodically become covered with a white, crystalline substance. European larch and Douglas fir, in particular, have been known to exude this material, often in great quantities. The name melezitose is derived from the French name for larch, ‘le mélèze’ (Hudson, 1946). It was later shown that melezitose, rather than being a product of the plants themselves, arises from the action of aphids and other insects on sucrose in the plants’ sap (Bacon and Dickinson, 1957). Although melezitose has been consumed by humans for many years as an ingredient of these natural substances, little is known of its nutritional or health effects. Theanderose and erlose Many D-fructofuranosidases are capable of transferring fructosyl units to various acceptors, producing in some cases fructofuranosyl analogues of sucrose. The acceptors may be monosaccharides, in which case the product is a disaccharide analogue of sucrose, or oligosaccharides, giving rise to glycosyl sucroses. Theanderose was first characterized by Barker et al. (1957) and was subsequently given its trivial name after one of its co-discoverers. Structurally, it is α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside (Fig. 12.5). It has also been referred to as isomaltosucrose. It is typically synthesized by the transferase action of β-D-fructofuranosidase, but can also be prepared by the action of levansucrase on sucrose and isomaltose. It has been proposed as a non-cariogenic sweetener (Kitahata and Fujita, 1993). According to a review article (Ogawa and Shimizu, 2002), theanderose and its higher homologues were manufactured in Japan, beginning in 1994, by Asahi Chemical Industry, using an immobilized enzyme bioreactor. Erlose, previously mentioned as a component of honey, was first identified at the Eastern Regional Research Laboratory (ERRL) of USDA, where it was produced by the action of honey invertase on sucrose (White and Maher, 1953).

Fig. 12.5

Theanderose.

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Fig. 12.6

249

Erlose.

The initials of the laboratory gave rise to its trivial name. Erlose, α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside (Fig. 12.6), is a linkage isomer of theanderose. Besides occurring in honey, presumably from the action of honey invertase on sucrose, it can also be formed via acceptor reactions of levansucrase with maltose (Côté and Ahlgren, 1993; Canedo et al., 1999). The author knows of no large-scale industrial application of erlose, although Hayashibara Biochemical Laboratories of Japan does offer it for sale in laboratory quantities. It is a major component of coupling sugar (see below). Xylsucrose Xylsucrose is an analogue of sucrose in which the α-D-glucopyranosyl unit has been replaced by an α-D-xylopyranosyl group, so that its structure is α-D-xylopyranosyl-(1↔2)-β-D-fructofuranoside (Fig. 12.7). It is formed by the action of levansucrase, which transfers D-fructofuranosyl units from sucrose to a xylose acceptor (Avigad et al., 1956). Like theanderose, it can also be formed by the transferase action of β-D-fructofuranosidase. It is useful as a non-cariogenic sweetener, where it can be substituted for sucrose on a 1:1 basis (Kitahata and Fujita, 1993).

Fig. 12.7

Xylsucrose.

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Fig. 12.8

Lactosucrose.

Lactosucrose Many studies have indicated that lactosucrose may be useful as a dietary ingredient for its prebiotic effect (Kitahata and Fujita, 1993; Kitahata, 2001; Rastall, 2004). It is a glycosylated derivative of sucrose, as are theanderose and erlose, but contains a galactose residue. Chemically, its structure is β-D-galactopyranosyl(1→4)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside. The trivial name derives from the fact that it contains both a sucrose and a lactose substructure (Fig. 12.8). It can be synthesized enzymatically via the fructosyltransferase reaction of either levansucrase (Avigad, 1957; Park et al., 2005) or β-D-fructofuranosidase; the latter is used for commercial production (Fujita et al., 1992; Taniguchi, 2004). The sweetness of lactosucrose is about half that of sucrose, as is its caloric value (Taniguchi, 2005). It is apparently not absorbed or digested in the upper gastrointestinal tract and is fermented by bacteria in the colon, particularly by Bifidobacterium spp. Several nutritional and health effects have been claimed, including the prevention of constipation and reduction of serum hyperlipidemia (Kitahata and Fujita, 1993). A significant market for lactosucrose is in pet foods, where it is claimed to reduce the odor of cat feces (Terada et al., 1993). At least 2000 tons are produced annually in Japan by the Bioresearch Corporation of Yokohama (Taniguchi, 2004). It has been marketed under the trade name Nyukaoligo, which is a mixture of lactosucrose with lesser amounts of sucrose, lactose and various trace sugars (Kitahata, 2001). Maltosyl sucrose (coupling sugar, glycosyl sucrose) Chemically related to erlose (Fig. 12.6), coupling sugar is sometimes considered to be a derivative of starch. However, it equally can be considered a sucrose-derived product, as it results from the coupling of starch-derived maltose-oligosaccharides with sucrose, hence the name coupling sugar. The term actually refers to a mixture of oligosaccharides thus derived. Coupling sugar is made enzymatically by the action of cyclomaltodextrin glucanotransferase (CGT) on starch in the presence of sucrose. Normally, CGT acts on starch to yield cyclomaltodextrins (cyclodextrins

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or Schardinger dextrins), which are cyclic α-(1→4)-D-gluco-oligosaccharides (French, 1957). However, in the presence of an acceptor, in this case sucrose, the gluco-oligosaccharides are transferred to the D-glucosyl portion of sucrose, forming an α-(1→4) linkage. The simplest structure formed in this way is α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside, or erlose (Fig. 12.6), which results from the transfer of a single glucosyl residue to sucrose. The next higher homologue in the series is the tetrasaccharide α-Dglucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1↔2)β-D-fructofuranoside, or maltosyl sucrose. Pentasaccharides and higher are also present in the mixture, although in lesser quantities (Okada and Kitahata, 1993; Kitahata, 2001; Monthieu et al., 2003). Coupling sugar is digestible by humans, mainly by amylases and sucraseisomaltase enzymes in the small intestines (Okada and Kitahata, 1993). Its caloric value is similar to that of starch or sucrose and its sweetness is about one-half that of sucrose (Taniguchi, 2005). It is non-cariogenic, and does not undergo Maillard browning reactions, owing to its nonreducing nature. Not only is it useful as a noncariogenic sweetener, but it can also prevent starch retrogradation and sugar crystallization in processed foods (Okada and Kitahata, 1993). Hayashibara Co. of Japan is a major manufacturer. Raffinose, stachyose and verbascose (soy oligosaccharides) Unlike the oligosaccharides discussed above, raffinose and its higher homologues stachyose and verbascose are not manufactured from sucrose, but are instead

Fig. 12.9

Raffinose.

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extracted from plants. However, since they are structurally and biologically derived from sucrose, a brief description is included here. The so-called raffinose family of oligosaccharides can be found in many plants, particularly in the seeds of legumes (French, 1954). Structurally, raffinose is α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside (Fig. 12.9), which may be considered to be an isomer of theanderose. Its higher homologues are the tetrasaccharide stachyose, which contains an additional galactosyl residue, and the pentasaccharide verbascose, which contains two additional galactosyl residues. Since these all contain galactosyl residues at one end, they cannot be hydrolysed by the same types of α-glucosidases that hydrolyse theanderose, erlose, and so on Instead, they pass undigested into the lower GI tract, where they are fermented by bacteria in the colon. Soy oligosaccharides are isolated from soy whey, the water-soluble by-product of soy protein production. The mixture typically contains sucrose, raffinose, stachyose and verbascose, with stachyose being the major oligosaccharide component. Cottonseed meal is also a good source of these oligosaccharides and raffinose is the predominant component from that source. There is evidence that soy oligosaccharides can function as useful prebiotics, serving as growth substrates for several species of Lactobacillus and Bifidobacterium (Koga et al., 1993). It should be pointed out, though, that raffinose oligosaccharides are also fermented by gasproducing bacteria in the colon, and are responsible for the so-called ‘flatus effect’ of beans (Rackis, 1975).

12.2.4 Fructo-oligosaccharides (FOS) Without a doubt, the most widely studied sucrose-derived oligosaccharide products on the market today are the fructo-oligosaccharides (FOS). This is actually a broad class of complex carbohydrates which includes short chain FOS (scFOS), inulin, levan and levan oligosaccharides. Care should be taken to differentiate between these products, as they can have significantly different chemical, physical and metabolic properties. Entire volumes have been devoted to the subject and the author refers the reader to one in particular (Suzuki and Chatterton, 1993) which contains a useful glossary of terms used in this field (Waterhouse and Chatterton, 1993). In general, the FOS can be classed into the three categories mentioned above: short chain FOS, which are derived from the action of a microbial fructosyltransferase on sucrose and contain approximately three to five monosaccharide units; inulin, which can be viewed as either a small polysaccharide or very large oligosaccharide derived directly via extraction from plants; and levan oligosaccharides, which are derived directly or indirectly from sucrose through the action of microbial levansucrase enzymes. We will discuss each of these separately. Inulin Inulin has been a part of human diets since prehistory, as it forms the primary storage carbohydrate of many plants, most notably the Jerusalem artichoke

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(Helianthus tuberosus) and chicory (Cichorium intybus) (McDonald, 1946; Frese, 1993). Inulin is a relatively water-soluble, short chain polysaccharide synthesized from sucrose and takes the place of starch as a carbohydrate storage pool in some plants (Fuchs, 1993). It typically consists of approximately 10–30 fructofuranoside units connected by β-(2→1) linkages. As a result of its biosynthesis from sucrose, the fructan chain is terminated at one end by a glucose unit in a sucrose linkage. It is usually extracted from the tuberous roots of the plants using water and may undergo further processing for various applications. Although the traditional and commercial source of inulin is from the plants mentioned above, it should be pointed out that certain bacteria are known to secrete enzymes capable of converting sucrose to inulin, most notably the food-grade lactic acid bacteria Lactobacillus reuteri (van Hijum et al., 2002) and Leuconostoc citreum (Olivares-Illana et al., 2003; Ortiz-Soto et al., 2004). In these instances, a single enzyme, inulosucrase, is responsible and the product tends to be of much higher molecular weight than plant inulins. There have been many studies on the utility of inulin in the diets of humans and animals. Any attempt to do this field justice in such a short review as this would certainly fall short, so I will just point out a few representative works. Undoubtedly, the most widely studied application of inulin is as a prebiotic. It is unfortunate that some studies fail to emphasize the difference between inulin and other types of FOS. It can be said that FOS in general are useful as prebiotics, where they enhance the growth of beneficial bacteria, especially Bifidobacterium and Lactobacillus spp. (Flickinger and Fahey, 2002; Kolida et al., 2002; Sobotka et al., 1997; Tungland, 2003). In fact, it can be fairly stated that inulin and FOS have become the standard against which all other prebiotics, current and proposed, are measured. Although some studies on the prebiotic effect of fructans do not take into account the difference between inulin and shorter chain FOS, more recent studies are aimed at differentiating between the two and better understanding the effect of molecular size on prebiotic activity (Biedrzycka and Bielecka, 2004; Sangeetha et al., 2005). Besides its utility as a prebiotic, inulin can contribute to the quality of foods as a texturizer, emulsion stabilizer and partial fat replacer (Franck, 2002). It also has seen medical uses, most notably in the measurement of kidney function and as a drug carrier (Fuchs, 1993). Inulin may be partially hydrolysed, either enzymatically or chemically, to yield inulooligosaccharides (Fuchs, 1991). These find uses similar to the scFOS described below. Orafti Co. of Belgium produces both native and partially hydrolysed inulin as Raftiline and Raftilose, respectively. Both possess prebiotic activity (Niness, 1999). Short chain fructo-oligosaccharides (scFOS) The amount of information available on short chain fructo-oligosaccharides, often referred to as simply FOS, is at least as voluminous as that on inulin, if not more so. Rather than attempt to address the functional and physiological studies on scFOS, the reader is referred to some reviews of their nutritional physiology (Roberfroid, 1999; Hirayama, 2002; Losada and Olleros, 2002; Oku and Nakamura, 2002; Wolf et al., 2003).

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Fig. 12.10 1-Kestose.

Unlike inulin, scFOS are produced in vitro by microbial or enzymatic action on sucrose. The most common method entails the use of a β-D-fructofuranosidase from Aspergillus niger (Hidaka et al., 1988, 1991; Kono, 1993). The enzyme catalyses the intermolecular transfer of fructosyl units from one sucrose molecule to another, resulting in a family of β-D-fructofuranosyl sucrose compounds. The predominant trisaccharide formed is 1-kestose, or β-D-fructofuranosyl-(2→1)-βD-fructofuranosyl-(2↔1)-α-D-glucopyranoside (Fig. 12.10), abbreviated GF2. The fructosyl chains can be built up beyond the trisaccharides initially formed, to give tetrasaccharide (nystose or GF3) and pentasaccharide (GF4) as well (Oku et al., 1984; Yun, 1996; Hidaka and Hirayama, 1991). The fructosyl units are linked β-(2→1) and so scFOS bear a structural relationship to inulin and inulooligosaccharides, despite their very different origins. Short chain FOS have been produced and marketed by several firms under various trade names, including Meioligo (also known as Neosugar, Meiji Seika Kaisha, Japan), Actilight (Beghin-Meiji Industries, France), Nutraflora (GTC Nutrition, USA) and Oligo-sugar (Cheiljedang Corporation, Korea). In the latter case, the company apparently uses a fructosyltransferase from Aureobasidium pullulans, rather than A. niger (Yun, 1996). Although FOS have garnered the lion’s share of the prebiotics market, there is an active movement to find newer prebiotics that do not possess some of the shortcomings of FOS. Among the more sought-after properties in the so-called second generation prebiotics are enhanced stability to acids and more specific utilization by Bifidobacterium or Lactobacillus spp. (Rastall and Maitin, 2002). Levan oligosaccharides Levans are extracellular polysaccharides produced by the bacterial enzyme levansucrase (E.C. 2.4.1.10; Côté and Ahlgren, 1993). These fructans differ from

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inulin in that they are generally of higher molecular weight and the fructosyl units are linked β-(2→6). Oligosaccharides may be obtained by partial hydrolysis of the polysaccharide, or by acceptor reactions of the enzyme. Since the structures are different from the inulin and scFOS discussed above, it may be expected that the levan oligosaccharides would have somewhat different properties. To date, very few studies have been carried out on this relatively new class of FOS. One study showed that oligosaccharides produced by partial acid hydrolysis of levan could be utilized by several Bifidobacterium species, and were particularly well utilized by Bifidobacterium adolescentis (Marx et al., 2000).

12.2.5 Gluco-oligosaccharides There are many sources of glucose oligosaccharides, but relatively few come from sucrose. We have already discussed the preparation known as coupling sugar, which is derived from starch and sucrose. Another type of glucose oligomer can be produced by the class of enzymes known as glucansucrases, which are glucosyltransferases that transfer α-D-glucopyranosyl units from sucrose into polymeric α-D-glucans. Similar in some ways to levansucrase and inulosucrase, glucansucrases are typically produced extracellularly by lactic acid bacteria growing on sucrose, resulting in the formation of copious amounts of slimy or gummy polysaccharide. These glucans will be discussed in more detail later. Like the fructosyltransferases, glucansucrases can also carry out acceptor reactions, resulting in the formation of various oligosaccharides. Many strains of L. mesenteroides produce glucansucrases and the structures of the resultant glucans vary in the relative distribution and amount of the predominant α-(1→6) linkages and the less predominant α-(1→2), α-(1→3) and α-(1→4) linkages (Jeanes et al., 1954). These different strains produce different types of acceptor products (Côté and Leathers, 2005). Branched gluco-oligosaccharides L. mesenteroides strain NRRL B-1299 produces at least two different glucans from sucrose, both of which contain α-(1→6) linkages in the main backbone of the glucan chains and α-(1→2) branch linkages (Jeanes et al., 1954; Kobayashi et al., 1973; Dols et al., 1997). In the presence of acceptor sugars such as glucose or maltose, a series of gluco-oligosaccharides are formed which contain both types of linkages (Paul et al., 1992; Remaud-Simeon et al., 1994; Dols et al., 1998). Maltose is a particularly good acceptor and the products have been characterized (Dols et al., 1998). The enzyme produces at least three different types of acceptor products in this case. The first type is simply linear oligodextrans, which contain only α-(1→6) linkages. The other types contain α-(1→2) branch linkages at either the non-reducing end or on the glucose residue penultimate to the non-reducing end. The average degree of polymerization (DP) of the oligosaccharide mixture depends on the conditions of synthesis. A higher ratio of sucrose donor to maltose acceptor results in a higher average DP. An oligosaccharide mixture from immobilized L. mesenteroides NRRL B-1299

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is produced in the order of several tonnes per year and is marketed in Italy under the name Bioecolians. The average DP is in the range of 5–6 and they are at least partially indigestible (Valette et al., 1993). The mixture appears to be a promising prebiotic (Djouzi et al., 1995) and is also useful for the regulation of carbohydrate metabolism (Boucher et al., 2003). Another, related type of branched gluco-oligosaccharide is produced by the glucansucrases from L. mesenteroides NRRL B-742. These differ from the B-1299 products in that they lack α-(1→2) linkages, but instead contain α-(1→3) branch linkages (Remaud et al., 1992). These oligosaccharides have been produced in two ways: by a mixed fermentation using L. mesenteroides NRRL B-742 and a dextranase-producing Lipomyces starkeyi strain (Yoo et al., 2001) and by acceptor reactions in the presence of maltose (Day and Yoo, 2001; Chung and Day, 2002). In vitro studies indicate that the oligosaccharides may be useful as prebiotics (Chung and Day, 2002), at least in poultry (Chung and Day, 2004). These oligosaccharides are not yet in commercial production. Alternan oligosaccharides Alternansucrase (E.C. 2.4.1.140) is related to the glucansucrases described above, but synthesizes an alternating sequence of α-(1→3) and α-(1→6)-linked glucose units (Côté and Robyt, 1982a). The oligosaccharide acceptor products formed by this enzyme have been studied for over 20 years (Côté and Robyt, 1982b; Pelenc et al., 1991) and are of interest because of their potential prebiotic activity. Recent in vitro studies have shown that they are capable of selectively supporting the growth of specific Bifidobacterium spp. (Côté et al., 2003; Holt et al., 2005; Sanz et al., 2005b), thus suggesting promise as novel prebiotics. A proprietary mixture containing these oligosaccharides has also shown potential as a low-glycemic index sweetener (Carlson and Woo, 2004). Isomalto-oligosaccharides (isomaltodextrins) Isomaltose is the glucose disaccharide α-D-glucopyranosyl-(1→6)-α-D-glucopyranose. Oligosaccharides of this series may be synthesized by at least three different enzymatic methods. Transglucosylation and condensation reactions catalysed by microbial α-glucosidases can convert glucose-containing syrups from starch to a mixture with a high percentage of α-(1→6)-linked oligosaccharides. Since these are not derived from sucrose, we will not consider them here. Instead, we will focus on the remaining two methods, namely, partial hydrolysis of the α(1→6)-linked glucan known as dextran (see below), and acceptor reactions by dextransucrase. Dextran is an α-(1→6)-linked glucan produced enzymatically from sucrose (Leathers, 2002). Partial chemical or enzymatic hydrolysis of dextran results in low-molecular weight dextrans and oligosaccharides. These α-(1→6)-linked oligosaccharides are known as isomaltodextrins or oligodextrans. Acid hydrolysis of polysaccharides tends to yield unacceptably high levels of the monosaccharide (Turvey and Whelan, 1957), so enzymatic approaches are generally preferred. In

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the case of dextran, endo-dextranases are used to yield a mixture of low-molecular weight dextran and isomaltodextrins. The hydrolysis can be done on preformed dextran (Mountzouris et al., 1999, 2001) or the synthesis and hydrolysis may be carried out simultaneously using a mixture of dextransucrase and dextranase, with sucrose as the sole substrate (Goulas et al., 2004a, 2004b). In either case, a purification step is required at some point in order to remove unwanted high or low-molecular weight components. Isomalto-oligosaccharides (IMO) may also be conveniently prepared using the acceptor reactions of dextransucrase. L. mesenteroides NRRL B-512F, which is used commercially for the production of dextran, is also used as the source of dextransucrase for IMO production. In the presence of a suitable acceptor, oligosaccharides are formed and fructose is released (Koepsell et al., 1953). Glucose may be used as the acceptor, in which case the acceptor products are isomalto-oligosaccharides containing solely α-(1→6)-linkages (Pereira et al., 1998; Tanriseven and Dogan, 2002), or maltose may be used, in which case the products contain an α-(1→4)-linkage at the reducing end (Buchholz and Seibel, 2003). Since the latter is a much more efficient acceptor, most studies have been performed using maltose (Paul et al., 1986; Castillo et al., 1992; Heincke et al., 1999). It is considered advantageous to use immobilized dextransucrase for largescale production (Tanriseven and Dogan, 2002; Berensmeier et al., 2004). When studying the literature on the nutritional properties of isomaltose oligosaccharides, one must be careful to differentiate between the three types of preparations, all of which have been referred to as IMO. Many studies that refer to IMO activity as a prebiotic have used the transglucosidase product from starch hydrolyzates. This is also sometimes referred to as anomalously linked oligosaccharide, or ALO (Yatake, 1993). Studies on the sucrose-derived product are more scarce. Buchholz and Seibel have reviewed the relevant literature (Buchholz and Seibel, 2003).

12.3 Polysaccharides from sucrose Microbial polysaccharides are finding increasingly widespread use in foods as well as in non-food applications. These include xanthan, gellan, curdlan, pullulan, microbial alginate and others. Sucrose may be used as a feedstock for the fermentative production of a wide variety of microbial products, including these polysaccharides. In these instances, the applications do not take advantage of the unique structural features of sucrose, but merely use it as a convenient carbohydrate or carbon source. There are some microbial polysaccharides that are synthesized uniquely from sucrose and we will consider those here. These examples all depend on a particular class of enzymes known as glycansucrases. We have already discussed some of these enzymes in the preceding sections, as they are useful for oligosaccharide synthesis as well as polysaccharide synthesis. The general reaction catalysed by glycansucrases is the transfer of either the α-Dglucopyranosyl units or the β-D-fructofuranosyl units from sucrose into

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homopolysaccharides. We will discuss some of the more well-known members of this class separately.

12.3.1 Dextran The term dextran refers to a large group of α-D-glucans in which the main, or backbone chain, consists of α-(1→6)-linked D-glucopyranosyl units. There may be branches linked through various secondary linkages such as α-(1→2), α-(1→3) or α-(1→4) (Jeanes et al., 1954). Dextran used in commercial applications is usually derived from L. mesenteroides NRRL strain B-512F, which produces a glucan with approximately 95% α-(1→6) linkages and 5% α-(1→3) branch linkages. The native product is of very high molecular weight, typically around twenty million (20 × 106). Dextran has been the subject of several reviews and the reader is referred to them for an in-depth discussion of its chemistry and biochemistry (Sidebotham, 1974; Alsop, 1983; Robyt, 1986; DeBelder, 1993; Leathers, 2002; Naessens et al., 2005). Dextran is reported to be digestible by humans, resulting in a slow increase in blood glucose levels (Jeanes, 1975). Hehre and Sery (1952) showed that Bacteroides spp. from human sources were capable of fermenting dextran, and more recently, Olano-Martin et al., (2000) showed that dextran fractions were fermentable by probiotic human gut bacteria. Dextran was initially studied in the 1950s as a food ingredient, particularly as a thickener, but appears not to have been used for this purpose. The US Food and Drug Administration currently lists dextran as GRAS (generally regarded as safe) for two applications: indirectly in human foods as an ingredient of food contact surfaces, such as packaging (21CFR186.1275), and low-molecular weight dextran (<100 000) as an ingredient of animal feeds and medicines (21CFR582.1275). In 2001, the European Commission approved a petition by Puracor to use dextran in baked goods, up to levels of 5%. According to the official decision statement, it is considered to have nutritional properties similar to starch (Byrne, 2001). Many non-food uses for dextran exist, including the clinical-sized dextrans used as blood plasma extenders, cross-linked dextran as molecular sieves and dextran sulfates for medicinal purposes. Industrially, dextran has been used for aluminum ore processing and in X-ray film emulsions (DeBelder, 1993).

12.3.2 Alternan and reuteran Until 1982, alternan was considered to be a dextran. At that point, it was shown that this polysaccharide is unique, and that a separate enzyme is responsible for its biosynthesis (Côté and Robyt, 1982a). The name ‘alternan’ was coined to differentiate it from true dextrans, owing to its alternating sequence of α-(1→3) and α-(1→6) linkages. Alternan possesses many properties that differ from commercial dextran, including lower viscosity and increased resistance to microbial and enzymatic degradation (Côté, 2002). Low-molecular weight fractions of alternan

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have been produced by enzymatic and physical treatments (Côté, 1992; Côté et al., 1997; Côté and Willett, 1999; Côté, 2002; Leathers et al., 2002; Leathers et al., 2003). These may be useful as bulking agents and low-calorie food ingredients (Côté et al., 1997). It is believed that protein conjugates of alternan or dextran may be useful as replacements for gum arabic (Dunlap and Côté, 2005; Leathers et al., 2003). Alternan has been found in the Asian fermented vegetable dish kimchi, where it is apparently produced by the L. mesenteroides strain responsible for the fermentation process (Jung et al., 1999). Other, non-food uses for alternan have also been proposed (Côté, 2002). Reuteran is the name given to an α-D-glucan from the organism L. reuteri, a lactic acid bacterium associated with fermented milk products. It is similar to branched dextrans or alternan, but contains a high percentage of α-(1→4) linkages (van Geel-Schutten et al., 1999). It is one of several novel glucans produced by dairy strains of Lactobacillus spp. (Kralj, 2004) and may play a role in the thickening of fermented dairy foods. Technical and food applications of the enzymes and polysaccharides are currently being investigated (Tieking et al., 2005).

12.3.3 Levan Like inulin, levan is a polymer of fructose (old name: levulose, hence the name levan). However, its β-D-fructofuranosyl units are linked (2→6), rather than (2→1). Levan is produced by bacterial enzymes from sucrose in a manner analogous to the dextrans (Côté and Ahlgren, 1993). We have already mentioned the use of levansucrase for the production of xylsucrose and other oligosaccharides via acceptor reactions. The polysaccharide levan is produced in the absence of acceptors, and can reach molecular weights in the millions (Clarke et al., 1991). Various bacteria are known to produce levan, including Streptococcus, Lactobacillus, Leuconostoc, Pseudomonas, Zymomonas, and Actinomyces spp. The best studied are those from Aerobacter levanicum (synonym: Erwinia herbicola) and Bacillus spp. The enzymes of these bacteria can be isolated from the extracellular culture broth, so the polysaccharide can be produced either by fermentation or by cell-free enzyme preparations. In addition to sucrose, raffinose can also serve as a fructosyl donor (Côté and Ahlgren, 1993). Levan has been proposed for a number of applications, including blood plasma extenders, food thickeners, aqueous two-phase partitioning, encapsulation and drug excipients, to mention only a few (Han, 1990; Rhee et al., 2002). It is also being investigated as a prebiotic (Probert and Gibson, 2002; Dal Bello et al., 2001). Some of these applications are limited owing to the higher acid-lability of the fructan relative to glucans. One promising use is as a biodegradable adhesive (Combie, 2003; Combie et al., 2004) and a cosmetic texturizer. Pilot-scale production of levan for these and other applications has been undertaken by Shanghai Cathay Biotechnology in China, as part of a joint undertaking with Montana Biotech SE. The company produces the polymer by fermentation of sucrose-containing feedstocks, using a proprietary strain of Bacillus.

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12.4 Other products In addition to the oligosaccharides and polysaccharides discussed above, sucrose can be used as the feedstock for many other food and non-food products (Lichtenthaler and Peters, 2004). We will discuss two of the more commonly used, namely, invert sugar and sucrose esters.

12.4.1 Invert sugar Invert sugar is simply the hydrolysis product of sucrose. The α1↔β2 linkage of sucrose is more acid labile than the glycosidic linkages of most saccharides. Heating with acids can result in splitting sucrose into an equimolar mixture of fructose and glucose. Since fructose is nearly twice as sweet as sucrose, invert sugar is typically sweeter than sucrose. The name ‘invert sugar’ arises from the fact that it is a mixture of fructose anomers, which rotates polarized light in a levo- or left-handed manner, and glucose anomers. The mixture is slightly levorotatory, compared with the dextrorotatory nature of sucrose, so the direction of rotation of polarized light is said to have been inverted. Several different types of invert sugar exist, depending on the degree of hydrolysis. Invert sugar is often used in candies as a combination sweetener, humectant and crystallization inhibitor. Traditionally, invert sugar is made by acid hydrolysis of heated sucrose syrups. In cooking or candy making, the acid is often citrus juice or cream of tartar (a form of tartaric acid). However, these methods are not as suitable for large-scale production of invert sugar, owing to the energy input required for heating and the impurities that result as by-products of the acid reaction. An enzymatic approach is often used, in which yeast cells or a yeast-derived enzyme hydrolyses the sucrose linkage. The most commonly used enzyme, yeast invertase, is a β- D fructofuranosidase. Yeast invertase is sold under such trade names as Maxinvert (DSM Food Specialties), Bioinvert (Quest International), Sucrovert (Chr. Hansen) and Validase (Valley Research). Current research is aimed at reducing the cost of producing invertase and at the use of immobilized enzyme technology for the efficient conversion of sucrose syrups.

12.4.2 Sucrose esters A number of different sucrose esters can be found in foods and other consumer products. Sucrose contains eight free hydroxyl groups and any number and combination can be combined with acid-containing compounds to yield a wide variety of sucrose esters. Some of the sucrose esters which may be found in consumer goods include sucrose octa-acetate, a bitter compound used to denature alcohol and some household chemicals, Olestra, a mixture of hexa- through octasubstituted fatty acid esters of sucrose used as a fat substitute in snack foods, and sucrose monoesters, used as emulsifiers. These esters are usually synthesized chemically, but developments in enzyme technology have led to the use of enzymes for some applications.

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Recent developments in enzyme technology have led to an increased interest in the use of lipases, proteases and related esterase-type enzymes for the synthesis of sugar esters. For example, Dean et al. (1992) synthesized sucrose monoesters using the protease subtilisin in organic solvents, and Plou et al. (1995) showed that sucrose monoesters can be converted to di- and tri-esters using commercially available protease. Other work has focused on the selectivity of such enzymatic reactions (Pedersen et al., 2002a,b; Raku et al., 2003). Advantages of the enzymatic approach include better product specificity and less likelihood of undesirable contaminants in the end product. It is expected that enzymatic processes will eventually replace chemical processes for some applications.

12.5 Future trends The last half of the 20th century saw many rapid developments in food technology, including many based on carbohydrate biotechnology. One of the most significant was the introduction of corn syrup-based sweeteners, especially high-fructose corn syrup. This product depends heavily on enzyme technology, including amylases and xylose isomerase. Another area that developed during this period was the introduction of microbial gums to replace the more traditional plant-based gums. Thus, we have seen xanthan and gellan take over a huge share of the market, with a resultant decrease in market shares for gums tragacanth, ghatti, karaya, and so on. We can already see trends that have developed over the last several years that can be expected to continue into the foreseeable future. Prebiotics have established themselves in the Asian markets and are becoming significant in western markets as well. Fructo-oligosaccharides, soy oligosaccharides, lactosucrose and others are considered to be just the first generation of carbohydrate-based prebiotics. The next decade will be likely to see an increased interest in prebiotics that exhibit greater selectivity for specific probiotic bacteria, especially in combinations known as ‘synbiotics’. We will also see more research into ways to enhance the persistence and fermentability of prebiotics in various populations such as the elderly, infants, and the immunocompromised (Rastall and Maitin, 2002; Rastall et al., 2005), as well as in livestock and pets. Whereas the latter half of the 20th century saw the introduction of artificial, non-caloric sweeteners such as acesulfame, aspartame, saccharine, and so on, the next decade will see an increasing interest in sweeteners that exhibit a slow release of carbohydrate into the bloodstream. The first of these low-glycemic index (lowGI) sweeteners have been introduced in the form of carbohydrates such as isomaltulose and sucromalt. We can expect more in the future. Another developing trend that will affect the food industry as much as it has affected the drug industry will be the increasing use of biocatalysts for more selective and ‘greener’ ways to synthesize ingredients. We are already seeing this in the enzymatic approaches to oligosaccharide synthesis. Another application where this is likely to take hold will probably be in the production of sugar esters as emulsifiers.

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12.6 Sources of further information and advice Although the sugar industry cannot be compared with the pharmaceutical industry and other more lucrative areas in terms of money available for research, there has been funding for sucrose utilization research through various government agencies. The US Department of Agriculture (USDA) and its Australian, Japanese and European counterparts have an interest in making sure that markets expand for sugar and related agricultural products. A primary purpose of the various Departments or Ministries of Agriculture is to support agriculture through research and regulation. In addition, there are various regulatory agencies throughout the world whose main function is to ensure the safety of the food supply. In the United States, this is primarily the job of the Food and Drug Administration (FDA), although USDA also has a role in certain aspects of food safety. In Europe, the European Commission has delegated this role to the European Food Safety Authority (EFSA) and in Japan it is the job of the Ministry of Health and Welfare (MHW). Internationally, the agencies work together through the Codex Alimentarious Commission, overseen by the United Nations World Health Organization (WHO) and the Food and Agriculture Organization (FAO). All of these agencies are sources of information on the regulatory status of food ingredients. For example, the Japanese MHW has issued a list known as FOSHU, or foods for specific health uses. The list includes several of the oligosaccharides discussed in this chapter. Various individual companies also support research into new food ingredients derived from sucrose. Research and lobbying efforts are also supported by public and private sector consortia such as the Sugar Processing Research Institute (SPRI, USA), the Sugar Research and Development Corporation (SRDC, Australia), The Sugar Association (USA) and others. From 2005, many of these entities had websites that were listed on the Sugar Association’s website: www.sugar.org. It is difficult to list websites in a book chapter such as this, owing to the dynamic and constantly changing nature of the Worldwide Web. However one review has attempted to give an overview of web resources for carbohydrate researchers (Berteau and Stenutz, 2004). The reader is probably best advised to make judicious use of the various search engines available and to read anything posted on the web with a critical attitude. The peer-reviewed literature, such as those papers listed at the end of this chapter, are still the most definitive source of information on sugarderived food ingredients.

12.7 References Alsop RM (1983). ‘Industrial production of dextrans’, in Bushell ME (ed.), Progress in Industrial Microbiology, Vol. 18, Microbial Polysaccharides, Elsevier, Amsterdam, 1– 44. Avigad G (1957). ‘Enzymatic synthesis and characterization of a new trisaccharide, αlactosyl-β-fructofuranoside’, J Biol Chem, 229, 121–129. Avigad G, Feingold DS and Hestrin S (1956). ‘An enzymic synthesis of a sucrose analog: α-D-xylopyranosyl-β-D-fructofuranoside’, Biochim Biophys Acta, 20, 129–134.

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Bacon JSD and Dickinson B (1957). ‘The origin of melezitose: a biochemical relationship between the lime tree (Tilia spp.) and an aphis (Eucallipterus tiliae L.)’, Biochem J, 66, 289–297. Barker SA and Bourne EJ, Theander O (1957). ‘Studies of Aspergillus niger. Part V. The enzymic synthesis of a new trisaccharide’, J Chem Soc, 2064–2067. Berensmeier S and Ergezinger M, Bohnet M, Buchholz K (2004). ‘Design of immobilised dextransucrase for fluidised bed application’, J Biotechnol, 114, 255–267. Berteau O and Stenutz R (2004). ‘Web resources for the carbohydrate chemist’, Carbohydr Res, 339, 929–936. Biedrzycka E and Bielecka M (2004). ‘Prebiotic effectiveness of fructans of different degrees of polymerization’, Trends Food Sci Technol, 15, 170–175. Boucher J, Daviaud D, Siméon-Remaud M, Carpéné C, Saulnier-Blache JS, Monsan P and Valet P (2003). ‘Effect of non-digestible gluco-oligosaccharides on glucose sensitivity in high fat diet fed mice’, J Physiol Biochem, 59, 169–174. Buchholz K and Seibel J (2003). ‘Isomaltooligosaccharides’, in Eggleston G and Côté GL (eds), Oligosaccharides in Food and Agriculture, American Chemical Society, Washington, ACS Symposium Series, 849, 63–75. Buchholz K, Nolls-Borcher M and Schwengers D (1998). ‘Production of leucrose by dextransucrase’, Starch/Stärke, 50, 164–172. Byrne D (2001). ‘Commission decision of 30 January 2001 on authorising the placing on the market of a dextran preparation produced by Leuconostoc mesenteroides as a novel food ingredient in bakery products under Regulation (EC) No 258/97 of the European Parliament and of the Council’, Official J Eur Comm, L44, 46. Canedo M, Jimenez-Estrada M, Cassani J and Lopez-Munguia A (1999). ‘Production of maltosylfructose (erlose) with levansucrase from Bacillus subtilis’, Biocat Biotransform, 16, 475–485. Carlson T and Woo A (2004). Use of Low Glycemic Index Sweeteners in Food and Beverage Compositions, US Patent Application 10/243,283. Castillo E, Iturbe F, Lopez-Munguia A, Pelenc V, Paul F and Monsan P (1992). ‘Dextran and oligosaccharide production with glucosyltransferases from different strains of Leuconostoc mesenteroides’, Ann NY Acad Sci, 672, 425–430. Cheetham PSJ (1984). ‘The extraction and mechanism of a novel isomaltulose-synthesizing enzyme from Erwinia rhapontici’, Biochem J, 220, 213–220. Cheetham PSJ (1987). ‘Production of isomaltulose using immobilized microbial cells’, Methods Enzymol, 136, 432–454. Chung C-H and Day DF (2002). ‘Glucooligosaccharides from Leuconostoc mesenteroides B-742 (ATCC 13146): A potential prebiotic’, J Ind Microbiol Biotechnol, 29, 196–199. Chung C-H and Day DF (2004). ‘Efficacy of Leuconostoc mesenteroides (ATCC 13146) isomaltooligosaccharides as a poultry prebiotic’, Poultry Sci, 83, 1302–1306. Clarke MA, Bailey AV, Roberts EJ and Tsang WS (1991). ‘Polyfructose: a new microbial polysaccharide’, in Lichtenthaler FW (ed.), Carbohydrates as Organic Raw Materials, VCH, Weinheim,169–181. Combie J (2003). ‘Natural polymer with adhesive properties produced by bacteria’, Adhesives Sealants Ind, 10, 26–27. Combie J, Steel A and Sweitzer R (2004). ‘Adhesive designed by nature’, Clean Technol Environ Policy, 6, 258–262. Côté GL (1992). ‘Low-viscosity α-D-glucan fractions derived from sucrose which are resistant to enzymatic digestion’, Carbohydr Polym, 19, 249–252. Côté GL (2002). ‘Alternan’, in Vandamme EJ, DeBaets S and Steinbüchel A (eds), Biopolymers, Vol. 5, Wiley-VCH, Weinheim, Germany, 323–350. Côté GL and Ahlgren JA (1993). ‘Metabolism in Microrganisms. 1. Levan and Levansucrase, Chapter 5’, in Suzuki M and Chatterton NJ(eds), Science and Technology of Fructans, CRC Press, Boca Raton, FL, 141–168. Côté GL and Leathers TD (2005). ‘A method for surveying and classifying Leuconostoc spp.

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glucansucrases according to strain-dependent acceptor product patterns’, J Ind Microbiol Biotechnol, 32, 53–60. Côté GL and Robyt JF (1982a). ‘Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1→6), (1→3)-α-D-glucan’, Carbohydr Res, 101, 57–74. Côté GL and Robyt JF (1982b). ‘Acceptor reactions of alternansucrase from Leuconostoc mesenteroides NRRL B-1355’, Carbohydr Res, 111, 127–142. Côté GL and Willett JL (1999). ‘Thermomechanical depolymerization of dextran’, Carbohydr Polym, 39, 119–126. Côté GL, Leathers TD, Ahlgren JA, Wyckoff HA, Hayman GT and Biely P (1997). ‘Alternan and highly branched limit dextrans: Low-viscosity polysaccharides as potential new food ingredients’, in Okai H, Mills O, Spanier AM and Tamura M (eds), Chemistry of Novel Foods, Chap 8, Allured Publishing, Carol Stream, Illinois, 95–109. Côté GL, Holt SM and Miller-Fosmore C (2003). ‘Prebiotic oligosaccharides via alternansucrase acceptor reactions’, in Eggleston G and Côté GL (eds), Oligosaccharides in Food and Agriculture, American Chemical Society, ACS Symposium Series, Washington, 849, 75–89. Dal Bello F, Walter J, Hertel C and Hammes WP (2001). ‘In vitro study of the prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis’, System Appl Microbiol, 24, 232–237. Day DF and Yoo SK (2001). ‘Natural glucans: production and prospects’, in Gross RA and Scholz C (eds), Biopolymers from Polysaccharides and Agroproteins, ACS Symposium Series, Washington, DC, 786, 292–300. Dean MA, de la Motte RS, Stryker VH, Torres MC and Wagner FW (1992). ‘Enzyme catalyzed transesterifications between sugars and fatty acyl derivatives’, in Clarke MA (ed), Carbohydrates in Industrial Synthesis, Verlag Bartens, Berlin, 27–36. DeBelder AN (1993). ‘Dextran’, in Whistler RL and BeMiller JN (eds), Industrial Gums – Polysaccharides and Their Derivatives, 3rd edition, Academic Press, San Diego, 399–425. Djouzi Z, Andrieux C, Pelenc V, Somarriba S, Popot F, Paul F, Monsan P and Szylit O (1995). ‘Degradation and fermentation of α-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats’, J Appl Bacteriol, 79, 117–127. Dols M, Remaud-Simeon M, Willemot R-M, Vignon M and Monsan PF (1997). ‘Characterization of dextransucrases from Leuconostoc mesenteroides NRRL B-1299’, Appl Biochem Biotechnol, 62, 47–59. Dols M, Remaud-Simeon M, Willemot R-M, Vignon M and Monsan PF (1998). ‘Structural characterization of the maltose acceptor-products synthesized by Leuconostoc mesenteroides NRRL B-1299 dextransucrase’, Carbohydr Res, 305, 549–559. Doner LW (1977). ‘The sugars of honey – a review’, J Sci Food Agric, 28, 443–456. Dunlap CA and Côté GL (2005). ‘β-Lactoglobulin-dextran conjugates; the effect of polysaccharide size on emulsion stability’, J Agric Food Chem, 53, 419–423. Flickinger EA and Fahey GC (2002). ‘Pet food and feed applications of inulin, oligofructose, and other oligosaccharides’, Br J Nutr, 87, S297–S300. Franck A (2002). ‘Technological functionality of inulin and oligofructose’, Br J Nutr, 87, S287–S291. French D (1954). ‘The raffinose family of oligosaccharides’, Adv Carbohydr Chem, 9, 149– 184. French D (1957). ‘The Schardinger dextrins’, Adv Carbohydr Chem, 12, 189–260. Frese L (1993). ‘Production and utilization of inulin. Part I. Cultivation and breeding of fructan-producing crops’, in Suzuki M and Chatterton NJ (eds), Science and Technology of Fructans, CRC Press, Boca Raton, FL, 303–317. Fuchs A (1991). ‘Current and potential food and non-food applications of fructans’, Biochem Soc Trans, 19, 555–560.

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Fuchs A (1993). ‘Production and utilization of inulin. Part II. Utilization of inulin’, in Suzuki M and Chatterton NJ (eds), Science and Technology of Fructans, CRC Press, Boca Raton, FL, 319–352. Fujita K, Kitahata S, Hara K and Hashimoto H (1992). ‘Production of lactosucrose and its properties’, in Clarke MA (ed.), Carbohydrates in Industrial Synthesis, Verlag Bartens, Berlin, 68–76. Goulas AK, Fisher DA, Grimble GK, Grandison AS and Rastall RA (2004a). ‘Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase’, Enzyme Microbial Technol, 35, 327–338. Goulas AK, Cooper JM, Grandison AS and Rastall RA (2004b). ‘Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase’, Biotechnol Bioeng, 88, 778–787. Hamada S (2002). ‘Role of sweeteners in the etiology and prevention of dental caries’, Pure Appl Chem, 74, 1293–1300. Han YW (1990). ‘Microbial levan’, Adv Appl Microbiol, 35, 171–194. Hehre EJ (1953). ‘The substituted-sucrose structure of melezitose’, Adv Carbohydr Chem, 8, 277–290. Hehre EJ and Sery TW (1952). ‘Dextran-splitting anaerobic bacteria from the human intestine’, J Bacteriol, 63, 424–426. Heincke K, Demuth B, Jördening H-J and Buchholz (1999). ‘Kinetics of the dextransucrase acceptor reaction with maltose – experimental results and modeling’, Enzyme Microbial Technol, 24, 523–534. Hidaka H and Hirayama M (1991). ‘Useful characteristics and commercial applications of fructo-oligosaccharides’, Biochem Soc Trans, 19, 561–565. Hidaka H and Hirayama M, Sumi N (1988). ‘A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611’, Agric Biol Chem, 52, 1181–1187. Hidaka H, Hirayama M and Yamada K (1991). ‘Fructooligosaccharides – Enzymatic preparation and biofunctions’, J Carbohydr Chem, 10, 509–522. Hirayama M (2002). ‘Novel physiological functions of oligosaccharides’, Pure Appl Chem, 74, 1271–1279. Holt SM, Miller-Fosmore CM and Côté GL (2005). ‘Growth of various intestinal bacteria on alternansucrase-derived oligosaccharides’, Lett Appl Microbiol, 40, 385–390. Hudson CS (1946). ‘Melezitose and turanose’, Adv Carbohydr Chem, 2, 1–36. Iizuka M, Hiyama M, Itaya K, Furuichi K, Ann Y-G, Minamiura N and Yamamoto T (1990). ‘Susceptibility of leucrose to carbohydrases’, J Ferment Bioeng, 70, 277–279. Irwin WE (1990). ‘Isomalt – a sweet, reduced-calorie bulking agent’, Food Technol, 44, 128. Jeanes A (1975). ‘Digestibility of food polysaccharides by man: a review’, in Jeanes A and Hodge J (eds), Physiological Effects of Food Carbohydrates, American Chemical Society, ACS Symposium Series, Washington, DC, 15, 336–347. Jeanes A, Haynes WC, Wilham CA, Rankin JC, Melvin EH, Austin MJ, Cluskey JE, Fisher BE, Tsuchiya HM and Rist CE (1954). ‘Characterization and classification of dextrans from ninety-six strains of bacteria’, J Am Chem Soc, 76, 5041–5052. Jung H-K, Kim K-N, Lee H-S and Jung S-H (1999). ‘Production of alternan by Leuconostoc mesenteroides CBI-110’, Korean J Appl Microbiol Biotechnol, 27, 35–40. Kitahata S (2001). R&D Trend Analysis – Biotechnology in Japan. No. 5: Current Industrial Production and Application of Saccharides in Japan, Kansai Research Institute, Kyoto, Japan. Kitahata S and Fujita K (1993). ‘Xylsucrose, isomaltosucrose and lactosucrose’, in Nakakuki T (ed), Oligosaccharides – Production, Properties and Applications, Gordon and Breach Science Publishers, Switzerland, 158–174. Kobayashi M, Shishido K, Kikuchi T and Matsuda K (1973). ‘Methylation analysis of fractions from the Leuconostoc mesenteroides NRRL B-1299 dextran’, Agric Biol Chem, 37, 2763–2769. Koepsell HJ, Tsuchiya HM, Hellman NN, Kazenko A, Hoffman CA, Sharpe ES and Jackson

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13 Production of structured lipids with functional health benefits Xuebing Xu, Janni B. Kristensen and Hong Zhang, BioCentrumDTU, Technical University of Denmark, Denmark

13.1 Introduction Structured lipids (SL) are broadly referred to as modified or synthetic oils and fats with functional or pharmaceutical applications. Some structured lipids are triglycerides that contain both long chain (mainly essential) fatty acids and medium or short chain fatty acids, with each group in specific locations, such as those illustrated in Fig. 13.1(A). A number of processed oils or fats are labelled as structured lipids, and exploit the chain length and saturation of fatty acids for functional or nutritional considerations, without regard to the locations of the fatty acids, as shown in Fig. 13.1(D). Artificial products that mimic the structure of natural materials with limited availability are also called structured lipids, and include human milk fat substitutes and cocoa butter equivalents, as illustrated in Fig. 13.1(B) and 13.1(C). Diglyceride oils, another form of structured lipid, do not occur naturally, and are claimed to help reduce body weight reduction or fat accumulation (see Fig. 13.2). Structured lipids can be produced in different ways, depending on the type. Some are produced using traditional lipid technology, such as chemical hydrolysis, physical fractionation, chemical interesterification or esterification. Certain other structured lipids cannot be synthesised using these methods, in particular products that require specific distributions of various fatty acids in particular positions. In these cases, enzymes demonstrate unique advantages over traditional methods. Selectivity is a unique and most important characteristic of enzymes used in

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Fig. 13.1 Typical structured lipids, with or without specific locations of fatty acids, with medium chains or long chain essential fatty acids or other fatty acids. R R R'

R'

Fig. 13.2 Diglyceride oils. R and R' are fatty acids.

lipid synthesis and modification, and includes stereo-, regio- and chemical specificity. The use of lipases as biocatalysts for the production of structured lipids has additional potential benefits apart from their specificity. The most important merits are (1) efficacy under mild reaction conditions for both simple and complex transformations; (2) utility in ‘natural’ reaction systems and products; (3) reduced environmental pollution; (4) availability from a wide range of sources; (5) no need for tedious protection and deprotection schemes for certain biocatalytic reactions; (6) simple and cheap refining and purification; (6) ability to improve lipases by genetic engineering; and, in special situations, (7) the production of particular biomolecules. For these reasons, many nutritional and functional structured lipids have been produced enzymatically and many studies have been published in the past 20 years (Xu, 2000a, 2004). In this chapter, we discuss novel diglyceride oils with nutritional benefits. This type of oil product has been on the market for several years. We then go on to discuss structured lipids but without region-positional requirements. Food and pharmaceutical companies have been manufacturing this type of structured lipid for a number of years using chemical processes. Nisshin Ollio has also used an enzyme process to synthesise a structured lipid marketed as a healthy oil.

13.2 Production of diglyceride oils In 1999 a new type of dietary oil was introduced in Japan, consisting of 80 wt% diglycerides (DAG) with the remainder being traditional triglyceride (TAG) oil

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(Flickinger and Matsuo, 2003). In 2003, this dietary DAG oil was the best selling brand in Japan at over 100 000 tonnes per year. DAG is a natural compound of various edible oils but only at a content of approximately 0.8–9.5 wt%. Intake of DAG oil, has been shown to have beneficial health effects with regard to the prevention and management of postprandial lipaemia (high blood lipid levels after a meal) and obesity, compared with traditional dietary TAG oils (Yasukawa and Katsuragi, 2004; Matsuo, 2001). Among the health benefits are less weight gain or even weight loss, suppression of body fat accumulation and lower postprandial blood lipid levels after intake of DAG oil (Flickinger and Matsuo, 2003). Hence, DAG oil has been suggested as a food element for reducing diet-induced obesity. In January 2005, DAG oil (Enova™ oil) was introduced nationwide in the USA. The use of DAG oil in food products has increased in recent years. DAG oil has been used in mayonnaise and salad dressings (Kawai, 2004; Saito et al., 2006), and in margarine, spreads, and butter blends (Masui, 2004; Kristensen et al., 2006). Moreover, DAG oil has also found applications in bread, cakes, nutritional bars, and so on. (Sikorski, 2004). ADM has a patent on food and drinks containing DAG oil as well (Boice et al., 2004). DAGs are naturally found in two isomeric forms; the sn1,3-DAG and sn1,2DAG (Fig. 13.3). The natural ratio of the two isomers is approximately 7:3. Studies have shown that the isomer responsible for the beneficial effects is sn1,3-DAG (Meng et al., 2004). Traditionally, partial glycerides, a mixture of monoglycerides (MAG) and DAG, are produced by a chemically catalysed glycerolysis reaction at temperatures >200 °C and using an alkaline catalyst such as sodium, potassium or calcium hydroxide (Sonntag, 1982). These DAG/MAG mixtures are primarily used as nonionic emulsifiers in the food, cosmetic and pharmaceutical industries. The use of more ‘green’ technology, lipase-catalysed reactions, is becoming more and more common in industry. Lipase-catalysed reactions have greatly improved the possibility of producing oils with high diglyceride content. 13.2.1 Lipase-catalysed reactions for production of diglyceride oil DAG oil can be produced in several ways using lipase-catalysed reactions. The simplest method is partial hydrolysis of an oil or fat (Fig. 13.4). Since the desired DAG isomer is 1,3-DAG, as mentioned above, a non-specific lipase is preferred

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Fig. 13.4 Partial hydrolysis of an oil or fat by a non-specific lipase. Only the initial steps in the reactions are shown. R = fatty acid chain. Acyl migration is not indicated.

Fig. 13.5 Esterification for synthesis of DAG by sn1,3-selective lipase-catalysis. Only the initial steps in the reactions are shown. R = fatty acid chain. The dashed arrow indicates the minor direction of the acyl migration.

because the acyl moiety in the sn-2 position in the TAG molecule can be hydrolysed by this type of lipase. A much more straightforward approach to obtaining high 1,3-DAG yields is by esterification of glycerol with free fatty acids (Fig. 13.5). This synthesis route has been intensively studied, using 1,3-specific lipases as catalysts (Rosu et al., 1999; Watanabe et al., 2003, 2005; Weber and Mukherjee, 2004). Another method for synthesising DAG is lipase-catalysed glycerolysis (Fig. 13.6). In several publications the authors have aimed to produce partial glycerides using glycerolysis (Rendon et al., 2001; Ferreira-Dias et al., 2003), but only a few have used this type of reaction with the aim of producing DAG oil in high yields (Kristensen et al., 2005a,b). In glycerolysis, either oil/fat or ethyl esters are used in reactions with glycerol. In the glycerolysis of oil/fat, DAG can be formed both by

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sn1,3- or non-specific lipase

Fig. 13.6 Glycerolysis for synthesis of DAG by sn1,3-selective or non-selective lipasecatalysis. Only the initial steps in the reactions are shown. R = fatty acid chain. Acyl migration reactions are not shown.

removal of an acyl moiety from the TAG molecule or by acylation of the MAG formed during the reaction. In the glycerolysis reaction, a non-specific lipase is optimal, since this type can remove an acyl moiety from the sn-2 position of the TAG molecule, preferably forming 1,3-DAG or 1(3)-MAG. With non-specific lipases, therefore, any acyl moiety on the TAG molecule can be transesterified to any position in the glycerol. With 1,3-specific lipases, only acyl moieties in the sn-1 and sn-3 positions of TAG molecules can be transesterified to the sn-1 and sn-3 positions of the glycerol. A recent method for producing high-purity 1,3-DAG in industry involves two reactions: the partial hydrolysis of a fat or oil to obtain a partial hydrolysate with a high FFA content, followed by 1,3-specific lipase-catalysed esterification of the FFA in the hydrolysate with glycerol (Sugiura et al., 1999).

13.2.2 Process technology for the production of diglycerides Once the type of lipase-catalysed reaction for DAG production is determined, several other process factors have to be taken into consideration. The esterification and glycerolysis reactions can be performed in an organic solvent or in solvent-free systems. The utilisation of a solvent can make the reaction faster owing to a better mass transfer. However, if the DAG oil is intended for the food industry, solvents are not desirable. The DAG synthesis can be performed as a liquid reaction or a solid-phase reaction. In solid-phase reactions, the melting point of the desired product is exploited so that it continuously solidifies out of the reaction mixture, thereby shifting the equilibrium towards a higher yield. In DAG synthesis, this approach may be difficult to apply since DAG has an intermediate melting point compared with TAG and MAG. With respect to lipases, both non-immobilised and immobilised lipases can be used. The advantage of immobilised lipases is their easy recovery and also the

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) TAG, (¡) 1,3-DAG, (●) 1,2-DAG, (¨) MAG, (■) total DAG. *No MAG detected (adapted from Kristensen et al., 2005a).

possibility of using them in a continuous process in which they are packed in a column (packed-bed reactor). Immobilisation may increase lipase stability, especially in a solvent system, but the immobilisation material may also promote acyl migration, which is unwanted, especially in esterification reactions. Again, if the DAG oil is targeted for the food industry, the lipase and possible carrier material have to be ‘food grade’. A very important factor is the selection of an appropriate lipase for the given reaction type and the chosen substrates. For example an sn1,3-selective lipase might be beneficial in one type of reaction but not necessarily good in another. Also fatty acid and glyceride specificity should be considered, as well as the effects of possible lipase carrier materials on the reaction. Screening potential lipases in the chosen reaction medium can be advantageous. Fig. 13.7 presents the results of lipase screening for DAG synthesis by solvent-free glycerolysis of rapeseed oil (Kristensen et al., 2005a). Consideration should also be given to the type of reactor used for the synthesis.

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Fig. 13.8 Main effects of factors on total DAG yield with 95% confidence intervals. The effect of each factor, when it is varied from a low to a high level and all other factors are kept at their averages, is displayed (adapted from Kristensen et al., 2005b).

The majority of laboratory work is carried out using batch reactors, where substrates and lipases are mixed in some type of container and stirred or shaken under controlled temperatures. However, if considering large-scale production, this batch type of bioreactor may not be optimal regarding production cost and efficiency. In commercial-scale operations, packed-bed reactors with immobilised enzymes are most frequently used and run as a continuous process (Xu, 2000b). Few publications describe the continuous production of DAG oil (Watanabe et al., 2005).

13.2.3 Optimizing the production of diglyceride oil by enzymatic means Reaction parameters can influence both yield and production costs considerably. Parameters that are important in batch reactions are discussed here.

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Lipases are the most costly component in the reaction, so the smallest amount of lipase that will give an acceptable DAG yield should be identified. Factors such as reaction time, temperature and the ratio of oil to glycerol can also influence DAG yield and production costs. Water content may also affect the outcome. Some water is, as mentioned, essential for the catalytic ability of lipases, but it will also affect the reaction kinetics, since hydrolysis is favoured over esterification when water is abundant. In esterification reactions, water removal is therefore crucial. Response surface methodology is an excellent tool for optimisation since it enables the evaluation of multiple parameters on response variables alone or in combination. Some papers have applied response surface methodology to the optimisation of a glycerolysis reaction (Ferreira-Dias et al., 2003; Kristensen et al., 2005b). Figure 13.8 shows the main effects of factors involved in the optimization of the solvent-free glycerolysis reaction of rapeseed oil using Candida antarctica lipase B (Novozym 435) (Kristensen et al., 2005b). Several process factors were optimized, including reaction time, enzyme load, reaction temperature, water content and substrate molar ratio.

13.3 Production of healthy oils containing medium chain fatty acids Medium chain fatty acids (MCFA), which contain 8–12 carbons, are easy for the body to absorb and digest (Papamandjaris et al., 1998). In recent rat experiments, Nisshin Oillio’s research group has discovered that MCFA raises the level of adiponectin in the blood. Adiponenctin is a type of fat cell hormone that plays an important role in preventing diabetes and arteriosclerosis. It is well known that MCFA is beneficial in prevention not only of obesity but also of lifestyle-related diseases. MCFA comes from different types of lipids, which normally exist in the form of triglycerides (TAG) in nature. TAG combines three fatty acids with different chain lengths in the glycerol backbone. The effects of structured and randomised medium and long chain triglycerides (MLCT) have been studied (Kasai et al., 2003; Nagata et al., 2003, 2004; Shinohara et al., 2005). These studies have shown that MLCT in the diet could result in a significant reduction in body weight and accumulation of body fat, as well as significant reduction in serum total cholesterol. LML (L-long chain fatty acids and M-medium chain fatty acids) types could effectively improve serum and liver lipid profiles and MLM types may be a preferable substrate for the pancreas and contribute to energy supply in rats (Nagata et al., 2004). However, one study concluded that the effects of the structured lipids containing caprylic acid and either eicosapentaenoic (EPA) or docosahexaenoic acid (DHA) were due to the fatty acids rather than the structural specificity. Further studies are needed to clarify this. MLCT oils have very wide applications. They can be used for cooking oils, mayonnaise, margarine, salad dressing, bread, ice cream, confectionary, gelatin capsules and fried foods (Aoyama, 2004). Industrial scale production has been

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Fig. 13.9 Reaction schemes of chemical randomisation and enzymatic interesterification between a long chain triglyceride (LCT) and a medium chain triglyceride (MCT). The triglyceride species in each of the rectangles have the same proportions if the sn-1,3 specific lipase has the same specificity towards different fatty acids and the sn-1 and sn-3 positions.

implemented by the Nisshin Oillio Group which produces about 10 000 tonnes per year (Negishi et al., 2003).

13.3.1 Reactions for lipase-catalysed interesterification between LLL (long chain triglycerides) and MMM (medium chain triglycerides) oil LCT–MCT interchange, in principle, gives different triglyceride (TAG) compositions in the end product, depending on the catalysts (Fig. 13.9). In most cases, interesterification between two different types of TAGs results a mixture of eight TAG species. This process can be carried out by either chemical or enzymatic interesterification. During enzymatic interesterification, by-products, such as diglycerides (DAG) and free fatty acids (FFA), are generated. The amount of byproduct can be minimised under optimal operating conditions (Zhang et al., 2001). This often entails minimising water in the reaction system, which is only needed to maintain enzyme activity. Chemical interesterification can be induced by alkali, such as sodium methylate, and randomised products are normally produced (Fig. 13.9). Enzymatic interesterification using sn-1,3 specific enzymes is better for producing positionally specific products.

13.3.2 Common process parameters To study enzymatic interesterification, different parameters, that is, enzyme type, dosage, and the effect of substrate ratio, reaction time, temperature, and water content in the system, are varied. They are all related each other (Fig. 13.10) and optimal conditions need to be found.

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100

2.5 60

2 1.5

40

1

DAG (wt%) and FFA (wt%)

Interesterification degree (%)

3 80

20 0.5 0

0 0

40

80

120

160

Residence time (min)

Fig. 13.10 Effect of residence time on the degree of reaction and DAG and FFA contents of lipozyme TL IM catalysed interesterification between fish oil and MCT. Reaction conditions: temperature 60 °C with no water added. (Adapted from Xu et al., 2002).

Enzyme screening. Enzymes are normally classified in three forms, liquid, powder (freeze-dried) and particle (immobilised) with different shapes. Immobilised enzymes, which are easy to handle and reuse, are commonly used for industrial applications. However, Nisshin Oillio uses powdered lipase (Alcaligenes sp., Meito) in a packed-bed reactor (PBR) for its industrial-scale production (Negishi et al., 2003). Some studies have shown that immobilised enzymes had higher activities than non-immobilised enzymes (Fomuso and Akoh, 1998) and are preferable for this application (see Chapter 4). Molar ratio effects. At a molar ratio of 1:2 (LLL:MMM) for lipozyme RM IMcatalysed interesterification, a high yield of di-medium chain, mono-long chain TAGs (53.5%) and di-long chain, mono-medium chain TAGs (22.2%) was achieved. However, in practice, the optimal molar ratio is decided by the end use of the product (Zhang et al., 2001; Zhang, 2005; Yokohama et al., 2004). Frying oil will have a low smoking point if there is a large amount of MCFA, and foams will form, making it unsuitable for frying. In this instance, a mass ratio for the substrates of about 71/29–97/3 between LLL/MMM is recommended (Yokohama et al., 2004). Temperature effects. Temperature normally affects lipase activity. High temperature usually increases the initial reaction rate. However, very high operating temperatures deactivate the enzyme owing to its temperature-labile protein nature (Godfrey and West, 1996). The optimal temperature is decided mainly based on the properties of feedstock, such as melting behaviour at different temperatures

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(Zhang et al., 2001) and reaction system, that is with solvent (Fomuso and Akoh, 1998) or solvent-free (Xu et al., 2002). Water effects. Lipase-catalysed interesterification involves water and is accompanied by the formation of new TAG products as well as DAG and FFA by-products (Zhang et al., 2000; 2001). The enzyme activity is related to water content in the reaction system. The optimal water content is highly dependent on the carrier types for some enzymes (Zhang et al., 2000; 2001), or dependent on the solvents used for others (Gorman and Dordick 1992). The activity of Lipozyme RM IM, immobilised on an ion exchange resin, is closely related to the water content in the system. Reusing Lipozyme RM IM without adjusting the amount of water in the reaction system leads to decreasing reaction activity (Zhang et al., 2000). On the other hand, Lipozyme TL IM, with a silica carrier, is not water dependent and activity during reuse will not be affected by water content. On the contrary, here the amount of water in the system should be minimised in order to reduce the amount of byproducts (Zhang et al., 2001).

13.3.3 Reactors for enzymatic interesterification Interesterification can be carried out either in a stirred tank reactor (STR) or a packed bed reactor (PBR). Most reactions in the laboratory are carried out in a batch reactor. These require fewer enzymes and facilitate process optimisation (Xu et al., 1999). However, they have some drawbacks, including the need for long reaction times, which leads to a high degree of acyl migration (Xu et al., 1998). The result is that the enzymes might partially or totally lose their specificity during operation. For these reasons, PBRs are recommended to preserve enzyme specificity. The relationship between these two reactors can be described as following without considering enzyme deactivation: Vb Fp = wp —— wb · t where wp is the amount of enzyme in the packed-bed reactor; Fp is the flow rate through the packed-bed reactor and wb, Vb and t are the enzyme dosage, the amount of oil and the reaction time in a batch reactor, respectively (Zhang et al., 2004a).

13.3.4 Determination of interesterified products Changes in chemical and physical properties are normally monitored during interesterification (Soumanou et al., 1997; Fomuso and Akoh, 1998; Zhang et al., 2001; Zhang, 2005). High-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), dropping point (DP) and nuclear magnetic resonance (NMR) for solid fat content (SFC) measurement can be used to monitor the extent of interesterification and product properties. During interesterification, the triglyceride profile changes and HPLC is a useful method for monitoring the reaction. If the change involves physical properties, DP

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or SFC can also be used. By-products can be monitored with a combination of HPLC, GC and TLC analyses. The reaction degree can be calculated as: Yt – Y0 X(%) = ——— × 100 = (1 – e–kt) *100 Y∞ – Y0 where X is the reaction degree and Y is the experimental value from analysis. The subscripts 0, t and ∞ of Y stand for the analytical values at reaction time 0, t and equilibrium. When the reaction is assumed to be a first order reaction for enzymatic interesterification, the reaction rate constant k can also be calculated (Zhang et al., 2004b). It can be simplified as: Y = Y0 – ∆Y(1 – e–kt) where ∆Y is the change between the initial and equilibrium stage.

13.4 Future trends DAG oil has been on the market for many years and intensive research is still improving our understanding of its production and uses. Industrial enzymatic production of DAG oil is claimed, but a mature processing technology using enzymes, on the other hand, has not been developed. The central issues are lowering costs and further improving production efficiency. Enzyme technology for developing functional lipid products will remain an area of interest for some years to come. The demand for better quality functional lipids is increasing. Continued improvement of enzymes in terms of cost and properties is likely to facilitate the application of enzyme processes in industry.

13.5 Acknowledgements Financial support is acknowledged from the Danish Research Council for Technology and Production, the Strategic Food and Health Programme, and the Centre for Advanced Food Studies, as well as other supports from industry and collaboration partners.

13.6 References Aoyama T (2004). Fats and Oils Composition for Reducing Lipids in Blood, US Patent 6,827,963, B2. Boice B, Egbert R, Sikorski D, Stuchell Y and Widlak N (2004). Foods and Drinks Containing Diglyceride, US Patent 0009284 A1. Ferreira-Dias S, Correia A C and da Fonseca M M R (2003). ‘Response surface modeling of glycerolysis catalyzed by Candida rugosa lipase immobilized in different polyurethane foams for the production of partial glycerides’, J Mol Catal B-Enzyme, 21(1), 71–80. Flickinger B D and Matsou N (2003). ‘Nutritional characteristics of DAG oil’, Lipids, 38(2), 129–132.

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Fomuso L B and Akoh C C (1998). ‘Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein’, J Am Oil Chem Soc, 75(3), 405–410. Godfrey T and West S (1996). Industrial Enzymology, 2nd edition, Macmillan Press, London. Gorman L S and Dordick J S (1992). ‘Organic solvent strip water off enzymes’, Biotechnol Bioeng, 39(4), 392–397. Kasai M, Nosaka N, Maki H, Negishi S, Aoyama T, Nakamura M, Suzuki Y, Tsuji H, Uto H, Okazaki M and Kondo K (2003). ‘Effect of dietary medium- and long-chain triacylglyerols (MLCT) on accumulation of body fat in healthy humans’, Asia Pacific J Clin Nutr, 12(2), 151–160. Kawai S (2004). ‘Oil in water foods: mayonnaise and salad dressing’, in Katsuragi Y, Yasukawa T, Matsuo N, Flickinger B D, Tokimitsu I and Matlock M G (eds), Diacylglycerol Oil, AOCS Press, Champaign IL, 208–214. Kristensen J B, Xu X and Mu H (2005a). ‘Diacylglycerol synthesis by enzymatic glycerolysis: Screening of commercially available lipases’, J Am Oil Chem Soc, 82(5), 329–334. Kristensen J B, Xu X and Mu H (2005b). ‘Process optimization using response surface design and pilot plant production of dietary diacylglycerols by lipase-catalyzed glycerolysis’, J Agric Food Chem, 53(18), 7059–7066. Kristensen J B, Nielsen N S, Jacobsen C and Mu H (2006). ‘Oxidative stability and sensory evaluation of diacylglycerol oil and butter blends containing diacylglycerols’, Eur J Lipid Sci Technol, 108(5), 336–350. Masui K (2004). ‘Water-in-oil type of emulsion foods: Margarine, spreads, and butter cream’, in Katsuragi Y, Yasukawa T, Matsuo N, Flickinger B D, Tokimitsu I and Matlock M G (eds), Diacylglycerol Oil, AOCS Press, Champaign IL, 215–222. Matsuo N (2001). ‘Diacylglycerol oil: an edible oil with less accumulation of body fat’, Lipid Technology, 11, 129–133. Meng X H, Zou D Y, Shi Z P, Duan Z Y and Mao Z G (2004). ‘Dietary diacylglycerol prevents high-fat diet-induced lipid accumulation in rat liver and abdominal adipose tissue’, Lipids, 39(1), 37–41. Nagata J, Kasai M, Watanabe S, Ikeda I and Saito M (2003). ‘Effects of high purified structured lipids containing medium-chain fatty acids and linoleic acid on lipid profiles in rats’, Biosci Biotechnol Biochem, 67(9), 1937–1943. Nagata J, Kasai M, Negishi S and Saito M (2004). ‘Effects of structured lipids containing eicosapentaenoic or docosahexaenoic acid and caprylic acid on serum and liver lipid profiles in rats’, BioFactors, 22(1–4), 157–160. Negishi S, Shirassawa S, Arai Y, Suzuki J and Mukataka S (2003). ‘Activation of powdered lipase by cluster water and the use of lipase powders for commercial esterification of food oils’, Enzyme Microb Technol, 32(1) 66–70. Papamandjaris A A, Macdougall D E and Jones P J H (1998). ‘Medium chain fatty acid metabolism and energy expenditure: Obesity treatment implications’, Life Sci, 62(14), 1203–1215. Rendon X, Lopez-Munguia A and Castillo E (2001). ‘Solvent engineering applied to lipasecatalyzed glycerolysis of triolein’, J Am Oil Chem Soc, 78(10), 1061–1066. Rosu R, Yasui M, Iwasaki Y and Yamane T (1999). ‘Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system’, J Am Oil Chem Soc, 76(7), 839–843. Saito S, Takeshita M, Tomonobu K, Kudo N, Shiiba D, Hase T, Tokimitsu I and Yasukawa T (2006). ‘Dose-dependent cholesterol-lowering effect of a mayonnaise-type product with a main component of diacylglycerol-containing plant sterol esters’, Nutrition, 22(2),174–178. Shinohara H, Ogawa A, Kasai M and Aoyama T (2005). ‘Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats’, Biosci Biotechnol Biochem, 69(10), 1811– 1818.

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Sikorski D (2004). ‘Application of diacylglycerol oil in baked goods, nutritional beverages/ bars, sauces, and gravies’, in Katsuragi Y, Yasukawa T, Matsuo N, Flickinger B D, Tokimitsu I and Matlock M G (eds), Diacylglycerol Oil, AOCS Press, Champaign IL, 223–252. Sonntag N O V (1982). ‘Fat splitting, esterification, and interesterification’, in Swern D (ed.), Bailey’s Industrial Oil and Fat Products, 4th edition, John Wiley and Sons, New York, Vol 2, 97–174. Soumanou M M, Bornscheuer U T, Menge U and Schmid R D (1997). ‘Synthesis of structured triglycerides from peanut oil with immobilized lipase’, J Am Oil Chem Soc, 74(4) 427–433. Sugiura M, Shimizu M, Yamada N and Yamada Y (1999). Process for Producing Diglycerides, WO Patent 9909119. Watanabe T, Shimizu M, Sugiura M, Sato M, Kohori J, Yamada N and Nakanishi K (2003). ‘Optimization of reaction conditions for the production of DAG using immobilized 1,3regiospecific lipase Lipozyme RM IM’, J Am Oil Chem Soc, 80(12), 1201–1207. Watanabe T, Sugiura M, Sato M, Yamada N and Nakanishi K (2005). ‘Diacylglycerol production in a packed bed bioreactor’, Process Biochem, 40(2), 637–643. Weber N and Mukherjee K D (2004). ‘Solvent-free lipase-catalyzed preparation of diacylglycerols’, J Agric Food Chem, 52(17), 5347–5353. Xu X (2000a). ‘Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a review’, Eur J Lipid Sci Technol, 102(4), 287–303. Xu X (2000b). ‘Enzyme bioreactors for lipid modifications’, Inform, 11, 1004–1012. Xu X (2004). ‘Biocatalysis for lipid modification’, in Dunford N T and Dunford H B (eds.), Nutritionally Enhanced Edible Oil Processing, AOCS Press, Champaign IL, 162–196. Xu X, Balchen S, Høy C-E and Adler-Nissen J (1998). ‘Pilot batch production of specificstructure lipids by lipase-catalyzed interesterification: Preliminary study on incorporation and acyl migration’, J Am Oil Chem Soc, 75(11), 301–308. Xu X, Mu H, Høy C-E and Adler-Nissen J (1999). ‘Production of specific structured lipids by enzymatic interesterification in a pilot enzyme bed reactor: process optimisation by response surface methodology’, Fett/Lipid, 101(6), 203–214. Xu X, Porsgaard T, Zhang H, Adler-Nissen J and Høy C-E (2002). ‘Production of structured lipids in a packed-bed reactor with Thermomyces lanuginose lipase’, J Am Oil Chem Soc, 79(2), 561–565. Yasukawa T and Katsuragi Y (2004). ‘Diacylglycerols’, in Katsuragi Y, Yasukawa T, Matsuo N, Flickinger B D, Tokimitsu I and Matlock M G (eds), Diacylglycerol Oil, AOCS Press, Champaign IL, 1–15. Yokohama H T, Fujisawa M A, Yokosuka F A and Yokosuka N T (2004). Oil and Fat Composition, US Patent 6,835,408 B2. Zhang H (2005), Lipase-catalyzed Interesterification for Margarine Fat Production, PhD Thesis, Technical University of Denmark, Lyngby, Denmark. Zhang H, Xu X, Mu H, Nilsson J, Adler-Nissen J and Høy C-E (2000). ‘Lipozyme IMcatalyzed interesterification for the production of margarine fats in a 1-kg scale stirred tank reactor’, Eur J Lipid Sci Technol, 102(6), 411–418. Zhang H, Xu X, Nilsson J, Mu H, Adler-Nissen J and Høy C-E (2001). ‘Production of margarine fats by enzymatic interesterification with silica-granulated Thermomyces lanuginosus lipase in a large-scale study’, J Am Oil Chem Soc, 78(1), 57–64. Zhang H, Smith P and Adler-Nissen J (2004a). ‘Effects of degree of enzymatic interesterification on the physical properties of margarine fats – Solid fat content, crystallization behavior, crystal morphology and crystal network’, J Agr Food Chem, 52(14), 4423–4431. Zhang H, Pedersen L S, Kristensen D, Adler-Nissen J and Holm H C (2004b). ‘Modification of margarine fats by enzymatic interesterification: Evaluation of a solid-fat-content-based exponential model with two groups of oil blends’, J Am Oil Chem Soc, 81(7), 653–658.

14 Lipase-catalyzed harvesting and/or enrichment of industrially and nutritionally important fatty acids George J. Piazza and Thomas A. Foglia, US Department of Agriculture, USA, and Xuebing Xu, BioCentrum-DTU, Technical University of Denmark, Denmark

14.1 Introduction The use of enzymes as catalysts in the processing of fats and oils into industrial and consumer products is a rapidly developing area. The primary barrier to increased development in this area is enzyme cost which is high owing to the cost of enzyme preparation and limited enzyme stability. The inability of some enzymes to work well in lipid systems at higher temperatures without added solvent is another barrier to adoption of enzyme technology. The fat and oil industry largely uses chemical methods to split fats and oils into glycerol and fatty acids (FA) and the subsequent physical refining of the liberated free FA. In general, industrial products are not formulated with unsaturated and polyunsaturated FA (PUFA) and their derivatives since they are considered to be a liability because of their slow oxidation, which promotes product degradation. As such, unsaturated FA are subjected to hydrogenation before further processing. An exception is made for those FA whose carbon–carbon double bonds are required for further chemical processes such as epoxidation or dimerization. As noted above, although enzymes generally are not currently used for the manufacture of industrial lipid products, one class of enzymes called lipases is now starting to be used to synthesize triacylglycerols (TAG) for use in nutritional and/ or nutraceutical type applications. Lipases are enzymes that catalyze the splitting

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of fats and oils to FA and glycerol. The splitting reaction is reversible and at low water levels many lipases also can catalyze the reverse reaction, namely ester formation. When TAG are synthesized using lipases they are commonly referred to as tailored or structured lipids depending upon the specificity, if any, of the lipase used for their syntheses. There are several reasons for the adoption of enzyme technology in this instance. First, PUFA are among the most desirable FA for human consumption because of their purportive beneficial effects for the user, such as lower rates of cardiovascular disease and degenerative diseases such as Alzheimers and cancer; lipases can be used for the recovery and incorporation of PUFA under milder processing conditions that do not cause extensive degradation of PUFA. Second, some lipases have positional specificity, and their use in synthesis allows a particular fatty acyl residue to be placed at either the primary (carbons 1 and 3) or the secondary hydroxy function (carbon 2) of the glycerol backbone. This type of hydroxy group selectivity is obtained using purely chemical methods only with great difficulty. The positional location of a fatty acyl group on the TAG affects how it is metabolized by the human digestive system and, by applying this knowledge, lipases can be used to prepare both high and low-energy type TAG. Third, the classical method for changing oils into more solid fats is partial hydrogenation, but unfortunately this procedure often causes isomerization of the remaining carbon–carbon double bonds in the fat or oil from the natural cis to the trans configuration as well as double bond migration along the fatty acid chain. The presence of the trans fatty acids in fats and oils has been associated with an increased risk of the incidence of cardiovascular disease. Lipases allow the chemical blending of fats and oils to give a product of intermediate hardness which has zero trans fat levels and is useful for a number of food applications. Hence, TAG that are produced using lipases are able to command a premium price in the marketplace because of their defined chemical structure and to provide unique dietary benefits that cannot be easily obtained with TAG synthesized by currently practiced chemical processes. This chapter will discuss three related areas of lipase chemistry: lipase selectivity, FA harvesting with lipases and structured lipid syntheses with lipases. Some commercial applications of structured lipids are also given. Only the more recent literature will be covered, and the reader is referred to the excellent review by Hayes (Hayes, 2004) for earlier references in these areas.

14.2 Lipase selectivity Enzyme kinetics is a field of study that attempts to classify enzymes by the sequence of the reactions necessary to form a given product and to understand which enzymatic step or steps of the process are rate limiting. When investigating enzymes that act on lipid substrates, classical kinetic techniques often cannot be used because the substrates usually are not fully soluble in the reaction medium. Two frequently encountered situations are that a two phase system of substrate and solvent is present or that the enzyme is added into a neat mixture of the substrates,

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which is usually kept in the liquid phase by elevating the temperature. Because of the special situations that apply to lipase reactions of lipids, the usual ways of comparing substrate selectivity such as comparisons of Vmax or Vmax/KM cannot be used. As an alternative, mixtures of substrates are used and an analytical technique is used either to measure the remaining composition of the substrate mixture or the composition of the product mixture formed over time. The various substrates are competing against each other for the enzyme; the substrates that interact most closely with the enzyme are those that tend to react the fastest. In the reactions of lipases with TAG, another feature that is of great practical interest is the position of the fatty ester that is hydrolysed most quickly by the lipase. There are two types of lipase: those that are non-specific with regard to position and those that are specific or selective for the sn-1 and -3 positions. Although there have been claims for lipases that are specific for the sn-2 position, none have been validated to date. From a practical perspective, the lipid scientist is usually trying to isolate a particular FA or fatty acid ester (FE) from a vegetable oil or animal fat or to prepare a structured lipid product. If FA harvesting is the goal, then a given fat or oil is subjected to hydrolysis using a variety of lipases to determine if the lipases express any positional or acyl selectivity towards the fatty acyl groups in the fat or oil. This is done by measuring the distribution of the liberated free FA after a given hydrolysis time and comparing to the original fatty acyl composition of the fat or oil under study. The lipase that either liberates the greatest amount of the desired FA or liberates the least amount of the desired FA is chosen by trial and error. In the latter case, the glycerides remaining are then treated with a non-specific lipase to liberate the desired FA. In some situations better results are obtained by adding an alcohol into the reaction mixture to give a FE as a product rather than a FA, since lipase selectivity is often more pronounced in the esterification mode than in the hydrolysis mode. One other method used to determine lipase specificity is to add a mixture of FA to an alcohol receptor. After a limited time the composition of the esters formed is determined to check whether some FA were incorporated selectively. Regardless of the specifics of the procedure used to determine selectivity, it is beneficial to have a body of knowledge about the enzyme specificity of a large number of lipases to guide research to the best choice of lipase to use for harvesting a particular FA or to create a structured lipid containing the ester of a particular fatty acid. This need for specificity data is the main driving force behind most studies of lipase specificity. Table 14.1 lists a summary of recent research results on lipase selectivity. Entries 12 and 13 deal with selectivity toward DHA [4,7,10,13,16,19(Z)-docosahexaenoic acid] and EPA [5,8,11,14,17(Z)-eicosapentaenoic acid] (Halldorsson and Haraldsson, 2004; Halldorsson et al., 2004). The remainder of the listed research is concerned primarily with saturated FA or FE. The emphasis on research with saturated fats is partly due to the large body of older research not covered here on the specificity of lipases toward unsaturated fats. In the publication by Hellyer et al., (1999), a large number of plant-derived lipases were tested on the hydrolysis of a fully randomized oil (Entry 1, Table 14.1). The oil was prepared by blending 1 part cocoa butter, 1 part soybean oil, 1 part

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coconut oil and 0.5 part medium-chain TAG and then randomizing the fatty acyl groups by chemical interesterification with sodium methoxide. All of the lipases tested either showed no selectivity (dashed entries) or some selectivity for medium chain fatty acyl groups. The lipase from Cuphea procumbans showed a 20-fold selectivity for C10:0. Another study of plant lipases from Euphorbia characias latex, pineapple and papaya also showed a preference for medium chain FA and FE during transesterification reactions (Entry 3; Caro et al., 2000). The selectivity of Candida antarctica lipase was assessed in reactions of FA and fatty acid methyl esters (FAME) with 1-propanol, 2-propanol and their acetate derivatives (Entry 2; Arsan and Parkin, 2000). The selectivity for FA and FAME chain length changed depending on the alcohol or alcohol acetate that was used as the co-substrate. The magnitude of any expressed selectivity, however, was not very high, although it was somewhat higher with FAME than with FA. The selectivity of lipases from C. antarctica, Pseudomonas cepacia, and Rhizomucor miehei was tested by reacting mixtures of FA with glycerol, 1,2-propanediol and 1,3-propanediol (Entry 4; Lee and Parkin, 2000). Lipase selectivity differences arising from esterification of the FA with different alcohols were weak. All the enzymes showed a preference for FA C8, but the lipase from P. cepacia showed higher selectivity for both C8 and C18 FA in the presence of 1,2- and 1,3propanediol. Potato lipase was also tested under similar conditions except that 2-propanol was substituted for 1,2-propanediol owing to insufficient reactivity of the latter (Entry 5; Pinsirodom and Parkin, 2000). Potato lipase showed the highest reactivity with C8 or C10 FFA in each case. The influence of water activity in a solvent of t-butyl methyl ether, and the effects of having the lipase fixed on a resin or as the free powder were tested on lipases from R. miehei, C. antarctica, and C. rugosa in the reaction of mixtures of saturated FA with 1,3-propanediol (Entry 6; Lee and Parkin, 2001). The lipase from R. miehei showed a preference for C8 FA, as in Entry 4, with little influence from the state of immobilization. However with C. antarctica, immobilization tended to broaden the selectivity so that C6–C10 had about the same reactivity. Lipases from porcine pancreas, Rhizopus japonicus, and Chromobacterium viscosum were modified with stearic acid, and TAG hydrolyses and acidolysis reactions were conducted in media containing hexane (Entry 8; Maruyama et al., 2002). Medium chain TAG were preferred as substrates, and in acidolysis reactions with oleic acid, medium chain TAG were preferred. In contrast to the medium chain specificity exhibited by the above lipases, the lipase purified from Trichosporon preferred C18 saturated and unsaturated FA, and in ester formation reactions the longer chain alcohol, oleyl alcohol, was preferred for ester formation with oleic acid, linoleic acid and linolenic acid (Entry 7; Song et al., 2001). In another example, the esterification activity of R. miehei and Burkholderia cepacia was tested in hexane using glycerol as the alcohol substrate, and it was found that palmitic (C16) was preferred (Entry 9, Fu and Parkin, 2004a). In a subsequent study with B. cepacia in hexane, it was found that long chain FA (C14–C18) in some instances reacted well with glycerol and monoacylglycerol (MAG) containing different length saturated FA (Entry 10, Fu and Parkin, 2004b). With β-MAG (fatty ester on the sn-2 position) there was little

Table 14.1 Selectivity of lipases Lipase

Physical form

Milieu

Preferred substrates

Reference

– –

Hellyer et al., 1999

– – – 12:0/14:0 – 12:0 10:0 10:0 10:0 10:0 12:0 14:0 14:0 14:0 8:0/10:0/12:0

Lipase-catalyzed harvesting and/or enrichment of fatty acids

10:0 TAGa + H2O

Oil/aq. buffer (1:1) 14:0

289

1. Brassica napus Cuphea racemosa Butyrospermum parkii Garcinia mangostana Theobroma cacao Arecastrum romanzoffianum Butia capitata Orbignya cohume Ulmus americana Cuphea procumbans Cuphea lanceolata Cuphea llavea Cocas nucifera Cuphea aequipetala Cuphea palustris Myristica fragrans Umbellularia californica Cinnamomum camphora Irvingia gabonensis Crude extracts

Substrate reaction

290

Table 14.1

(cont)

Lipase

Substrate reaction

Milieu

Preferred substrates

Commercial Immobilization

FA + 1-propanol FAME + 1-propanol FA + propyl acetate FAME + propyl acetate FA + 2-propanol FAME + 2-propanol FA + isopropyl acetate FAME + isopropyl acetate Other alcohols + hexanoic acid TAG + H2O

Hexane with different [water] (salt hydrates)

6:0 Arsan and Parkin, 2000 6:0 6:0 4:0 6:0 6:0 14:0 14:0 n-butyl and iso-butyl alcohol 4:0 Caro et al., 2000

3. Euphorbia Latex characias Bromelain Crude Papain Crude 4. Pseudomonas Free powder cepacia Resin-fixed Crosslinked to a polyacrylic resin Rhizomucor miehei Candida antarctica

TAG + tributyrin FA (C4:0-C18:0) + Glycerol 1,2-propanediol 1,3-propanediol Glycerol 1,2-propanediol 1,3-propanediol Glycerol 1,2-propanediol 1,3-propanediol

aq NaCl

4:0 Hexane 6:0 t-butyl methyl ether with different [water] (salt hydrates) C:8 C:8 and C:16 C:8 and C:16 C:8 C:8 C:8 C:8 C:8 C:8

Reference

Lee and Parkin, 2000

Novel enzyme technology for food applications

2. Candida antarctica

Physical form

5. Potato Patatin

Free powder

FA (C4:0-C18:0) + 1-propanol 2-propanol 1,3-propanediol Glycerol FA (C4:0-C18:0-C18:3) + 1,3-propanediol

Free powder, resin-fixed Free powder, resin-fixed Free powder

Iso-octane with different [water] (salt hydrates)

Pinsirodom and Parkin, 2000 C:8b C:10 C:10 C:8 Lee and Parkin, 2001

t-butyl methyl ether with different [water](salt hydrates)

C:8b C:8 C:8 C:6, C:8 C:6, C:8, C:10 C:4, C:8

Free

FA + alcohol

Alcohol

Oleyl alcohol

Song et al., 2001

Fatty acid-modified

TAG + FA

Hexane

C:6 (TAG)

Maruyama et al., 2002

Fatty acid-modified

C:6 C:10 TAG + H2O

Hexane/water

C:6 C:10

Resin-fixed Celite-fixed

FA (C4:0-C18:0-C18-1) + glycerol

Hexane with different [water] (salt hydrates)

C:10 C:16 C:16

Fu and Parkin, 2004a

Lipase-catalyzed harvesting and/or enrichment of fatty acids 291

6. Burkholderia cepacia Rhizomucor miehei Candida antarctica Candida rugosa 7. Trichosporon sp. 8. Porcine pancreas Rhizopus japonicus Chromobacterium viscosum Porcine pancreas Rhizopus japonicus C. viscosum 9. Rhizomucor miehei Burkholderia cepacia

Celite-fixed

292

Table 14.1

(cont)

Lipase

Milieu

Preferred substrates

Reference

10. Burkholderia Celite-fixed cepacia

FA (C4:0-C18:0-C18-1) + glycerol

C:16

Fu and Parkin, 2004b

11. Rhizomucor miehei

α-MAG (C:4 and C:10) α-MAG (C:16) β-MAG (C:4) β-MAG (C:10) β-MAG (C:16) FA (C4:0-C18:0C18:1) + glycerol

Hexane with different [water] (salt hydrates)

C:8 C:18 C:10-C:18 C:12-C:18 C:10-C:14, C:18 C:16

Fu and Parkin, 2004c

Resin-fixed

Powder

α-MAG (C:4) α-MAG (C:10) α-MAG (C:16) β-MAG (C:4) β-MAG (C:10) β-MAG (C:16) FAEE + H2O

Hexane with different [water] (salt hydrates)

Ester/aq. buffer (1:2)

C:8-C:10 C:8 C:18 C:10-C:18 C:12-C:18 C:10-C:14, C:18 Halldorsson et al., 2004 All except last two entries showed discrimination against esterified EPA and DHA, and more EPA was released than DHA except for Pseudomonas lipases which released more DHA.

Novel enzyme technology for food applications

Substrate reaction

12. Candida rugosa Aspergillus niger Penicillium roqueforti Penicillium camembertii Rhizopus delemar Humicola lanuginosa

Physical form

14. Rhizopus arrhizus

Free

TAG + H2O

Fish TAG/aq. buffer (1:25)

Released EPA and DHA rapidly.

Halldorsson and Haraldsson, 2004

FA esters of Astaxanthin + H2O

Emulsifier in aq. buffer

POP + H2O

Water

Candida lipase showed the highest activity, and there was discrimination against EPA and DHA. Salmo and Oncorhyncus lipases released EPA and DHA rapidly. Palmitic acid Tan and Yin, 2005 released faster than oleic acid. Mechanism of hydrolysis given.

Abbreviations: α-MAG, sn-1 or -3-monoacylglycerol; β-MAG, sn-2-monoacylglycerol; DHA, 4,7,10,13,16,19(Z)-docosahexaenoic acid; EPA, 5,8,11,14,17(Z)eicosapentaenoic acid; FA, fatty acid; FAEE, fatty acid ethyl ester; FAME, fatty acid methyl ester; TAG, triacylglycerol; POP, 1,3-palmitin-2-olein. b Aw equal to 0.69.

Lipase-catalyzed harvesting and/or enrichment of fatty acids

Rhizopus oryzae Geotrichum candidum Candida lipolytica Rhizomucor jayanicus Pseudomonas cepacia Pseudomonas fluorescens Salmo salar Crude Oncorhyncus mykiss 13. As above As above

a

293

294

Novel enzyme technology for food applications

discrimination between FA of varying chain length in reactions with β-MAG containing FE with different chain lengths. In contrast, esterification reactions of FA to α-MAG showed increased sensitivity to the length of the FA chain; saturated C18 FA reacted most quickly only with α-MAG containing saturated C16 FE. Similar experiments were performed using a lipase from R. miehei (Entry 11, Fu et al., 2004c). As with the lipase from B. cepacia, the selectivity of esterification of different length saturated FA to MAG was found to be highest with α-MAG containing different length saturated FE. The last entry in Table 14.1 concerns a lipase from Rhizopus arrhizus that shows mostly positional specificity (Entry 14; Tan and Yin, 2005). Here it was shown that palmitic acid is released most rapidly from 1,3-palmitin-2-olein. During the course of hydrolysis, individual molecular species of acylglycerides were followed. It was shown that the processes of hydrolysis, esterification, and isomerization must be occurring to explain the observed species, and a kinetic model was designed to explain the results obtained.

14.3 Fatty acid harvesting We use the term FA harvesting to mean the physical isolation of a particular FA or its chemical derivative such as a FE. Because FA of different lengths and degrees of unsaturation or their esters have similar physical properties, it is difficult to separate them fully using a single fractional distillation. Full separation using chromatography is possible but is generally expensive. Over the last decade, techniques to obtain fairly high purity FA have been developed that take advantage of the natural selectivity of lipases. Each technique is different. However, the following steps are frequently used: (1) hydrolysis of a fatty acyl residue from a glyceride to give a FA fraction that is selectively re-esterified; (2) transesterification of a glyceride with an alcohol to give an ester fraction; or (3) alcoholysis of one ester to give a second ester. Each of these steps has the potential to be selective when using a lipase as the catalyst. However, unless there is a body of previous published work, the lipase must be selected by trial and error. Often enzymatic steps are coupled to more traditional purification techniques such as distillation or fractionation to give high purity FA or FE fractions. In Table 14.2 are shown summaries of several recent publications on FA harvesting. It can be seen that most interest is concentrated in the area of harvesting FA or their derivatives whose consumption is connected with improved health or a decrease in the incidence of disease: arachidonic acid (AA), γ-linolenic acid (GLA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), conjugated linoleic acid (CLA) and an unsaturated C20:3, n-9 FA called mead acid which may be anti-inflammatory (Entry 8; Shimada et al., 2003). The only entry concerned with non-food ‘industrial’ FA is entry 2 which gives an enzymatic technique for separating the functionally unusual FA from coriander, meadowfoam and castor oils (Foglia et al., 2000). Entry 4 is concerned with the purification of an acetylenic FA. The starting TAG were synthetically prepared, although the purification

Lipase-catalyzed harvesting and/or enrichment of fatty acids

295

technique might be applied to a naturally occurring TAG containing acetylenic FA (Lie Ken Jie et al., 2002). Entry 10 is concerned with the isolation of the ethyl ester of palmitic acid (Chen et al., 2004). Although palmitic acid is usually considered to be a FA for industrial use because it is saturated and therefore stable to oxidation, in this example palmitate was used to prepare a lipid that mimics the TAG found in human milk. In entry 1 of Table 14.2, DHA, the ethyl ester of DHA, GLA and AA were all purified using selective esterification, or selective alcoholysis coupled to more traditional purification techniques (Shimada et al., 2001). In each example shown in this entry, the enzyme did not act on the desired FA or FE as fast as it reacted with other FA or FE. Thus the desired FA or FE was concentrated in the unreacted fraction. The other examples shown in Table 14.2 generally follow the same pattern in which the desired FA or FE reacts more slowly than the other FA or FE. In recent years, enzymatic enrichment of FA has become the method of choice for the isolation of high quality PUFA because they are relatively unstable to oxidation and degrade during separation procedures that require heat such as distillation. These isolated FA or their derivatives are often used to produce structured lipids and the enzymatic syntheses of these will be discussed in the following section.

14.4 Structured triacylglycerols Structured triacylglycerols (STAG) with particular distributions of specific fatty acids have received wide attention since the late 1970s and early 1980s, when patents were issued to Unilever and Fuji Oil for the processing of confectionery fats or cocoa butter equivalents (Unilever, 1977). The processes were later implemented in both companies for commercial production, even though production was greatly affected by changes in the confectionery fats market. For nutritional purposes, STAG are synthesized for use as functional foods, infant formulas, dietary supplements and as treatment for disease or maintenance of good health (nutraceuticals). Physical properties of STAG are important when intended for applications such as spreads, cooking and baking fats, frying oils, creams, and so on (Akoh, 2002). A nutritional product (Betapol) was introduced by Loders Croklaan as a human milkfat substitute for infant formula (Quinlan and Moore, 1993; Kavanagh, 1997). This represents the most common application for STAG at the present time. The need to put different groups of fatty acids in different locations along the glycerol backbone comes from our understanding of the absorption or metabolism of oils and fats (Høy and Xu, 2001; Mu and Porsgaard, 2005). The degradation of oils and fats in the human body is regiospecific and results in the formation of sn-2 monoglycerides (MAGs) and free fatty acids. Free fatty acids liberated from dietary lipids during digestion are metabolized more rapidly if they are medium or short chain, whereas long chain fatty acids can be absorbed directly in the form of MAGs. This implies that the fatty acids located at the sn-2 position may have a different metabolic pathway compared with those at the

296

Desired FA or FE

Enrichment steps

Fraction with desired FA or FE

Lipase source

Degree of Reference purity (%)

FA Pseudomonas sp – Shimada et al., 2001 (i) Borage oil + H2O =FA (ii) FA + LauOH FA Rhizopus delemar 70 =FE (iii) Purification of FA FA Rhizopus delemar 94 (iv) FA + LauOH =FE 1(a) DHA (i) Tuna oil + EtOH FE Candida antarctica – =FE (ii) Distill FE FE 57 (iii) FE + LauOH =FE + FLE FE 87 (AA from Mortierella single-cell oil purified using identical enzymatic steps) Pseudomonas cepacia 70 Foglia et al., 2000 2. Petroselinic acid (i) Coriander oil + H2O FA = FA (ii) FA + 1-butanol FA Geotrichum candidum 90 (Ricinoleic acid from caster oil and 5-eicosenoic, 13-docosaenoic and 5,13-docosadienoic acids from meadowfoam oil purified using identical steps) 3. DHA + EPA Tuna oil + EtOH Glycerides Pseudomonas fluorescens 74 Rakshit et al., 2000 = glycerides + FE Glycerides Candida rugosa 36 Lie Ken Jie et al., 2002 4. 13-Docosynoic acid Synthetic oil + H2O = glycerides + FA 1. GLA

Novel enzyme technology for food applications

Table 14.2 Fatty acid harvesting

6. GLA 7. DHA 8. n-9 LnA 20:3 n-9; MA

9. (c9, t11)-CLA (t10, c12)-CLA 10. Palmitate

Salmon oil + H2O = glycerides + FA Borage oil + H2O = glycerides + FA Tuna oil FA + glycerol = glycerides + FA (i) Mortierella alpina oil + H2O = glycerides + FA (ii) n-9 LnA + LauOH =FE (2X) (iii) MA glycerides =MA (base catalyst) (iv) MA + LauOH =FE (2X) Mixture of CLA isomers + LauOH =FE, then distillation of the ester fraction. Palmitic acid + ethanol =FE

Glycerides

Candida rugosa

27

Sun et al., 2002

Glycerides

Candida rugosa

45

Kawashima et al., 2002

FA

Rhizomucor miehei

78

Halldorsson et al., 2003a

FA glycerides

Pseudomonas aeruginosa

20 24

Shimada et al., 2003

FA

Candida rugosa

54

FA

Candida rugosa

60

FE FA

Candida rugosa

85 78

Nagao et al., 2003

FE

Pseudomonas cepacia

98

Chen et al., 2004

FA

Abbreviations: AA, arachidonic acid; CLA, conjugated linoleic acid; DHA, 4,7,10,13,16,19(Z)-docosahexaenoic acid; EPA, 5,8,11,14,17(Z)-eicosapentaenoic acid; FA, fatty acid; FLE, fatty lauryl ester; GLA, γ-linolenic acid (C18:3, n-6); LauOH, lauryl alcohol; MA, mead acid (C20:3; n-9); n-9 LnA, 6,9-octadecadiencoic acid.

Lipase-catalyzed harvesting and/or enrichment of fatty acids

5. DHA + EPA

297

298

Novel enzyme technology for food applications

1,3-positions. This has led to interest in the production of tailor-made fats with particular triglyceride structures. To attain the maximum benefits of STAG, the structural/compositional modification of TAG often is carried out with a selective lipase, since chemical modification does not selectively replace fatty acyl residues because of its random nature. Thus, STAG with defined characteristics can be used to provide specific metabolic effects for nutritive or pharmaceutical purposes, and thereby hold promise for wider usage in nutritional, medical or food applications (Lee and Akoh, 1998). A variety of FA are used in the synthesis of STAG, taking advantage of the functions and properties of each to obtain maximum benefits for a given STAG. These FA include short, medium, saturated, monounsaturated and PUFA (Willis et al., 1998). STAG produced from short and long chain FA are designated as low-calorie fats with intended use as coatings and confectionery fats (Gunstone, 2002). The nutritional advantage of medium-chain TAG in the treatment of certain metabolic disorders is well documented (Porsgaard, 2006). The enzymatic combination of a medium-chain and saturated or unsaturated long-chain fatty acid into a single STAG species has received interest because of their advantages in parenteral and enteral nutrition as well as reduced calorie fats (Lee et al., 2005). Another active area of STAG research is the synthesis of STAG containing n-3 PUFA and medium chain FA which have been shown to have the ability to improve immune function and reduce cholesterol concentration (Lee et al., 1999). As noted above, the main drawback to chemical transesterification is that it is not selective and, after the procedure is complete, the distribution of the fatty acyl groups is random. Another problem is that the double bonds of sensitive polyunsaturated fatty acyl groups such as those derived from the nutritionally important FA such as EPA and DHA may undergo isomerization by the alkali transesterification catalyst commonly used (e.g., sodium methoxide). In addition, during chemical transesterification, FA soaps are also produced and these, along with residual catalyst, must be removed from the product. There has been a resurgence of interest in the lipase-catalyzed synthesis of STAG with the recent advent of several commercially available lipases that are useful for this purpose. When proper reaction conditions and the proper lipases have been identified empirically, TAG that have very specific structures can be synthesized relatively easily compared with chemical methods of synthesis. These structured TAG are intended to be used as components of oils and fats principally for three purposes: to impart to the oils and fats particular physical properties without incorporating trans configuration double bonds in the fatty acyl groups, to contain fatty acyl groups that can increase or decrease the nutritive and caloric value of the fat or oil, and to contain fatty acyl groups that have putative impacts on human health. We briefly will discuss each of these items below. Partial hydrogenation has been the method of choice for modifying the properties of natural oils to make them into solids or to increase the melting point for use in margarines and shortenings. Partial hydrogenation usually introduces trans double bonds into the product. Because there is accumulating evidence linking these trans fats to increased risk of heart disease, the US Food and Drug Admin-

Lipase-catalyzed harvesting and/or enrichment of fatty acids

299

istration now requires the disclosure of the level of trans fat in food products (Moss, 2005). By using enzyme technology to prepare a STAG, the composition of the final product may be precisely controlled to such a degree that a product with desirable melting point can be obtained that contains no trans lipids. Enzyme technology can be used to create STAG that have increased or decreased nutritive value. The position of the fatty acyl groups in a consumed TAG is related to lipid digestion for two reasons (Linderborg and Kallio, 2005; Huiling and Porsgaard, 2005). First, the human digestive apparatus contains a 1,3 specific lipase which leaves intact a 2-MAG. This may be absorbed directly and will contribute to fat reserves and ultimately to the structure of phospholipids, the main component of cellular membranes. Second, the fatty acyl groups that initially resided at the sn-1 and sn-3 positions of glycerol are only poorly absorbed after being cleaved by the digestive lipase owing to formation of FA salts by calcium and magnesium ions in the digestive tract. In order to create high energy TAG for specialized foods, advantage may be taken of the mechanism of digestion. One example is to locate palmitate in the sn-2 position to create a human milk substitute. Another example would be to locate medium chain fatty acyl groups in the sn-1 and -3 positions because lipase cleavage during digestion gives FA that are relatively easily absorbed and rapidly metabolized compared with longer chain FA. A TAG containing a mixture of long chain and medium chain acyl groups is advantageous because pure medium chain TAG alone cannot provide essential FA. Simple physical mixtures of medium chain TAG and long chain TAG may be thought to meet nutritional requirements, however, the absorption efficiency has been reported to be low (Mogi et al., 2000). A structured lipid with very short chain fatty acyl groups in positions sn-1 and -3 contains much lower calorie levels than an unmodified lipid. Finally, enzyme technology can also be used to introduce healthy FA into structured TAG. Most of these are n-3 PUFA such as DHA and EPA, but lipids containing GLA are also of interest. PUFA are partially destroyed using traditional methods of isolation, but can be isolated using enzyme technology as was outlined in the previous section. Enzyme technology can then be utilized to prepare SL in which the healthy FA is located in only the sn-2 position from which it will be almost totally absorbed. There is also some evidence that sensitive PUFA such as DHA are oxidized more slowly when their esters are found in the central position of a glyceride. A review of structured lipids containing n-3 very long chain polyunsaturated fatty acyl groups has recently been published (Wijesundera, 2005). The main barrier to the increased adoption of enzymatically prepared STAG is cost. In addition to the cost of the enzyme, a source of relatively pure FA or FE must be obtained. Since the processes of acidolysis and esterification are equilibrium processes, it is necessary to add an excess of FA or FE to drive the process of TAG formation to completion. Thus the excess FA or FE must be removed and this too drives up the cost of the STAG. For this reason, some workers have chosen to develop enzymatic transesterification processes in which two different fats and/or oils are reacted. These reactions can be conducted under mild conditions and

300

Novel enzyme technology for food applications

sensitive PUFA are preserved. However, some FA forms during the transesterification process and this is usually removed. In Table 14.3 we have summarized a variety of publications from the last five years dealing with the enzymatic production of STAG. We have tried to group these according to intended composition or which special fatty acyl group was introduced into the structured TAG. A precise grouping was not possible here because in some publications two or more ‘functional’ fatty acyl groups were introduced into the same triglyceride. In addition we included two publications dealing with the enzymatic insertion of specific fatty acids into phospholipids. Many of the procedures include non-enzymatic steps, but these are not included except in two cases where non-enzymatic steps were mentioned to increase the clarity of the procedure. In Table 14.3 we have also used the trade names of the enzymes instead of naming their biological source as was done in the other tables. This is to show that most research on STAG is being conducted using commercial enzymes. The first section in Table 14.3 is ‘Preparation of TAG with very long chain fatty acids (VL)’. The structures of the TAG are given on the left where in addition to VL, L stands for long chain and M for medium chain FA. Note that most of the structures have a single type of fatty acyl group on positions sn-1 and -3, and a different type at position sn-2. This is caused by the use of a 1,3 specific lipase in the synthesis of these STAG. If only one kind of FA is indicated by the procedure, then the second FA was added by a chemical procedure. The addition of FA to glycerol or to a TAG is a favorite procedure. However, to get high incorporation of the FA, an excess must be added. If a TAG is used as the starting material rather than glycerol, the excess FA plus the displaced FA must be removed when the procedure is complete. Some of the STAG entries in this section contain M and VL, although the next section deals with incorporation of M because we wanted all of the research on VL incorporation into STAG to be grouped together to show that this area of STAG research is currently very active. As just noted the next section of Table 14.3 is devoted to the incorporation of M into STAG. Fractions from palm and coconut oils, as well as copra oil are among the favorite starting materials because they are readily available and relatively inexpensive. Most of the entries use pure or relatively pure sources of the starting FA, and this requires an extra purification procedure before STAG synthesis can occur. We want to note here that Entry 24 (Wongsakul et al., 2004) is somewhat unique in its approach. Usually a TAG containing a desirable fatty acyl residue at position n-2 is used as the starting material and then the fatty acyl groups from the end positions are displaced by the desired groups using a sn-1,3 specific lipase. However, in the case of Entry 24, a 1,3-DAG containing the esters of lauric or caprylic acid were prepared and then an ester of oleic acid was inserted into the middle position by an enzyme that had specificity for oleic acid but would not act to remove the laurate or caprylate groups. Examples of enzymes that use FA selectivity instead of positional specificity to achieve the synthesis of STAG are quite rare. Enzymes with good FA specificity have been known for many years, but the reason they are rarely used has not been discussed in the literature. It is possible

Lipase-catalyzed harvesting and/or enrichment of fatty acids

301

that these enzymes showed good FA specificity in aqueous media, but the specificity lessened in the neat media preferred for STAG synthesis. This issue could benefit from more investigation. The next section in Table 14.3 contains a single reference concerned with the enrichment of chicken fat and menhaden oil with the esters of monounsaturated fatty acids (MUFA) and PUFA obtained from fractionated chicken fat and menhaden oil (Entry 27; Lee et al., 2001). This is followed by two references for the preparation of STAG containing the esters of CLA (Entries 28 and 29; Arcos and Hill, 2000; Hirose et al., 2006). The various isomers of CLA have been reported to have numerous beneficial effects on human health and thus it is likely that research into CLA-containing lipids will continue. The next sections in Table 14.3 report research publications concerned with the preparation of a STAG for specific nutritional purposes: TAGs for human milk fat substitute, low calorie TAG, TAG for butter or margarine replacement and TAG for the improvement of butterfat. The last two entries concern the modification of the FE profile of phospholipids. The area of nutrition derived from ingested phospholipids has seen relatively little activity and this area needs more attention.

14.5 Single reaction step process for the production of STAG Figure 14.1 shows a simplified reaction scheme for producing STAG using a regiospecific lipases. The reaction shown represents either the acidolysis between triglycerides and fatty acids or an ester–ester exchange between triglycerides and fatty acid ethyl/methyl esters (Xu, 2000a,b). Generally, diglyceride intermediates are formed by enzymatic deacylation or hydrolysis of the triglycerides with water participation. The diglycerides are re-esterified with new fatty acids or their ethyl/ methyl esters in the reaction system and new triglycerides are formed. The reaction proceeds until equilibrium is reached at which point the final reaction mixture will contain newly formed triglycerides that contain either one or two newly incorporated fatty acyl groups and the original triglycerides. The triglycerides (BBB) are partially hydrolysed first into diglycerides (Fig. 14.1), which in turn react with fatty acyl donors, indicated as A in Fig. 14.1, to form the mono-incorporated triglycerides (ABB/BBA). The mono-incorporated triglycerides are transformed by the same procedure into di-incorporated triglycerides (ABA). The reaction can be monitored by determining the amount of ABA and ABB/BBA using highperformance liquid chromatography or by analyzing the amount of A incorporated into the glycerides using gas chromatography. The latter is often referred to as acyl incorporation. The reaction from triglyceride BBB to ABA seen in Fig. 14.1 is reversible. Therefore, there is a dynamic balance between the three triglyceride species involved in the reaction, BBB, ABB/BBA, and ABA, at equilibrium. Biocatalysts and other reaction parameters do not affect the reaction equilibrium, but only change the equilibration rate. Higher A content will push the reaction toward the ABA side, favoring higher product yield or acyl incorporation.

302

Table 14.3 Structured glyceride synthesis Enzymatic reaction(s)

Preparation of TAG with very long chain fatty acids (VLa): 1. L-VL-L Lipozyme FA + glycerol = 1,3-DAG VL-L-L Novozyme 435 FA + glycerol = TAG Lipozyme TAG + FA = TAG 2. M-VL-M Novozyme 435 FE = FA Novozyme 435 FA + glycerol = TAG Lipozyme IM TAG + FE = TAG 3. M-VL-M Lipase AP, TAG + FA = TAG Lipase P, Lipase AY, Lipase AK, Lipase F, Lipase D 4. M-VL-M Lipoxyme IM TAG + FA = TAG M-L-M 5. M-VL-M Lipozyme FA + glycerol = 1,3-DAG 6. M-VL-M Novozyme 435 Lipozyme IM 7. M-VL-M Lipozyme IM 8. M-VL-M Novozyme 435 M-L-M Ta-lipase 9. L-VL-L Novozyme 435 VL-L-VL 10. VL-L-VL PS-30

Reactor type/milieu

Lipid reactants

Reference

Batch/diethyl ether Batch/neat Batch/neat Batch/water Batch/neatb Batch/neat Batch/neat

Stearic acid Stearic acid, EPA, DHA Ethyl ester EPA, EPA, Ethylcaprylate Fish oil, caprylic acid

Haraldsson et al., 2000

Packed-bed/neat

Menhaden oil, canola oil, caprylic acid Caprylic, capric, lauric acids Esterified DHA, ethylcaprylate Caprylic acid, esterified EPA

Batch/diethyl ether

TAG = 2-MAG 2-MAG = TAG FA = FE FE + TAG = 2-MAG + 1,2-DAG = TAG FA + glycerol = TAG

Batch/ethanol Batch/neat Batch/ethanol Batch/water Water removal Batch/neat

TAG + FA = TAG TAG + TAG = TAG

Batch/neat Batch/neat

TAG + FA = TAG

Batch/neat

Irimescu et al., 2000 Zhou et al., 2000

Akoh et al., 2001 Halldorsson et al., 2001 Irimescu et al., 2001a Irimescu et al., 2001b

γ-linolenic acid, arachi- Kawashima et al., 2001 donic acid, EPA, DHA Caprylic acid Palm oil stearin, soyOsório et al., 2001 bean oil, TAG with EPA and DHA Borage oil, evening Senanayake and Shaihidi, 2002 primrose oil, EPA, DHA

Novel enzyme technology for food applications

Goal Lipase

11. L-VL-L

Novosyme 398

TAG + FA = TAG

15. M-VL-M Novozyme 435 TAG + FA = TAG Mucor miehei PS-30 AP-12 AY-30 16. L-VL-L Lipozyme TL IM TAG + TAG = TAG VL-L-VL 17. M-VL-M Lipoxyme IM

TAG + FA = TAG

Preparation of TAG with medium chain fatty acids (M): 18. M-L-M Candida rugosa TAG + FA = TAG Geotrichum candidum 19. M-L-M Saiken 100 TAG + TAG = TAG L-M-L (modified) 20. M-L-M

Lipozyme IM

TAG + FA = TAG

Batch/neat Batch/CH2Cl2 Batch/neat Batch/neat Packed bed/ neat or hexane Batch/neat

Batch/neat

Packed bed with recirculation/ neat or hexane Batch/neat Batch/neat Batch/neat

Salmon oil

Linder et al., 2002

Vinyl ester of C8:0-C16:0 EPA-rich TAG

Halldorsson et al., 2003

Ethyl stearate, DHA ethyl ester, CLA ethyl ester EPA, DHA-rich TAG, caprylic acid TAG with DHA, capric acid

Mogi et al., 2000 Zhou et al., 2001

303

14. M-VL-M Lipoxyme IM

Batch/water

Lipase-catalyzed harvesting and/or enrichment of fatty acids

TAG + H2O = DAG + MAG + FA filter to enrich VL Lipozyme IM + glycerol = TAG 12. M-VL-M Lipozyme RM IM Vinyl-FE + glycerol L-VL-L = DAG + acetaldehyde 13. L-VL-L Chirazyme L2 TAG + glycerol VL-L-VL = TAG + DAG + MAG L-L-VL Chirazyme L2 + FE = TAG VL-L-L

Torres et al., 2003

González Moreno et al., 2004 Hamam and Shaihidi, 2004

TAG with EPA and DHA, palm stearin, palm kernel oil EPA and DHA-rich TAG, caprylic acid

Nascimento et al., 2004

MUFA-rich TAG, caprylic acid Oleic acid-rich TAG, caprylic and capric acid-rich TAG Rapeseed oil, caprylic acid

Lee and Foglia, 2000

González Moreno et al., 2005

304

Table 14.3

(cont.) Enzymatic reaction(s)

Reactor type/milieu

21. M-L-M

Ta-lipase

TAG + FA = TAG

22. M-L-M

TAG + FA = TAG

23. M-L-M

Lipozyme IM Lipoxyme TL Lipozyme IM

24. M-L-M

Amano PS-D

1,3-DAG + FA = TAG 1,3-DAG + FE = TAG

25. M-M-M

Papain lipase

γ-linolenic acid-rich TAG, caprylic acid Batch/neat or hexane Perilla oil, caprylic acid Packed bed/neat Sunflower oil, caprylic acid Batch/neat Lauric and caprylic Batch/hexane acid-rich DAG, oleic acid,oleate vinyl ester Batch/neat Copra oil, copra Packed-bed butyl esters Batch/water Coconut and palm kernel distillate

TAG + FA = TAG

TAG + butanol = FE FE + TAG = FE + TAG 26. M-M-M Amano 30 lipase TAG + H2O = FA hydrolyzed distillate is distilled Lipozyme IM New distillate + glycerol = TAG Enrichment in MUFA and PUFA fatty esters: 27. L-L-L Lipozyme IM TAG + FA = TAG

Preparation of TAG with conjugated linoleic acid (CLA): 28. L-L-L Chirazyme L-9 FA + glycerol = TAG 29. L-L-L Rhizomucor FA + glycerol = TAG miehei + Alcaligenes sp. Penicillium camembertii + Alcaligenes sp.

Lipid reactants

Fixed bed/neat

Reference Kawashima et al., 2002 Kim et al., 2002 Jacobsen et al., 2003 Wongsakul et al., 2004 Capro et al., 2004 Nandi et al., 2005

Batch/hexane

Fractionated chicken Lee et al., 2001 fat FA, fractionated menhaden oil FA, plus unfractionated fat or oil

Packed bed/neat Batch/neat

CLA CLA

Acros and Hill, 2000 Hirose et al., 2006

Novel enzyme technology for food applications

Goal Lipase

Preparation of low calorie TAG: 35. L-S-L Chirazyme L-2 TAG + FA = TAG Preparation of TAG for replacement for margarine/butter: 36. M-L-M Lipoxyme IM TAG + TAG = TAG L-M-L Improvement of TAG in Butterfat: 37. M-L-M Lipoxyme TL TAG + TAG = TAG L-M-L Lipxyme RM Novozyme 435 Lipase PS-C Lipase PS-D Modification of phospholipid FE profile: 38. X-L-L Lipozyme RM PL + FA = PL X-L-M Lipozyme TL Novozyme 435 39. X-L-L Lecitase PL + FA = PL Lipozyme TL Lipozyme RM

Batch/neat Batch/hexane Batch/neat Batch/hexane Batch/neat Batch/neat hexane

Tripalmitin, oleic acid Palmitate-enriched palm oil fraction Ethyl palmitate, tripalmitin Lard, soybean oil FA Tripalmitin, CLA, CLA, caprylic acid

Nagao et al., 2001 Chen et al., 2004

Yang et al., 2003 Yang et al., 2005

Batch/hexane

Tripalmitin, hazelnut oil FA, stearic acid

Sahin, 2005

Batch/neat

Triacetin stearic acid

Yang et al., 2001

Batch/neat

Palm stearin, palm kernel olein

Batch/neat packed bed/neat

Butterfat, rapeseed oil

Rønne et al., 2005

Batch/neat Batch/hexane

Caprylic acid, CLA, EPA, DHA

Peng et al., 2002

Batch/hexane

CLA

Hossen and Hernandez, 2005

Chu et al., 2002

Lipase-catalyzed harvesting and/or enrichment of fatty acids

Preparation of TAG for human milk fat substitute: 30. L-L-L Fusarium TAG + FA = TAG heterosporum 31. L-L-L PS-30 TAG + ethanol = FE Lipozyme IM Novozyme 435 FE + glycerol = TAG Lipozyme IM TAG + oleic acid 32. L-L-L Lipozyme IM TAG + FA = TAG 33. L-L-L Lipozyme IM TAG + FA = TAG M-L-M (change in temperature to reduce acyl migration) 34. L-L-L Lipozyme IM TAG + FA = TAG

a

305

Abbreviations: CLA, conjugated linoleic acid; DAG, diacylglycerol; DHA, 4,7,10,13,16,19(Z)-docosahexaenoic acid; EPA, 5,8,11,14,17(Z)-eicosapentaenoic acid; FA, fatty acid; FE, fatty ester; L, long chain FA C14–18; M, medium chain FA C8–12; MAG, monoacylglycerol; MUFA, monounsaturated fatty acid; PL, phospholipid; PUFA, polyunsaturated fatty acid; TAG, triacylglycerol; VL, very long chain FA C≥20; X, glycerol carbon atom containing phosphate or phosphorylated head group. b Small amounts of water added to enhance enzyme activity not mentioned. The water is bound tightly to the enzyme preparation and does not participate in the reaction.

306

Novel enzyme technology for food applications A B B B Substrate triglyceride

Fig. 14.1

via diglyceride A

A

B

B

A B B Intermediate triglyceride

B

via diglyceride A

B

A B A Product triglyceride

Reaction scheme of sn-1,3-lipase catalyzed interesterification between a triglyceride (BBB) and an acyl donor (A).

Besides the reactions from BBB to ABA in Fig. 14.1, acyl migration also occurs owing to the existence of diglycerides that arise from the lipase-catalyzed hydrolysis. The hydrolysis cannot be fully avoided since there is often a small amount of water associated with the lipase. This process can lead to the formation of nonspecific triglycerides such as AAA, AAB, and so on. Since regiospecificity is one of the major aims of the process and determines product quality, this side reaction has to be minimized. Diglycerides play a very important role in the induction of acyl migration (Xu, 2003). The greater the amount of diglycerides produced in the reaction, the greater the extent of acyl migration found in the products. Acyl migration, on the premise of diglycerides being the precursor, takes place via the formation of an unstable cyclic intermediate and is initiated by the nucleophilic attack of a lone pair of electrons from the free hydroxyl oxygen on the ester carbonyl carbon, which results in a five-member ring intermediate. Subsequently, the ring opens and results in two products, the original diglyceride and a migrated diglyceride. 1,2(2,3)-Diglycerides are thermodynamically unstable and tend to rearrange to 1,3-diglycerides. The ratio of 1,2(2,3)-diglycerides and 1,3-diglycerides is about 2:3 at equilibrium. A good quality STAG should have high acyl incorporation or content of monoand/or di-incorporated triglycerides and low acyl migration and diglyceride content. In many cases, factors such as temperature, water content, reaction time, and so on that favor higher acyl incorporation also favor a higher degree of acyl migration and diglyceride content. In this case, the reaction has to be optimized and a compromise has to be made. The process for such an operation uses a simple stirred tank reactor or a plug flow reactor. There is not much difference in process productivity between the two processes (Xu, 2003). However, there is some impact on product quality, process cost and ease of operation. Since the system is often a homogenous system, solvents can be avoided. This makes the process much simpler than a process that requires solvents. Such processes have been implemented in pilot plants and industrial production (Quinlan and Moore, 1993). Many case studies have been published concerning different STAG (Chang et al., 1990; Xu et al., 1998, 1999; Yang et al., 2003; Nielsen et al., 2006).

Lipase-catalyzed harvesting and/or enrichment of fatty acids

LLL + ethanol

Novozym 435

307

2-MAG + L-EE + ethanol 1. Remove enzyme 2. Remove ethanol

Lipozyme RM IM 8/L/8 + L-EE + 8:0

2-MAG + L-EE Vacuum

1. Remove Lipozyme RM IM 2. Remove 10:0 and L-EE 8:0/L/8:0 up to 90%

8:0

Fig. 14.2 Scheme of the two-step reaction process for the production of ABA-type structured lipids, where A is caprylic acid and B is a long chain essential fatty acid (L), EE are ethyl esters and MAG monoglycerides.

14.6 Multiple reaction step processes for the production of STAG For products with higher purity, a more sophisticated procedure for the production of STAG with a regiospecific distribution of fatty acids has been developed. A schematic presentation of the procedure is given in Fig. 14.2 (Soumanou et al., 1998; Kim and Yoon, 2003; Iwasaki and Yamane, 2004). The first step is ethanolysis of LLL to produce sn-2 monoglycerides. This reaction can reach 100% in 2–3 h. The monoglycerides produced can either react with a fatty acid or its ethyl ester, using another enzyme to synthesize into ALA structured lipid. The intermediate monoglycerides can either be isolated from the system or used directly for further synthesis without isolation but with over-abundance of acyl donors (A) to minimize reverse reactions with the acyl donors released in the first step. The synthesis step can be very fast, less than one hour. This sophisticated procedure has, so far, only been applied on a laboratory scale. Purity can reach 90% with a 500 g production run. Previous studies in the laboratory have reported 95% purity (Iwasaki and Yamane, 2004). Acyl migration of the monoglycerides produced is a central issue for this process and it is critical to develop a process technology that will ensure high product quality.

14.7 Nutritional and other uses of structured lipids There are few if any industrial applications for which STAG have been claimed, other than partially hydrogenated products, presumably because of the costs associated with producing STAG. Hence most applications claimed for STAG usage have been as nutritional or nutraceutical type lipids. STAG products that

308

Novel enzyme technology for food applications

have been commercialized include reduced-calorie fats that are composed of at least one VL (C16–C18) and a short chain fatty acid (C2–C3) as characterized by the Benefat series of STAG lipids (Cultor Food Science). Similar type reduced-calorie STAG can be prepared by interesterification of long-chain FA and M (C6–C8), which are marketed under the name Caprenin (Procter and Gamble). While a targeted STAG product Behenin (Fuji Oil) is produced by enzymatic esterification of behenic and oleic acid in a 2:1 ratio. These reducedcalorie fats, which typically have half the caloric density of natural fats (∼5 kcal g–1) have been used for confections, baking, compound coatings and dairy applications. Medium-chain TAG are another common form of STAG that are composed of mixed medium-chain FA either alone or in combination with a VL. Examples of the former products include Captin (Stepen Co.) and Captex (Abitec Corp.), which are mixtures C8–C12 FA that are used in such applications as sport drinks, energy bars and infant formulas. In the latter instance, the medium chain TAG is interesterified in combination with a long-chain FA (usually an unsaturated C18) and includes products such as Neobee (Stepan Corp.) Impact (Novartis Corp.), and Laurical (Calgene Inc.) all of which have claimed medical and/or nutritional benefits in such applications as nutritional beverages, parenteral nutrition and intravenous fat emulsions. Finally, recently enacted labeling laws both in the USA and Europe have prompted the food and ingredient industry to manufacture foods with low or zero trans fat content. Such products originally were produced by interesterification of a full saturated fat (typically fully hydrogenated oil) with a non-hydrogenated oil. Similar type oils have been produced enzymatically by ADM Corp and are marketed as a series of products under the trade name NovaLipid. These zero or reduced trans structured fats and oils are intended for use as shortenings and margarines.

14.8 Summary and future trends In this chapter we have attempted to provide an overview of recent literature on the selectivity of selected lipases, the synthesis of STAG, materials used in their synthesis, their current availability and SL applications. An understanding of the functional and nutritional properties of the fatty acyl groups in STAG will also provide new products with beneficial end-use properties. With these perspectives in mind, the outlook and potential for further commercialization of enzymatic produced STAG should continue to meet consumer needs and hence increase their potential commercialization. Factors that need to be considered for continued commercial success of these enzymatic processes include: identification of new markets for food ingredients or fine chemicals, process scale-up, catalyst reuse and cost of enzymatic processes versus chemical routes, consumer preferences for natural versus synthetic products and meeting government regulations for new food ingredients.

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14.9 References Akoh C C (2002). ‘Structured Lipids’, in Akoh C K and Min D B (eds), Food Lipids, Marcel Decker, New York, 877–908. Akoh C C and Moussata C O (2001). ‘Characterization and oxidative stability of enzymatically produced fish and canola oil-based structured lipids’, J Am Oil Chem Soc, 78, 25–30. Arcos J A and Hill, Jr. C G (2000). ‘Rapid solvent-free esterification of conjugated linoleic acid and glycerol in a packed bed reactor containing an immobilized lipase’, Studies Surface Sci Catal, 130, 3405–3410. Arsan J and Parkin K L (2000). ‘Selectivity of Candida antarctica B lipase toward fatty acid and (iso)propanol substrates in esterification reactions in organic media’, J Agric Food Chem, 48, 3738–3743. Capro Y, Turon F, Villeneuve P, Pina M and Graille J (2004). ‘Enzymatic synthesis of medium-chain triacylglycerols by alcoholysis and interesterification of copra oil using a crude papain lipase preparation’, Eur. J Lipid Sci Technol, 106, 503–512. Caro Y, Villeneuve P, Pina M, Reynes M and Graille J (2000). ‘Lipase activity and fatty acid typoselectivities of plant extracts in hydrolysis and interesterification’, J Am Oil Chem Soc, 77, 349–354. Chang M K, Abraham G and John V T (1990). ‘Production of cocoa butter-like fat from interesterification of vegetable oils’, J Am Oil Chem Soc, 67(4), 832–834. Chen M-L, Vali S R, Lin J-Y and Ju Y-H (2004). ‘Synthesis of the structured lipid 1,3dioleoyl-2-palmitoylglycerol from palm oil’, J Am Oil Chem Soc, 81, 525–532. Chu B S, Ghazali H M, Lai O M, Che Man Y B and Yusof S (2002). ‘Physical and chemical properties of a lipase-transesterified palm stearin/palm kernel olein blend and its isopropanol-solid and high melting triacylglycerol fractions’, Food Chem, 76, 155–164. Foglia T A, Jones K C and Sonnet P E (2000). ‘Selectivity of lipases: isolation of fatty acids from castor, coriander, and meadowfoam oils’, Eur. J Lipid Sci Technol, 102, 612–617. Fu X and Parkin K L (2004a). ‘Selectivity of fatty acid incorporation into acylglycerols in esterification reactions using Rhizomucor miehei and Burkholderia cepacia lipases’, Food Res Int, 37, 651–657. Fu X and Parkin K L (2004b). ‘Reaction selectivity of Burkholderia cepacia (PS-30) lipase as influenced by monoacylation of sn-glycerol’, J Am Oil Chem Soc, 81, 33–44. Fu X and Parkin K L (2004c). ‘Reaction selectivity of Rhizomucor miehei lipase as influenced by monoacylation of sn-glycerol’, J Am Oil Chem Soc, 81, 45–55. González Moreno P A, Robles Medina A, Camacho Rubio F, Camacho Páez B and Molina Grima E (2004). ‘Production of structured lipids by acidolysis of an EPA-enriched fish oil and caprylic acid in a packed bed reactor: analysis of three different operation modes’, Biotechnol Prog, 20, 1044–1052. González Moreno P A, Robles Medina A, Camacho Rubio F, Camacho Páez B, Esteban Cerdán L and Molina Grima E (2005). ‘Production of structured triacylglycerols in an immobilized lipase packed-bed reactor: batch mode operation’, J Chem Technol and Biotechnol, 80, 35–43. Gunstone F D (2002). ‘Food Applications of Lipids’, in Akoh C C and Min D B (eds), Food Lipids, New Marcel Decker, York, 729–750. Halldorsson A and Haraldsson G G (2004a). ‘Fatty acid selectivity of microbial lipase and lipolytic enzymes from salmonid fish intestines toward astaxanthin diesters’, J Am Oil Chem Soc, 81, 347–353. Halldorsson A, Magnusson C D and Haraldsson G G (2001). ‘Chemoenzymatic synthesis of structured triacylglycerols’, Tetrahedron Lett, 42, 7675–7677. Halldorsson A, Kristinsson B, Glynn C and Guðmundur G H (2003a). ‘Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol’, J Am Oil Chem Soc, 80, 915–921. Halldorsson A, Magnusson C D and Haraldsson G G (2003b). ‘Chemoenzymatic synthesis of structured triacylglycerols by highly regioselective acylation’, Tetrahedron 59, 9101– 9109.

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Index

acetolactate decarboxylase 206 acylglycerols, modification by immobilized enzymes 62 cocoa butter equivalents 65 diacylglycerols 67 modified triacylglycerols 65–6 trans-free oils 62–5 aflatoxins 50–1 alternan oligosaccharides 256, 258–9 alternansucrase–maltose acceptor oligosaccharides 224–5 amino acids, flavour characteristics 184 α-amylase 206 β-amylase 206 angiotensin I-converting enzyme (ACE) inhibitors 161–3 milk-derived 165 structural implications 163–5 antibiotic resistance markers 53–4 antihypertensive peptides 162, 165 antimicrobial peptides 168–9 antioxidant peptides 169 arabinanoligosaccharide (AOS) 230 (arabino)galacto-oligosaccharide (ATOS) 230 arabinoxylan (AX) 228–9 arabinoxylo-oligosaccharide (AXOS) 231 aromatic amino acids (AAA) 74 artichoke inulin 219–20 aspartame 76–7, 190–1 Atlantic cod enzymes 205–8 cryotin 209–11

Bacillus subtilis 7 baking, applications of crosslinking enzymes 109–10 protein-based fat replacers 149 transglutaminase (TG) 110–12 biocatalysis 16 biochemical assays 8–9 bioinformatics 6–7 bioreactors chromatographic 176 membrane 173–6 bovine chymosin see chymosin, bovine branched chain amino acids (BCAA) 74 carbohydrates, health-functional 215, 237 alternansucrase–maltose acceptor oligosaccharides 224–5 arabinoxylan (AX) 228–9 dietary fibre 215–17 gluco-oligosaccharides (GOS) 223–4 inulin 219–22 oligodextran 234–7 oligosaccharides from non-starch polysaccharides (NSP) 230–2 pectins 232–4 prebiotics 217–18 resistant starch (RS) 226–8 transgalacto-oligosaccharides (TOS) 222–3 carbohydrates, modification by immobilized enzymes functional oligosaccharides 69–72

316

Index

high-fructose corn syrup (HFCS) 68–9 lactose hydrolysis 72–3 tagatose 73 Cascade system 199, 200 cellulases 206 cereals, applications of crosslinking enzymes 112–14 cheese applications of crosslinking enzymes 121 protein-based fat replacers 150 enzyme modified cheese (EMC) 194–8 flavours 196 chicory inulin 219–20 chondroitin sulphate 188–90 chromatographic bioreactors 176 chymosin 206 bovine 44, 45 chymotrypsin 171 classical strain improvement (CSI) 27 cloning 10–11, 18–19 cocoa butter equivalents 65 colonic diseases 218 combinatorial libraries enhanced by recombination in yeast (CLERY) 23 computational protein engineering 28–9 combined with laboratory evolution 34–3 consumer attitudes 85–6 future trends 95 implications 94–5 information sources 95 studies 88 attitude formation 88–9 effect of information on attitudes 89–90 effect of product experience on attitudes 92–4 intention to buy 91–2 theoretical approaches to attitude formation 86–7 impact on buying behaviour 87–8 Corning Glass process 72 crosslinking enzymes 101–3, 126 baking and pasta applications 109–10 oxidative enzymes in cereals 112–14 transglutaminase in baking 110–12 transglutaminase in pasta manufacture 112 chemistry of crosslinks 122–3 carbohydrates, feroylylated 124 proteins 123–4 dairy applications 118–20 cheese manufacture 121

milk stability 121 set and stirred yoghurts 121 effect on nutritional properties 124–6 meat and fish processing 114–15 heated meat products 115–18 restructured meat products 118 other applications 122 types oxidative enzymes 104–9 transglutaminase 103–4 cryotin 209–11 cyclodextrin glucano transferases (CGTases) 32 dairy products, applications of crosslinking enzymes 118–20 cheese manufacture 121 milk stability 121 protein-based fat replacers 149 set and stirred yoghurts 121 degenerate homoduplex gene family recombination (DHR) 23 degenerate oligonucleotide gene shuffling (DOGS) 23 dextran 258 dietary fibre 215–17 diglyceride (DAG) oils 67, 271–2 enzymatic optimization 277–8 lipase-catalysed production 272–4 process technology 274–7 discovery of industrial enzyme systems background 3–4 technologies 4 method selection 13 screening methodologies 8 expression cloning 10–11 functional biochemical assays 8–9 molecular screening 11–12 primary and secondary screening 10 screening strategies 4 bioinformatics and genomics 6–7 natural diversity 5–6 protein optimization 7–8 1,3-distearoyl-2-mono-olein (SOS) 65 DNA microarrays 12 DNA shuffling 19, 22, 26–8 enantioselectivity 26 enzyme modified cheese (EMC) 194–8 erlose 248–9 expressed sequence tags (EST) 11–12 expression cloning 10–11, 18–19 extracellular enzymes 55

Index fatty acids 62–5 fatty acids, lipase-catalysed harvesting/ enrichment 285–6 future trends 308 harvesting 294–5, 296–7 lipid selectivity 286–94 structured triacylglycerols (STAG) 295–301 multiple reaction step production 307 nutritional and other uses 307–8 single reaction step production 301–6 fermentation techniques 54–6 fish processing, applications of crosslinking enzymes 114–15 flavours and flavour enhancers see also sucrose-derived flavourings future trends 200–2 information sources 202–3 production 183–4 aspartame 190–1 cheese flavour 193–8 chondroitin sulphate 188–90 industrial proteases 185–6 monosodium glutamate (MSG) 186–7 protease classification 184–5 savoury flavours 198–9 vanilla extraction 191–3 yeast extract 199–200, 201 production by immobilized enzymes aspartame 76–7 ester flavour synthesis 75–6 fluorescence activated cell sorting (FACS) 21 fructo-oligosaccharides (FOS) 71, 217–18, 252–3 short-chain (scFOS) 253–4 β-galactosidase 72 ‘generally regarded as safe’ (GRAS) 47 gene mutations 7–8 genetically modified (GM) foods, consumer attitudes 88–9 genome shuffling 26–8 genomics 6–7 β-glucanase 206 glucoamylase 206 gluco-oligosaccharides (GOS) 223–4 glucose isomerase 48 glucose oxidases 102, 108–9, 113, 206 glutathione oxidase 102 glycoside hydrolases 32 hexose oxidases 102, 109, 113 high fructose corn syrup (HFCS) 48, 68–9

317

high-throughput screening (HTS) 24 historical background 3–4, 43–4 honey oligosaccharides 246–7 hydrolytic membrane bioreactors 175 ice-cream, applications of crosslinking enzymes protein-based fat replacers 150–1 immobilized enzyme assay (IDEA) system 74 immobilized enzymes 60–2 flavour production aspartame 76–7 ester flavour synthesis 75–6 future trends 77–8 modification of acylglycerols 62 cocoa butter equivalents 65 diacylglycerols 67 modified triacylglycerols 65–6 phospholipids 66 trans-free oils 62–5 modification of carbohydrates functional oligosaccharides 69–72 high-fructose corn syrup (HFCS) 68–9 lactose hydrolysis 72–3 tagatose 73 modification of proteins protein functionality 74–5 protein hydrolysates 73–4 immunomodulatory peptides 169 improving enzyme performance 16–17 future trends 35 information sources 35–6 laboratory evolution 17–18 enantioselectivity 26 enzyme stability 24–6 genome shuffling 26–8 selection and screening 20–4 techniques 18–20, 22–3 laboratory evolution combined with computational design 34–3 rational and computational protein engineering 28–9 construction of designed sequences 29 enzyme stability 30–2 ligand and substrate specificity 32–3 reaction mechanism 32 in vitro recombination 19 in vitro selection 20–1 in vivo random mutagenesis 22 incremental truncation for the creation of hybrid enzymes (ITCHY) 19–20, 23

318

Index

SCRATCHY 23 intracellular enzymes 54–5 inulin 71, 219–20 isolation of high chain lengths 221–2 novel production 220–1 plant synthesis 220 invert sugar 260 isomalto-oligosaccharides (IMO) 71, 256–7 isomaltulose (palatinose) 71, 244–5 kestose 254 koji process 45, 47 laboratory evolution of enzymes 17–18 combined with computational design 34–3 improvement examples enantioselectivity 26 enzyme stability 24–6 genome shuffling 26–8 selection and screening 20–4 techniques 18–20, 22–3 laccases 102, 105–7, 113 applications in meat and fish processing 117 lactase 206 lactobacilli 27 lactoferricin 167, 168 lactoferrin 167 β-lactoglobulin 74–5 lactose hydrolysis 72–3 lactosucrose 250 lecithin 66 leucrose 246 levan oligosaccharides 254–5, 259 lipases 206 selectivity 286–94 lipids, structured see structured lipids lipoxygenases (LOX) 102, 108, 113 low-fat foods 149–52 lysozyme 206 maltosyl sucrose 250–1 marine proteases 211–12 meat processing, applications of crosslinking enzymes 114–15 heated meat products 115–18 laccases 117 protein-based fat replacers 149 restructured meat products 118 transglutaminase (TG) 116–17 tyrosinases 117 medium chain fatty acids (MCFA) 278–9

common process parameters 279–81 determination of products 281–2 lipase-catalysed interestification 279 reactors for interestification 281 medium chain triacylglycerols (MCT) 65–6 melezitose 247–8 membrane bioreactors 173–6 microbial fermentation 43–4 milk applications of crosslinking enzymes 121 bioactive peptides 170 ACE inhibitors 165 production processes and bioreactors 172–6 proteolytic enzymes 170–2 miso process 44, 45 molecular screening 11–12 molecular weight cut-off (MWCO) membranes 174 monosodium glutamate (MSG) 186–7 mutation of genes 7–8 natural diversity 5–6 nitrosoguanidine 27 non-digestible oligosaccharides (NDO) 217–18 non-starch polysaccharide (NSP) oligosaccharides 230–2 nutritional properties of foods, effects of crosslinking 124–6 oils, trans-free 62–5 oligodextran 234–7 oligonucleotides, synthetic 20 oligosaccharides 69–72 from non-starch polysaccharides (NSP) 230–2 production enzymes 70 opioid peptides 168 oxidative enzymes 104 applications cereals 112–14 glucose and hexose oxidases 108–9 laccases 105–7 lipoxigenases (LOX) 108 peroxidases 107 sulphydryl oxidases 107–8 tyrosinases 104–5 palatinose (isomaltulose) 71, 244–5 1(3)-palmitoyl-3(1)-stearoyl-2-mono-olein (POS) 65 papain 44, 206, 208

Index pasta, applications of crosslinking enzymes 109–10 transglutaminase (TG) 112 pectic-oligosaccharides 233 pectinase 206 pectins 232–4 pepsin 170 peptides, bioactive 160–1 ACE inhibitors 161–3 milk-derived 165 structural implications 163–5 future trends 177 information sources 177 production from milk and whey proteins 170 processes and reactors 172–6 proteolytic enzymes 170–2 types and benefits 165–7 antimicrobial peptides 168–9 antioxidant peptides 169 immunomodulatory peptides 169 opioid peptides 168 periplasmic binding protein (PBP) 32–3 peroxidases 102, 107, 113 phospholipids 66 plant carbohydrates 216 polymerase chain reaction (PCR) random point mutagenesis 18–19, 22 polyunsaturated fatty acids (PUFA) 285, 286 prebiotics 217–18 production of enzymes 43–4 expression system design 46–7 antibiotic resistance markers 53–4 host strain safety 47–8 host strain sporulation 51–2 secretion/accumulation of product 48 side activities and potential contamination 48–9 toxic metabolites 49–51 vector design 52–3 future trends 56 information sources 56 process development 54–6 traditional sources 44–6 promoters 52–3 proteases, cold adapted 205–8 applications 208–9 applications: serine proteases 209–11 future trends 212 modification of marine proteases 211–12 proteases, classification 184–5 protein hydrolysates 73–4

319

protein optimization 7–8 proteinase K 171 protein-based fat replacers 140–2 applications in low-fat foods 149–52 enhancing fat-mimicking properties of proteins 142–3 protein crosslinking 146–8 proteolysis 143–6 future trends 152 proteins, modification by immobilized enzymes protein functionality 74–5 protein hydrolysates 73–4 public opinion see consumer attitudes raffinose 251–2 random chimeragenesis on transient templates (RACHITT) 22 random point mutagenesis 18–19, 22 rational protein engineering 28–9 improvement examples enzyme stability 30–2 ligand and substrate specificity 32–3 reaction mechanism 32 recombinant DNA technology 46 resistant starch (RS) 226–8 reuteran 259 saturation mutagenesis 22 SCRATCHY 23 secretomics 11 sequence homology-independent protein recombination (SHIPREC) 23 sequence independent site-directed chimeragenesis (SISDC) 23 Snamprogetti process 72 soy oligosaccharides 252 soy sauce 198–9 soya oil 62 sporulation of host strains 51–2 stability of enzymes 24–6 laboratory evolution 24–6 rational protein engineering 30–2 stachyose 252 staggered extension process (StEP) 22 starch, resistant (RS) 226–8 structure based combinatorial protein engineering (SCOPE) 23 structured lipids (SL) 270–1, 295–301 diglyceride (DAG) oils 271–2 enzymatic optimization 277–8 lipase-catalysed production 272–4 process technology 274–7 future trends 282

320

Index

medium chain fatty acids (MCFA) 278–9 common process parameters 279–81 determination of products 281–2 lipase-catalysed interestification 279 reactors for interestification 281 multiple reaction step production 307 nutritional and other uses 307–8 single reaction step production 301–6 submerged fermentation 54 subtilisin 171–2 sucrose esters 260–1 sucrose-derived flavourings 244–5 see also flavours and flavour enhancers fructo-oligosaccharides (FOS) 252 inulin 252–3 future trends 261 gluco-oligosaccharides (GOS) 255 alternan oligosaccharides 256 branched 255–6 isomalto-oligosaccharides (IMO) 256–7 glycosyl sucroses erlose 248–9 lactosucrose 250 maltosyl sucrose 250–1 melezitose 247–8 raffinose 251–2 soy oligosaccharides 252 stachyose 252 theanderose 248 verbascose 252 xylsucrose 249 honey oligosaccharides 246–7 information sources 262 levan oligosaccharides 254–5 linkage isomers and polyols 244 isomaltose (palatinose) 244–5 leucrose 246 trehalulose 245–6 other products 260 invert sugar 260 sucrose esters 260–1

polysaccharides 257–8 alternan 258–9 dextran 258 levan 259 reuteran 259 sulphydryl oxidases (SOX) 102, 107–8 suppression subtractive hybridization (SSH) 12 surface fermentation 54 synthetic oligonucleotides 20 tagatose 73 theanderose 248 thermolysin 171 trans-free oils 62–5 transgalacto-oligosaccharides (TOS) 217–18, 222–3 transglutaminase (TG) 102, 103–4 applications baking 110–12 dairy products 119, 120 enhancing fat-mimicking properties of proteins 147 heated meat products 115–18 meat and fish processing 114–15, 116–17 pasta manufacture 112 restructured meat products 118 transposon assisted signal trapping (TAST) 11 trehalulose 245–6 triacylglycerols, modified 65–6 trichothecenes 51 trypsin 170 tyrosinases 102, 104–5, 113 applications in meat and fish processing 117 vanilla 191–3 vanillyl-alcohol oxidase (VAO) 33 verbascose 252 xylsucrose 249