Handbook of Hydrocolloids

Handbook of hydrocolloids Related titles from Woodhead’s food science, technology and nutrition list: New ingredients...

Author: Glyn O. Phillips | Peter A. Williams

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Handbook of hydrocolloids

Related titles from Woodhead’s food science, technology and nutrition list: New ingredients in food processing – Biochemistry and agriculture (ISBN: 1 85573 443 5) G Linden and D Lorient The food industry is now seeing a rapidly expanding primary processing industry manufacturing tailor-made ingredients (or Intermediate Food Products) for the secondary sector. This major new text is an essential reference offering a comprehensive guide to the range of IFPs available, their key benefits (greater flexibility, functionality and more consistent quality) and the ways in which their manufacture can be tailored to the requirements of the food industry. Functional foods – Concept to product (ISBN: 1 85573 503 2) Glenn R Gibson and Christine M Williams Bringing together a range of international experts this book first defines and classifies this complex and evolving field. It then summarises the key work on functional foods and the prevention of disease, from coronary heart disease to cancer and gastrointestinal disorders. Finally, there are a series of chapters on the commercial exploitation of the health benefits of these functional ingredients. Bender’s dictionary of nutrition and food technology Seventh edition (ISBN: 1 85573 475 3) David A Bender and Arnold E Bender The seventh edition provides succinct, authoritative definitions of over 5000 items in nutrition and food technology (an increase of 25% from the previous edition). In addition there is a nutrient composition data for 287 foods. ‘This valuable book continues to fulfil the purpose of explaining to specialists in other fields the technical terms in nutrition and food processing.’ Chemistry and Industry.

Details of these books and a complete list of Woodhead’s food science, technology and nutrition titles can be obtained by: • visiting our web site at www.woodhead-publishing.com • contacting Customer Services (e-mail: [email protected]; fax: +44 (0)1223 893694; tel.: +44 (0)1223 891358 ext. 30; address: Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge CB1 6AH, England) If you would like to receive information on forthcoming titles in this area, please send your address details to: Francis Dodds (address, tel. and fax as above; e-mail: [email protected]). Please confirm which subject areas you are interested in.

Handbook of hydrocolloids Edited by

G. O. Phillips and P. A. Williams

Published by Woodhead Publishing Limited Abington Hall, Abington Cambridge CB1 6AH England Published in North and South America by CRC Press LLC 2000 Corporate Blvd, NW Boca Raton FL 33431 USA First published 2000, Woodhead Publishing Limited and CRC Press LLC ß 2000, Woodhead Publishing Limited The authors have asserted their moral rights. Conditions of sale 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 prior permission in writing from the publishers. The consent of Woodhead Publishing Limited and CRC Press LLC 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 or CRC Press LLC 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 Limited ISBN 1 85573 501 6 CRC Press ISBN 0-8493-0850-X CRC Press order number: WP0850 Cover design by The ColourStudio Project managed by Macfarlane Production Services, Markyate, Hertfordshire Typeset by MHL Typesetting Limited, Coventry, Warwickshire Printed by TJ International, Cornwall, England

Contents

Preface

xiii

List of contributors

............................................................

xv

1 Introduction to food hydrocolloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. A. Williams and G. O. Phillips, North East Wales Institute, Wrexham

1

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thickening characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viscoelasticity and gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synergistic combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrocolloid fibres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 4 7 12 15 16 18 19

2 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Armise´n and F. Galatas, Hispanagar S A, Madrid

21

2.1 2.2 2.3 2.4 2.5 2.6 2.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agar manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical structure of agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gellification of agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21 23 26 28 33 38 39

vi

Contents

3 Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Murphy, National Starch and Chemical, Manchester 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8

41

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status: European label declarations . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 41 42 46 50 54 64 65

4 Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. A. Ledward, University of Reading

67

4.1 4.2 4.3 4.4 4.5 4.6 4.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New gelatin derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 70 76 79 84 85 86

5 Carrageenan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Imeson, FMC Corporation (UK) Ltd

87

5.1 5.2 5.3 5.4 5.5 5.6 5.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 87 89 91 95 101 101

6 Xanthan gum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Sworn, Monsanto (Kelco Biopolymers, Tadworth)

103

6.1 6.2 6.3 6.4 6.5 6.6 6.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103 103 103 105 112 115 115

Contents 7 Gellan gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Sworn, Monsanto (Kelco Biopolymers, Tadworth) 7.1 7.2 7.3 7.4 7.5 7.6 7.7

vii 117

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 117 117 118 124 134 134

8 Galactomannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. C. Wielinga, Maehall AG (Rhodia Food, Kreuzlingen)

137

8.1 8.2 8.3 8.4 8.5 8.6 8.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137 140 144 146 153 153 153

9 Gum arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. A. Williams and G. O. Phillips, North East Wales Institute, Wrexham

155

9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supply and market trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155 155 157 158 161 164 166 168

10 Pectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. D. May, Consultant

169

10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The chemical nature of pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commercial pectin: properties, modification and function . . . . . . . . . . Nutritional and safety aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Legal status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169 169 171 172 175 176 188 188

viii

Contents

11 Milk proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. P. Ennis and D. M. Mulvihill, University College, Cork 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8

189

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The milk protein system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture of milk protein products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional properties of milk protein products . . . . . . . . . . . . . . . . . . . . Food uses of milk protein products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

189 189 193 202 205 213 214 215

12 Cellulosics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. C. F. Murray, Hercules Limited, Reigate

219

12.1 12.2 12.3 12.4 12.5 12.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

219 219 220 220 222 229

13 Tragacanth and karaya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. Weiping, Arthur Branwell and Co. Limited, Epping

231

13.1 13.2 13.3

Gum tragacanth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gum karaya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

231 238 245

14 Xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Nishinari (Osaka City University), K. Yamatoya and M. Shirakawa (Dainippon Pharmaceutical Co. Limited)

247

14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin and manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modification of tamarind seed xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

247 247 250 253 259 261 262 266

15 Curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Nishinari and H. Zhang, Osaka City University

269

15.1 15.2 15.3 15.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Native curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269 270 270 271

Contents 15.5 15.6 15.7 15.8

ix

Functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

271 283 285 285

16 Cereal -glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Morgan, Industrial Research Limited, New Zealand

287

16.1 16.2 16.3 16.4 16.5 16.6 16.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

287 288 291 297 301 305 305

17 Soluble soybean polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Maeda, Fuji Oil Co. Limited, Osaka

309

17.1 17.2 17.3 17.4 17.5 17.6 17.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic material properties and characteristics . . . . . . . . . . . . . . . . . . . . . . . Functions and applications of SSPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

309 309 310 312 314 319 319

18 Bacterial cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Omoto, Y. Uno and I. Asai, San-Ei Gen FFI Inc., Japan

321

18.1 18.2 18.3 18.4 18.5 18.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

321 321 322 324 326 330

19 Microcrystalline cellulose: an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Iijima and K. Takeo, Asahi Chemical Industry Co. Limited

331

19.1 19.2 19.3 19.4 19.5 19.6 19.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of colloidal type microcrystalline cellulose to food systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

331 331 333 335 341 345 346

x

Contents

20 Gums for coatings and adhesives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Nussinovitch, The Hebrew University of Jerusalem 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9 20.10 20.11 20.12 20.13

347

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Today’s edible protective films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parameters to be considered before, during and after food coating Hydrocolloid non-food coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Film-application techniques and stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for testing coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adhesives: non-food uses and applications . . . . . . . . . . . . . . . . . . . . . . . . Adhesives: food uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioadhesives: uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrocolloid adhesion tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrocolloids as wet glues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coatings and adhesion: future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

347 347 351 352 353 353 354 355 356 357 357 361 362

21 Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Terbojevich (University of Padua) and R.A.A. Muzzarelli (University of Ancona)

367

21.1 21.2 21.3 21.4 21.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chitosan chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of chitosans and derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

367 368 370 375 376

22 Alginates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. I. Draget, Norwegian University of Science and Technology

379

22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical and physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties and applications of alginate in the liquid phase . . . . . . . . Gels and gelling technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

379 380 381 384 387 387 393 393

23 FrutafitÕ-inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensus Operations CV, The Netherlands

397

23.1 23.2 23.3 23.4 23.5 23.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FrutafitÕ-inulin and hydrocolloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FrutafitÕ-inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of FrutafitÕ-inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FrutafitÕ-inulin as a gelling agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parameters affecting gel characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . .

397 397 398 398 399 399

Contents 23.7 23.8

xi

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrocolloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

401 403

24 CRC emulsifying biopolymer: a new emulsifier for the soft-drinks industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Lane (Food Science Australia) and E. Chai (University of Melbourne)

405

24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CRC process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product specifications for CRC Biopolymer . . . . . . . . . . . . . . . . . . . . . . . Functional properties and efficacy of CRC Biopolymer . . . . . . . . . . . Emulsion stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composition of CRC Biopolymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patent status of CRC Biopolymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

405 406 406 408 409 410 411 411

25 Konjac mannan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Takigami, Gunma University

413

25.1 25.2 25.3 25.4 25.5 25.6 25.7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

413 415 418 419 422 423 423

26 Philippine Natural Grade or semi-refined carrageenan . . . . . . . . . . . . . . . . . . H. J. Bixler and K. D. Johndro, Ingredients Solutions Inc., Searsport

425

26.1 26.2 26.3 26.4 26.5 26.6 26.7 Index

History of product development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Processing of PNG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical analysis of PNG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The regulatory status of PNG as a food ingredient . . . . . . . . . . . . . . . . Applications of PNG in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

425 426 428 430 432 440 442

.......................................................................

443

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Preface

The growth of the Food Ingredient Exhibitions over the past few years has been quite remarkable. In Paris in 1999 there were 1011 companies exhibiting, which was 6% more than the previous year. When Food Ingredients Central and Eastern Europe, Asia, Latin America and the long standing Institutes of Food Technology, USA are thrown in, there are literally thousands of companies, tens of thousands of technical staff all innovating with new technology to produce healthy food with better texture, shape, visual appeal and taste, and in the process attracting and hopefully satisfying literally millions of customers. The bases of this revolution in food fabrication are the hydrocolloids. Without their ability to achieve scientific water control it would not be possible to improve the body and mouthful of beverages, improve or modify texture, pouring properties and cling in a range of food products. Taking one such hydrocolloid at random, the company producing it claims it can achieve the following properties: • • • • • • • • • • • •

thickening gelling syneresis control emulsion stabilisation pH stability heat stability salt tolerance suspension ability ease of use clarity coating binding.

Not all the hydrocolloids function equally at different pH solutions, electrolyte concentration, thermal treatment or have the same storage ability, etc. Therefore the selection of the particular hydrocolloid for a specific purpose is the task of the food fabricator. In this respect he/she will get endless advice and information through marketing visits and the scores of glossy brochures lauding the company’s products and

xiv

Preface

philosophy. All very helpful of course, but this is no substitute for getting to know in an unbiased manner the properties of the various available hydrocolloids. That is the purpose of this handbook. It is not a textbook. There are plenty of those available, which describe at length the academic aspects and various interactions of the individual hydrocolloids and mixtures of them. This is meant to be a convenience reference to provide the relevant information readily and at the same time authoritatively. The chapters have all been written by top specialists in their fields. The subject does not stay still for a moment. The customer is now not only demanding convenience but also healthier foods. The functional foods, or nutraceuticals, have now come of age. Sales of such foods have tripled in value between 1990 and 1997 to be worth now £3 billion, and the market is forecast to grow from its current level to take 10% of the food sales by the year 2006. In recognition of this growth the year 2000 sees the launch of the exhibition ‘Health Ingredients Europe’ in Germany, which is currently the leading European market in this field, with sales worth around DM3.5 billion annually. Here again the growth would not be possible without the core functionality of the hydrocolloids described in this book. Soluble and non-soluble fibre, which will ensure healthy functioning of the colon through fermentation and/or physical action, can reduce cholesterol, control diabetes, reduce cancer incidence of the colon and give rise to numerous other health claims. How appealing it is to have bakery fillings, dairy drinks and desserts, sauces and gravies, puddings and pie fillings, syrups and toppings – and all healthy and low in calories. To achieve this calls for a deep understanding of the rheology of single and mixed hydrocolloids, with new technologies such as particulated gels or sheared gel providing mixed gelling systems with novel texture, shape, flavour release and visual appeal from two or more gelling phases. The prize is indeed great for the food companies because the European chilled dessert market alone is predicted to grow at 2– 3% per annum from a current value of US$ 12.8 billion. Without question it can prove rewarding to become well aquainted with the hydrocolloids described in this book. The tumultuous growth associated with the revolution already referred to is both confusing and perplexing. The choice of hydrocolloids is larger but fewer and fewer companies are in a position to provide them because as they become larger each company tries to provide the widest possible range of hydrocolloids. Due to the rash of recent mergers a single company can now provide galactomannans, guar and locust bean gum, pectins, alginates, carrageenans, xanthan and gelatin. Added to this, the technological developments are leading to the crossing of traditional boundaries. Carrageenan is challenging the functionality of gelatin. Starch is trying to replicate the behaviour of gum arabic, and so on. Therefore, there is no substitute for studying the hydrocolloids individually and objectively, in order to avoid this over-concentration of expertise and supply. We hope that this handbook will assist in this respect. We welcome any comments or suggestions. We certainly hope that it will assist all levels of reader, from the student to the food scientist, to understand this rapidly growing, enjoyable yet challenging subject. Glyn O. Phillips and Peter A. Williams Centre for Water Soluble Polymers The North East Wales Institute

Contributors

Chapters 1 and 9 Glyn Phillips is currently Chairman of Research Transfer Ltd, Editor of Food Hydrocolloids and Chairman of the Food Hydrocolloids Trust, which organises the so called ‘Wrexham Conferences’ on Gums and Stabilisers for the Food Industry. His research work has centred on polysaccharides and he has published more than 500 papers in leading scientific journals and contributed as Editor or Author to 29 books. He is at present Scientific Adviser to the International Association for the Promotion of Gums and Consultant to a number of industries associated with food additives, medical hydrocolloids and tissue banking. Professor G O Phillips 2 Plymouth Drive Radyr Cardiff CF4 8BL Tel: 01222 843298 Fax: 01222 843298 E-mail: [email protected] Professor Peter Williams Faculty of Science The North East Wales Institute Plas Coch Mold Road Wrexham LL11 2AW Tel: 01978 293083 Fax: 01978 290008 E-mail: [email protected] Chapter 2 Dr Rafael Armisen R + D Director Hispanagar S A Pedro de Valdivia 34 Madrid 28006 Spain

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Tel: 34-91-4117911 Fax: 34-91-5624645 E-mail: [email protected] and [email protected] Chapter 3 Pauline Murphy is European Technical Manager at National Starch and Chemical, responsible for the European Technical Centre, Application Laboratory and Technical Service Group. Pauline has extensive experience of advising and training R&D, product development and production groups on food formulation and processing. She has written technical articles and given many presentations in this area across Europe. A graduate from Kings College, University of London, where she studied food science specialising in chemistry, Pauline is currently focusing on innovation and troubleshooting techniques within sauces, dressings, low fat and organic products – particularly using the functional native starch range NOVATION. Mrs Pauline Murphy National Starch & Chemical Prestbury Court Greencourts Business Park 333 Styal Road Manchester M22 5LW Tel: 0161 435 3200 Fax: 0161 435 3221 Chapter 4 Professor David Ledward is the head of the Department of Food Science & Technology at the University of Reading, UK. His current research interests include the use of high pressures to modify protein functionality and protein-polysaccharide interactions. He has published over 100 papers concerned with aspects of protein chemistry and edited and contributed to several books in the same area. Professor D A Ledward The University of Reading Department of Food Science & Technology PO Box 226 Whiteknights Reading RG6 6AP Tel: 0118 9318715 Fax: 0118 931 0080 E-mail: [email protected] Chapter 5 Dr Alan Imeson is responsible for new business development and technical support in the UK and Ireland for FMC BioPolymer, a major innovative manufacturer of carrageenan, alginates and microcrystalline cellulose. He has edited two internationally renowned books on thickening and gelling agents for food and he is the author of several articles on different hydrocolloids in food. Dr A Imeson 12 Langley Close Epsom Surrey KT18 6HG Tel: 01372 279025 Fax: 01372 279025 E-mail: [email protected]

Contributors xvii Chapters 6 and 7 Dr Graham Sworn is a Senior Research Scientist with Kelco Biopolymers which specialises in the manufacture of hydrocolloids through fermentation. He is the author of a number of papers on hydrocolloids. Current areas of development include high sugar/polysaccharide systems and mixed polysaccharide gels. Dr Graham Sworn Monsanto Waterfield Tadworth Surrey KT20 5HQ Tel: 01737 377000 Fax: 01737 377100 E-mail: [email protected] Chapter 8 Willem Wielinga is a Dutch chemical engineer and textile chemist. From 1959 to 1962 he worked in the potato starch industry (textile sizing). Since 1962 he has worked in the gum industry in development research and applications. He specialised in the field of textile printing and stabilisation of food products including frozen products. He is the author of a number of articles on galactomannan applications. Mr Willem Wielinga Hinterdorfstrasse 41 CH-8274 Taegerwilen Switzerland Tel: +41 71 6692 145 Chapter 10 Dr Colin May was Chief Scientist of Citrus Colloids Ltd, the sole UK pectin producer, until closure in 1998. He is now Executive Secretary of the International Pectin Producers’ Association and a consultant on food additives and quality systems. Dr C D May PO Box 151 Wellington Hereford HR4 8YZ Tel: 01432 830529 Fax: 01432 830716 E-mail: [email protected] Chapter 11 Dr Daniel Mulvihill is Associate Professor of Food Chemistry in the Department of Food Science & Technology, University College Cork. He teaches at undergraduate and postgraduate level in Food Chemistry, and he directs a basic and applied research programme on the physio-chemical characteristics and food applications of proteins and hydrocolloids. He is the author of approximately 100 research and review articles in his research area. Professor D Mulvihill Dept of Food Chemistry University of Cork Cork Ireland Tel: 353-21-276871 (direct dial 353-21-902650) Fax: 353-21-270001 E-mail: [email protected]

xviii

Contributors

Chapter 12 Dr Charles Murray is Sales Manager – Food Gums at Hercules Limited. He has been involved in sales and technical service in food applications of a range of hydrocolloids for some 20 years. Dr J C F Murray Hercules Limited 31 London Street Reigate Surrey RH2 9YA Tel: 01737 242434 Fax: 01737 224287 Chapter 13 Dr Weiping Wang is the technical consultant of Arthur Branwell Company which specialises in hydrocolloid applications. He is the author of 30 articles on exudate gums and their applications. He received his Ph.D degree in Polysaccharide Chemistry from Edinburgh University in 1992 and M.S degree in Food Technology from WXULI, China in 1985. He is also a regular visiting professor of hydrocolloid applications at Fuzhou University, China. Dr Wang Weiping Arthur Branwell & Co Limited Bronte House 58-62 High Street Epping Essex CM16 4AE Tel: 01992 577333 Fax: 01922 561138 E-mail: [email protected] Chapters 14 and 15 Professor Katsuyoshi Nishinari is interested in the gels and gelling processes of polysaccharides and proteins. He is the author of articles and chapters in books on these problems based mainly on rheology, DSC and related physico-chemical methods. Ms Mayumi Shirakawa is the manager of the Food, Food Additives and Chemicals Division of Dainippon Pharmaceutical Co. Ltd which specialises in the manufacture of food polysaccharides. She is the author of articles and chapters in books on enzymology, and structural and rheological properties of xyloglucan. Dr Kazuhiko Yamatoya is the researcher in the Food, Food Additives and Chemicals Division of Dainippon Pharmaceutical Co. Ltd which specialises in the manufacture of food polysaccharides. He is the author of articles and chapters in books on dietary fibre and food structure. Professor Katsuyoshi Nishinari Department of Food and Nutrition Faculty of Human Life Science Osaka City University Sumiyoshi Osaka 558-8585 Japan Tel: +81 6 605 2818 Fax: +81 6 605 3086 E-mail: [email protected]

Contributors

xix

Chapter 16 Dr Keith Morgan was formerly a research scientist with the Carbohydrate Chemistry Team at Industrial Research in New Zealand. He now runs his own business. He has published a number of papers on -glucans and solid-state NMR studies of polysaccharides. Dr Keith Morgan Industrial Research Limited PO Box 31-310 Lower Hutt New Zealand Tel: +64 (0)4 569 0264 Fax: +64 (0)4 569 0055 E-mail: [email protected] Chapter 17 Dr Hirokazu Maeda is the Deputy General Manager of the New Ingredients Division of Fuji Oil Company Limited, manufacturer of many foods and food-ingredients. He researches organic synthesis and polymer chemistry. Current development work includes creation of soluble soybean polysaccharide. Dr Hirokazu Maeda Fuji Oil Co Limited 1 Sumiyoshi-cho Izumisano-shi Osaka 599-8540 Japan Tel: +81 724 63 1751 Fax: +81 724 63 1943 E-mail: [email protected] Chapter 18 Dr Iwao Asai is the Corporate Officer for the Hydrocolloids Division of San-Ei Gen F.F.I., Inc. which specialises in the manufacture of food ingredients such as thickeners, emulsifiers, flavours, colours, sweeteners and stabilisers. He is the author of a number of articles on food gum applications. Dr Iwao Asai San-Ei Gen FFI Inc 1-1-11 Sanwa-cho Toyonaka Osaka 561-8588 Japan Fax: +81 6 333 2076 E-mail: [email protected] Chapter 19 Dr Hideki Iijima is the Senior Research Manager of Asahi Chemical Industry Company Limited, which is one of the leading companies manufacturing microcrystalline cellulose (MCC) for both food, pharmaceutical and industrial uses. He researches the structure and properties of MCC and cellulose membranes. He currently works in the field of interaction between biomaterials and synthetic and cellulose membranes for medical use. Dr Hideki Iijima Senior Research Manager Analytical Research & Computer Science Center Analytical Research Laboratory Asahi Chemical Industry Company Limited

xx

Contributors

2-1 Samejima Fuji 416-8501 Japan Tel: +81 545-62-3186 Fax: +81 545-62-3189 E-mail: [email protected] Mr Kimihiko Takeo is the General Manager of Tohmi Chemicals Plant of Asahi Chemical Industry Company Limited. Microcrystalline cellulose (MCC) is one of the products manufactured in this plant. He has been dedicating himself to the study of physico-chemical properties of MCC since the dawn of MCC and has developed several original MCC grades for Asahi Chemical. He is an expert in powder technology and rheology and the practical application of MCCs in the fields of pharmaceutics and foods. Mr Kimihiko Takeo General Manager Tohmi Chemicals Plant Asahi Chem Ind Company Limited 304 Mizushiri Nobeoka Miyazaki 882-0015 Japan Tel: +81 982-22-6272 Fax: +81 982-22-6277 E-mail: [email protected] Chapter 20 Professor Amos Nussinovitch is the Director of the Biochemistry and Food Science Department at the Hebrew University of Jerusalem. He is the author of more than 85 articles, 15 patents and books most of them dealing with theoretical and practical aspects of hydrocolloid glues, food and cell coatings, cellular solids, immobilization, special textures, capsules and food biotechnology. Professor Amos Nussinovitch Institute of Biochemistry, Food Science & Human Nutrition Faculty of Agricultural Food & Environmental Quality Sciences The Hebrew University of Jerusalem PO Box 12 Rehovot 76100 Israel Tel: 972-8-9489016 Fax: 972-8-9363208 E-mail: [email protected] Chapter 21 Maria Terbojevich is Associate Professor of Organic Chemistry, Faculty of Sciences, University of Padova (Italy). She did her post-doctoral work at the University of Mainz and Max Planck Institut in Munich (Germany), dealing with electron microscopy and protein separation techniques. She has written numerous articles on macromolecular characterisation and chemical modification of biopolymers, such as poly--aminoacids and polysaccharides. In particular her current areas of investigation include cellulose, chitin-chitosan, and natural and semi-synthetic polyelectrolytes. Professor Maria Terbojevich Universita Degli Studi di Padova Dipartimento di Chimica Organica Via Marzolo 1 - 35131 Padova Italy

Contributors

xxi

Fax: +39 (049) 8275239 Dr Riccardo Muzzarelli is Professor of Enzymology in the Faculty of Medicine at the University of Ancona. He has published over 275 articles in international journals, and has written or edited 11 books. He is the Past Chair of the European Chitin Society. Professor Riccardo Muzzarelli Centre for Innovative Biomaterials Faculty of Medicine Univerity of Ancona Via Ranieri 67 IT-60100 Ancona Italy Fax: +39 (071) 2204683 E-mail: [email protected] Chapter 22 Dr Kurt Ingar Draget is a senior research scientist at the Norwegian Biopolymer Laboratory (NOBIPOL), NTNU, a department with 50 years of continuous alginate research. His research experience ranges from authorship of several articles covering basic aspects of alginate functionality through numerous alginate based industrial development projects. His core alginate interest is in the characterisation of gels and gelling kinetics. Dr Kurt Ingar Draget Norwegian Biopolymer Laboratory (NOBIPOL) Department of Biochemistry Norwegian University of Science and Technology Sem Saelands vei 6/8 N-7491 Trondheim Norway Tel: +47 73598260 Fax: +47 73591283 E-mail: [email protected] Chapter 23 Sensus is a Dutch company which markets FrutasunÕ fructose syrups and FrutafitÕ-inulin. FrutafitÕ-inulin is Sensus’ innovative food ingredient. FrutafitÕ-inulin is a prebiotic soluble dietary fibre with texturising properties. Sensus is also part of the Dutch COSUN co-operation. The core business of Cosun are sugar and customer specific food ingredients. Sensus Operations Oostelijke Havendijk 15 4704 RA Roosendaal PO Box 1308 4700 BH Roosendaal The Netherlands Tel: +31 165 582 500 Fax: +31 165 55 13 52 E-mail: [email protected] Web: www.sensus.nl Chapter 24 Dr Alan Lane has had extensive experience in developing commercial fermentation-based processes for a variety of products, including animal and human vaccines, antibiotics and anticancer compounds. He was instrumental in the establishment of the CRC for Industrial Plant Biopolymers in 1992 and was its Director of Process Development Research, until his retirement in 1999.

xxii

Contributors

The CRC for Industrial Plant Biopolymers School of Botany University of Melbourne Parkville 3052 Australia Chapter 25 Dr S Takigami Associate Professor Technical Research Center for Instrumental Analysis Gunma University Kiryu Gunma 376-8515 Japan E-mail: [email protected] Chapter 26 Dr Harris J. Bixler and Dr Kevin D. Johndro Ingredient Solutions, Inc. 33 Mt Ephraim Road Searport ME 04974 USA Tel: 207 548 2636 Fax: 207 548 2921 E-mail: [email protected]

1 Introduction to food hydrocolloids P. A. Williams and G. O. Phillips, North East Wales Institute, Wrexham

1.1

Introduction

The term ‘hydrocolloids’ refers to a range of polysaccharides and proteins that are nowadays widely used in a variety of industrial sectors to perform a number of functions including thickening and gelling aqueous solutions, stabilising foams, emulsions and dispersions, inhibiting ice and sugar crystal formation and the controlled release of flavours, etc. The commercially important hydrocolloids and their origin are given in Table 1.1. Table 1.1

Source of commercially important hydrocolloids

Botanical trees cellulose tree gum exudates gum arabic, gum karaya, gum ghatti, gum tragacanth plants starch, pectin, cellulose seeds guar gum, locust bean gum, tara gum, tamarind gum tubers konjac mannan Algal red seaweeds agar, carrageenan brown seaweeds alginate Microbial xanthan gum, curdlan, dextran, gellan gum, cellulose Animal Gelatin, caseinate, whey protein, chitosan

2

Handbook of hydrocolloids

Fig. 1.1

Examples of food products containing hydrocolloids.

The food industry, in particular, has seen a large increase in the use of these materials in recent years. Even though they are often present only at concentrations of less than 1% they can have a significant influence on the textural and organoleptic properties of food products. Some typical examples of foods containing hydrocolloids are shown in Fig. 1.1. The baked beans and hoi-sin sauce contain modified corn starch as a thickener while guar gum is used to thicken the sweet and sour sauce. The Sunny Delight fruit drink contains modified starch as an emulsifier with carboxymethyl cellulose (CMC), and xanthan gum as thickeners. The Italian dressing includes xanthan gum as a thickener and the ‘Light’ mayonnaise contains guar gum and xanthan gum as fat replacers to enhance viscosity. The yoghurt incorporates gelatin as a thickener rather

Introduction to food hydrocolloids Table 1.2

3

Price of the major hydrocolloids

Hydrocolloid

Principal function

Cost $/kg Cost $/kg Cost $/kg in 1983* in 1993** in 1999***

Agar Alginate Arabic Carrageenan Processed euchema seaweed Carboxymethyl cellulose Hydroxypropyl cellulose Methyl cellulose

Gelling agent Gelling agent Emulsifier Gelling agent Gelling agent Thickener Thickener and emulsifier Thickener, emulsifier and gelling agent Thickener and gelling agent Gelling agent Thickener Thickener Thickener Gelling agent Gelling agent Emulsifier and foam stabiliser Thickener and gelling agent Thickener and gelling agent Thickener Thickener

15.0–15.4 19.72 7.7–9.9 6.58 2.64 3.69 5.5–13.2 7.35

Microcrystalline cellulose Gelatin Guar gum Karaya Locust bean gum Pectin Pectin (low methoxy) Propylene glycol alginate Starch Starch (modified) Tragacanth Xanthan gum

3.5–4.4 6.6–8.3 6.6

3.18

3.9–4.3 4.4 4.04 1.0–1.1 0.77 4.6 2.89 4.6 6.40 7.6 9.19 10.6 9.1 0.5 1.3 26.4–35.2 9.60 13.4

8–17.6 8 4.8–8 9.6–11.2 5.72–9.02 2.86 11.2–16

13.64

Sources: * R. G. Morley, in Gums and Stabilisers for the Food Industry 2 eds G. O. Phillips, D. J. Wedlock and P. A. Williams, Pergamon Press (1984) p. 211. ** US Department of Commerce. *** Suppliers.

than a gelling agent while the mousse contains modified maize starch as a thickener with guar gum, carrageenan and pectin present as ‘stabilisers’. The Bramley apple pies contain modified maize starch with sodium alginate as a gelling agent. The fruit pie bars contain gellan gum and the blackcurrant preserve and redcurrant jelly contain pectin as gelling agents. The trifle contains xanthan gum, sodium alginate and locust bean gum as ‘stabilisers’, modified maize starch as a thickener and pectin as a gelling agent. The changes in modern lifestyle, the growing awareness of the link between diet and health and new processing technologies have led to a rapid rise in the consumption of ready-made meals, novelty foods and the development of high fibre and low-fat food products. In particular, numerous hydrocolloid products have been developed specifically for use as fat replacers in food. This has consequently led to an increased demand for hydrocolloids. Today the world hydrocolloids market is valued at around $4.4 billion p.a. with a total volume of about 260,000 tonnes. Growth through the 1990s has been at the rate of 2–3%. Hydrocolloid selection is dictated by the functional characteristics required but is inevitably influenced by price (Table 1.2) and security of supply. It is for these reasons that starches are the most commonly used thickening agents. It is interesting to note here, however, that xanthan gum, since its introduction in the early 1970s, has become the thickener of choice in many applications despite its high price. This is due to the fact that xanthan gum has unique rheological behaviour. It forms highly viscous, highly shear

4

Handbook of hydrocolloids

Fig. 1.2

(a) Value of world market for individual hydrocolloids. (b) Volume of world market for individual hydrocolloids.

thinning solutions at very low concentrations and the viscosity is not influenced to any great extent by changes in pH, the presence of salts and temperature. The high viscosity at low shear enables the gum to prevent particle sedimentation and droplet creaming and the shear thinning characteristics ensure that the product readily flows from the bottle after shaking. Hence its widespread application in sauces and salad dressings. Gelatin is by far the most widely used gelling agent although with the increasing demand for non-animal products, and in particular the recent BSE outbreak in the UK, prices have increased significantly over the last few years. There is currently much activity in the development of a gelatin replacement. The carrageenan market has also been unstable over recent years due to the introduction of cheaper lower-refined grades (Processed Euchema Seaweed, PES), which can compete effectively with the traditional purified grades in applications where gel clarity is not important. It is likely that the price difference between carrageenan and PES will decrease in time with the development of cheaper purification processes. The gum arabic market has been particularly erratic due to extreme price fluctuations and security of supply and much effort has been directed at finding alternatives. A number of starch-based substitutes are now available. Gellan gum, approved for food use in Japan in 1988 and later in the USA and more recently in Europe, is beginning to establish its own niche market. An overview of the hydrocolloids market is given in Figs 1.2(a) and 1.2(b).

1.2

Regulatory aspects

Food hydrocolloids do not exist as a regulatory category in their own right, that is, referring strictly to the use and marketing of any given hydrocolloid. Rather they are regulated either as a food additive or as a food ingredient. With the exception of gelatin, however, the vast majority of hydrocolloids are currently regulated as food additives.

1.2.1 International The most widely accepted fully international system to regulate the safety of food additives is that set up by a Joint FAO/WHO Conference on Food Additives in September 1955, which recommended that the two organisations collect and disseminate information on food additives. Since that time more than 600 substances have been evaluated and provided with specifications for purity and identity by the Joint/WHO Expert Committee on Food Additives (JECFA).

Introduction to food hydrocolloids

5

JECFA was first established in the mid-1950s by FAO and WHO to assess chemical additives in food on an international basis. In the early 1960s the Codex Alimentarius Commission (CAC), an international inter-governmental body, was set up with the primary aim of protecting the health of the consumer and facilitating international trade in food commodities. When CAC was formed, it was decided that JECFA would provide expert advice to Codex on matters relating to food additives. A system was established whereby the Codex Committee on Food Additives and Contaminants (CCFAC), a general sub-committee, identified food additives that should receive priority attention, which were then referred to JECFA for assessment before being considered for inclusion in Codex Food Standards. Specialists invited to serve as Members of JECFA are independent scientists who serve in their individual capacities as experts and not as representatives of their governments or employers. The objective is to establish safe levels of intake and to develop specifications for identity and purity of food additives. The reports of the JECFA meetings are published in the WHO Technical Report Series. The toxicological evaluations, which summarise the data that serve as the basis for safety assignments, are published in the WHO Food Additive Series. The specifications are published in the FAO Food and Nutrition Paper Series. The procedure, therefore, is for JECFA to consider the specification of any given additive and to recommend to the Codex Committee on Food Additives and Contaminants (CCFAC) that this be adopted. If they agree after further consideration by all Member States at a Plenary Session, the specification can be confirmed by the full Codex Commission. The ultimate for a food additive, therefore, is to be included into the Codex General Standard for Food Additives. The procedure can prove lengthy, controversial and expensive, since all interested parties can input objections or amendments. However, once accepted the food additive has world-wide currency. The detailed procedures are described in Codex Alimentarius (General Requirements) second edition (revised 1995). Thus Codex Alimentarius is a collection of internationally adopted food standards presented in a uniform manner. The food standards aim at protecting consumers’ health and ensuring fair practice in the food trade. Once accepted, an international number is allocated to the additive which is an acknowledgement of its acceptability. It must be stressed that there are food additives which stay at the JECFA specification level and that this advisory specification is the authority for its use, at the conditions given, until the full acceptance by Codex is given. Gum arabic is one such example, a food hydrocolloid which has been used for more than 2000 years but only finally gained full Codex specification in June 1999. 1.2.2 European system Clearance of food hydrocolloids by the European Commission was first introduced in 1995 under Directive 95/2/EU for Food Additives other than colours and sweeteners. This is known as the Miscellaneous Additives Directive (MAD), which provides authorisation for a large number of additives from the hydrocolloid group. The majority of these are authorised for general use in foods to Quantum Satis (QS) levels given in Annex 1 of the directive. Starches, the vast majority of gums, alginates and celluloses enjoy this wide authorisation. Almost immediately following adoption of the original Directive the Commission began working on proposed amendments, largely to take account of market developments that had not been taken into account in the last stages of the lengthy and complicated legislative process. The historical development of the process must be referred to in order

6

Handbook of hydrocolloids

to understand the almost unintelligible machinery adopted by the European Commission in its work. The ground rules for food additives harmonisation were set out in the form of a framework Directive, 89/107/EEC adopted in 1988 (and amended by the European Parliament and Council Directive 94/34/EC). It instructs the Council to adopt in subsequent follow-up Directives • a list of additives to be authorised • a list of foods to which the additives may be added and the levels of use, which gives delegated powers to the Commission to adopt • specifications for each additive • where necessary, methods of analysis and procedures for sampling. A number of general criteria for the use of additives in food are also set out. According to the criteria, food additives may be authorised only if • a reasonable technological need can be demonstrated • they present no hazard to health at the levels proposed • they do not mislead the consumer. Evidence of the need for an additive which, incidentally, plays no part in approvals in the USA, must be provided by the user of the additive, that is, the food manufacturer, not the supplier or manufacturer of the additive. The criteria also stipulate that all food additives must be kept under ‘continuous observation and re-evaluated whenever necessary in the light of changing conditions of use and new scientific observation’. The EC process acknowledges the JECFA system and in the most unintelligible legal language adopted by the Commission adds the following: • Whereas Directive 78/663/EEC should be repealed accordingly: • Whereas it is necessary to take into account the specification and analytical techniques for food additives as set out in the Codex Alimentarius as drafted by JECFA: • Whereas food additives, if prepared by production methods or starting materials significantly different from those included in the evaluation of the Scientific Committee for Food, or if different from those mentioned in this Directive, should be submitted for evaluation by the Scientific Committee for Food for the purposes of a full evaluation with emphasis on the purity criteria. It is surprising that any progress was made at all with all the accompanying bureaucracy. After a most highly political first amendment to the MAD intended to clear Processed Euchema Seaweed (E407a) (see Directive 96/85/EC of 19 December 1996), a second amendment was introduced which affected many hydrocolloids. Member States are required to implement the provisions of this Directive by 4 May and some by November 2000. At this time the new authorisation introduced by the current Directive 98/72/EC will come into force in all EU Member States. This Directive provides for E401 Sodium alginate, E402 Potassium alginate and E407 Carrageenan, E440 Pectin, E425 Konjac and E412 Guar. Each has controlling conditions associated with the approval.

1.2.3 Other trade blocks While the Codex Alimentarius Commission is the ultimate specification and can provide for approval throughout the world, each country (outside the EU) is free to adopt its own standards. In the USA, for example, the United States Food Chemical Codex also has

Introduction to food hydrocolloids

7

currency. The Food Chemicals Codex (FCC) is an activity of the Food and Nutrition Board of the Institute of Medicine that is sponsored in the United States Food and Drug Administration (FDA). The current specification of hydrocolloids is to be found in the fourth edition (1996). Japan too has its own specifications which include many of the food additives particular to Japan.

1.2.4 International numbering system for food additives (INS) INS is intended as an identification for food additives for use in one or more member countries. The criteria for INS inclusion (para 90, Alinorm 91/12–22nd FAC) are: • The compound must be approved by a member country as a food additive. • The compound must be toxicologically cleared for use by a member country. • The compound must be required to be identified on the final product label by a member country. There is an equivalence with the EU system of E numbers, albeit that the EU system is more restricted. Where both INS and E numbers are available they are interchangeable. The current position is given below in Tables 1.3 and 1.4 for the hydrocolloids given in this handbook.

1.3

Thickening characteristics

Hydrocolloids are widely used to thicken food systems and a much clearer understanding of their rheological behaviour has been gained over the last twenty years or so particularly through the development of controlled stress and strain rheometers capable of measuring to very low shear rates (< 10ÿ3 sÿ1). The viscosity of polymer solutions shows a marked increase at a critical polymer concentration, commonly referred to as C*, Table 1.3

INS and E numbers for modified starches

Modified starch* Dextrin (roasted starch) Acid treated starch Alkali treated starch Bleached starch Oxidised starch Monostarch phosphate Distarch phosphate Phosphated distarch Acetylated starch Starch acetate Acetylated distarch adipate Hydroxypropyl starch Hydroxypropyl distarch phosphate Starch sodium octenyl succinate Starch, enzyme treated

INS/E number 1400 1401 1402 1403 1404 1410 1412 1413 1414 1420 1422 1440 1442 1450 1405

The Codex General Standard for labelling of pre-packaged foods specifies that modified starches may be declared as such in a list of ingredients. However, certain countries require specific identification and use these numbers.

8

Handbook of hydrocolloids

Table 1.4

INS and E numbers for hydrocolloids

Hydrocolloid

INS/E number* Function

Carrageenan (including 407 Thickener, gelling agent, stabiliser, emulsifier furcelleran) Processed euchema seaweed 407a Thickener, gelling agent, stabiliser, emulsifier Xanthan gum 415 Thickener, stabiliser, emulsifier, foaming agent Gellan gum 418 Thickener, gelling agent and stabiliser Guar gum 412 Thickener, stabiliser and emulsifier Locust bean gum 410 Thickener, gelling agent Gum arabic 414 Emulsifier, stabiliser, thickener Pectin 440/E440 (i) Gelling agent, thickener, stabiliser Amidated pectin E440 (ii) Gelling agent, thickener, stabiliser Microcrystalline cellulose INS460 (i) Anticake, emulsifier, stabiliser and dispersing agent Powdered cellulose INS460(ii)/E460 Anticake, emulsifier, stabiliser and dispersing agent Cellulose 460 Anticake, emulsifier, stabiliser and dispersing agent Tragacanth gum 413 Emulsifier, stabiliser, thickening agent Karaya gum 416 Emulsifier, stabiliser and thickening agent Konjac flour Gelling agent, thickener, emulsifier, stabiliser Konjac mannan E425 Gelling agent, thickener, emulsifier, stabiliser Propylene glycol alginate 405 Thickener, emulsifier Sodium alginate 401 Thickening agent, stabiliser Potassium alginate 402 Thickening agent, stabiliser Alginic acid 400 Thickening agent, stabiliser Calcium alginate 404 Thickening agent, stabiliser Ammonium alginate 403 Thickening agent, stabiliser Methyl cellulose 461 Thickening agent, emulsifier, stabiliser Hydroxypropyl cellulose 463 Emulsifier, thickener, stabiliser, binder, suspension agent, film coating Hydroxypropyl methyl cellulose 464 Emulsifier, thickening agent, stabiliser Sodium carboxymethyl cellulose 466 Thickening agent, stabiliser, suspending agent Agar 406 Thickening agent and stabiliser Oat gum 411 Thickener, stabiliser * Where no distinction is made the INS and E numbers are identical. When only an E number is given, there is no equivalent INS number. Those hydrocolloids not included are either classed as food ingredients (e.g. gelatin) or have yet to complete the regulatory process.

corresponding to the transition from the so-called ‘dilute region’, where the polymer molecules are free to move independently in solution without interpenetration, to the ‘semi-dilute region’ where molecular crowding gives rise to the overlap of polymer coils and interpenetration occurs. Polysaccharide solutions normally exhibit Newtonian behaviour at concentrations well below C*, i.e., their viscosity is independent of the rate of shear. However, above C* nonNewtonian behaviour is usually observed. A typical viscosity–shear rate profile for a polymer solution above C* is given in Fig. 1.3 and shows three distinct regions: (a) a lowshear Newtonian plateau; (b) a shear-thinning region, and (c) a high-shear Newtonian plateau. At low shear rates the rate of disruption of entanglements is less than the rate of re-entanglement and hence viscosity is independent of shear. Above a critical shear rate, disentanglement predominates and the viscosity drops to a minimum plateau value at infinite shear rate. A number of empirical mathematical models have been developed to describe the flow characteristics. The most widely used model to describe the whole shear rate range is the Cross equation.

Introduction to food hydrocolloids

Fig. 1.3

Fig. 1.4

9

Typical viscosity–shear rate profile for a polymer solution above C*.

Viscosity–shear rate profiles for 1% solutions of guar gum of varying molecular mass.

1

0 ÿ 1 m

1

where is the viscosity at infinite shear rate, 1 is the infinite shear viscosity, 0 is the zero shear viscosity, is a shear-dependent constant denoting the onset of shear thinning,

is shear rate and m is an exponent quantifying the degree of shear thinning. m has a value of 0 for a Newtonian solution and increases to 1 with increased shear thinning. The viscosity of polymer solutions is influenced significantly by the polymer molecular mass as illustrated in Fig. 1.4 which gives the shear viscosity of a series of guar

10

Handbook of hydrocolloids

Fig. 1.5 Viscosity–shear rate profiles for 1% CMC, 1% HEC, 20% dextran and 30% gum arabic.

gum samples as a function of shear rate. The viscosity shear rate dependency increases with increasing molecular mass and the shear rate at which shear thinning occurs shifts to lower values. C* is also a function of the molecular mass of the polymer and is given by the following relationship: C a=

2

where a is an integer which varies for different polysaccharides. For random coils a has a reported value of between 1 and 4. In addition to molecular mass effects, the hydrodynamic size of polymer molecules in solution is significantly influenced by molecular structure. Linear, stiff molecules have a larger hydrodynamic size than highly branched, highly flexible polymers of the same molecular mass and hence give rise to a much higher viscosity. This is illustrated in Fig. 1.5 which shows the viscosity shear rate profiles for CMC, hydroxyethylcellulose (HEC), dextran and gum arabic. The cellulosic polymers are relatively stiff molecules and at 1% are close to or above C*. Consequently they have a high viscosity at low shear rates with the viscosity decreasing with increasing shear to a low minimum value. Dextran is slightly branched and has a very flexible structure due to the (1, 6) glycosidic linkages while gum arabic is very highly branched. Both, therefore, are compact and have relatively small radii of gyration for their molecular mass compared to the linear cellulosic polymers. Significant interpenetration does not occur even at concentrations of 20–30% and hence their viscosity–shear rate profiles exhibit Newtonian characteristics. It should be noted, however, that although the viscosity of these concentrated solutions is much less than the viscosity of the 1% cellulosic solutions at low shear, they are greater at high shear rates. The stiffness of the polysaccharide chains also has a very pronounced influence on the shear thinning characteristics of the polysaccharide solutions as illustrated in Fig. 1.6 which gives the viscosity–shear rate profile for 1% xanthan gum (persistence length, q,

Introduction to food hydrocolloids

Fig. 1.6

11

Viscosity–shear rate profiles for solutions of 1% xanthan gum, 1% CMC and 5% sodium polyacrylate.

> 100nm), 1% CMC (q ~ 10–30nm), and 5% sodium polyacrylate (q < 10nm). The slopes of the shear thinning section of the curves decrease in the order xanthan > CMC > sodium polyacrylate indicating that the extent of shear thinning increases dramatically with increasing polymer persistence length. Charged polymers have a higher viscosity than non-ionic polymers of similar molecular mass due to the fact that their molecular coils are expanded due to intramolecular charge repulsions. Addition of electrolyte or adjustment of the pH to reduce the degree of dissociation of the charged groups normally leads to compaction of the coils and a significant drop in viscosity. Interestingly, CMC and xanthan gum show atypical behaviour with viscosity actually showing an increase on addition of electrolyte. The main hydrocolloid thickeners used in food products are listed in Table 1.5. Table 1.5

Main hydrocolloid thickeners

Xanthan gum Very high low-shear viscosity (yield stress), highly shear thinning, maintains viscosity in the presence of electrolyte, over a broad pH range and at high temperatures. Carboxymethyl cellulose High viscosity but reduced by the addition of electrolyte and at low pH. Methyl cellulose and hydroxypropyl methyl cellulose Viscosity increases with temperature (gelation may occur) not influenced by the addition of electrolytes or pH. Galactomannans (guar and locust bean gum) Very high low-shear viscosity and strongly shear thinning. Not influenced by the presence of electrolyte but can degrade and lose viscosity at high and low pH and when subjected to high temperatures.

12

1.4

Handbook of hydrocolloids

Viscoelasticity and gelation

Hydrocolloid solutions are viscoelastic and can be characterised by the magnitude and frequency dependence of the storage and loss modulii, G0 and G00 respectively. In dilute solutions below C* where intermolecular entanglement does not occur, most polymers show G00 greater than G0 over much of the frequency range. Both G0 and G00 show significant frequency dependence, G00 is proportional to the frequency, !, while G0 is proportional to !2, hence at higher frequencies G0 > G00 . At higher concentrations, in the entanglement region above C*, G0 and G00 are still frequency dependent but G0 is greater than G00 over a broader range of frequencies. This is illustrated in Fig. 1.7 which shows the frequency dependence for guar gum solutions at concentrations of 0.5% and 2.0%. Some polysaccharides, notably xanthan gum, have a tendency to undergo weak intermolecular chain association in solution leading to the formation of a threedimensional network structure. The junction zones formed can be readily disrupted even at very low shear rates and the network structure is destroyed. In these systems G0 > G00 over a broad frequency range and both have a reduced frequency dependence. This is illustrated in Fig. 1.8(a) which shows the mechanical spectrum for a 1% solution of xanthan gum and is typical for ‘weak gels’. Other polysaccharides, for example amylose, agarose, carrageenan and gellan gum, can form stable intermolecular regions of association (referred to as junction zones) and as a consequence strong gel structures are produced. For these systems G0 G00 and both are virtually independent of frequency (Fig. 1.8(b)). Hydrocolloid gels are referred to as ‘physical gels’ because the junction zones are formed through physical interaction, for example, by hydrogen bonding, hydrophobic association, cation-mediated crosslinking, etc., and differ from synthetic polymer gels which normally consist of covalently crosslinked polymer chains. Some hydrocolloids form thermoreversible gels and examples exist where gelation occurs on cooling or heating. Some form non-thermoreversible gels. In such cases gelation may be induced by crosslinking polymer chains with divalent cations. Gels may be optically clear or turbid and a range of textures can be obtained. Gel formation occurs above a critical minimum concentration which is specific for each hydrocolloid. Agarose,

Fig. 1.7 G0 and G00 of 0.5% and 2.0% guar gum solutions as a function of frequency.

Introduction to food hydrocolloids

Fig. 1.8

13

(a) G0 and G00 of 1% xanthan gum solution as a function of frequency. (b) G0 and G00 of 1.5% amylose gels as a function of frequency.

for example, will form gels at concentrations as low as 0.2%, while for acid-thinned starch, a concentration of ~15% is required. Typically, however, concentrations of less than 1% are required. Gel strength increases with increasing concentration. Molecular mass is also important. It has been shown that gel strength increases significantly as molecular mass increases up to ~100,000 but then becomes independent of molecular mass at higher values. The principal hydrocolloid gelling agents are listed below in Table 1.6 and a comparison of their relative gel textures is illustrated in Fig. 1.9. An increase in

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Handbook of hydrocolloids

Table 1.6

Main hydrocolloid gelling agents

1. Thermoreversible gelling agents Gelatin Gel formed on cooling. Molecules undergo a coil-helix transition followed by aggregation of helices. Agar Gel formed on cooling. Molecules undergo a coil-helix transition followed by aggregation of helices. Kappa Carrageenan Gel formed on cooling in the presence of salts notably potassium salts. Molecules undergo a coilhelix transition followed by aggregation of helices. Potassium ions bind specifically to the helices. Salts present reduce electrostatic repulsion between chains promoting aggregation. Iota Carrageenan Gel formed on cooling in the presence of salts. Molecules undergo a coil-helix transition followed by aggregation of helices. Salts present reduce electrostatic repulsion between chains promoting aggregation. Low methoxyl (LM) pectin Gels formed in the presence of divalent cations, notably calcium at low pH (3–4.5). Molecules crosslinked by the cations. The low pH reduces intermolecular electrostatic repulsions. Gellan gum Gels formed on cooling in the presence of salts. Molecules undergo a coil-helix transition followed by aggregation of helices. Salts reduce electrostatic repulsions between chains and promote aggregation. Multivalent ions can act by crosslinking chains. Low acyl gellan gels are thermoreversible at low salt concentrations but non-thermoreversible at higher salt contents (> 100mM) particularly in the presence of divalent cations. Methyl cellulose and hydroxypropyl methyl cellulose Gels formed on heating. Molecules associate on heating due to hydrophobic interaction of methyl groups. Xanthan gum and locust bean gum or konjac mannan Gels formed on cooling mixtures. Xanthan and polymannan chains associate following the xanthan coil-helix transition. For locust bean gum the galactose deficient regions are involved in the association. 2. Thermally irreversible gelling agents Alginate Gels formed on the addition of polyvalent cations notably calcium or at low pH (< 4). Molecules crosslinked by the polyvalent ions. Guluronic acid residues give a buckled conformation providing an effective binding site for the cations (egg box model). High methoxyl (HM) pectin Gels formed at high soluble solids (e.g. 50% sugar) content at low pH < 3.5. The high sugar content and low pH reduce electrostatic repulsions between chains. Chain association also encouraged by reduced water activity. Konjac mannan Gels formed on addition of alkali. Alkali removes acetyl groups along the polymer chain and chain association occurs. Locust bean gum Gels formed after freezing. Galactose deficient regions associate.

Introduction to food hydrocolloids

Fig. 1.9

15

Qualitative comparison of the textures of gels produced by different hydrocolloids.

brittleness is usually accompanied by an increase in the tendency to undergo syneresis and is attributed to an increase in the degree of aggregation of molecular chains.

1.5

Synergistic combinations

Mixtures of hydrocolloids are commonly used to impart novel and improved rheological characteristics to food products and an added incentive is a reduction in costs. Classic examples include the addition of locust bean gum to kappa carrageenan to yield softer more transparent gels and also the addition of locust bean gum to xanthan gum to induce gel formation. The nature of the synergy can be due to association of the different hydrocolloid molecules or to non-association. The various effects that can occur are summarised schematically in Fig. 1.10. If the two hydrocolloids associate then precipitation or gelation can occur. Oppositely charged hydrocolloids (e.g., a protein below its isoelectric point and an anionic polysaccharide) are likely to associate and form a precipitate while there is evidence to show that for some stiff polysaccharide molecules (e.g., the examples noted above) association results in gel formation. If the two hydrocolloids do not associate, as is commonly the case, then at ‘low’ concentrations they will appear to exist as a single homogeneous phase while at higher concentrations they will separate in time into two liquid phases each enriched in one of the hydrocolloids. The phase separation process involves the formation of ‘water-in-water’ emulsions which consist of droplets enriched in one hydrocolloid dispersed in a continuous phase enriched in the other. Whether the hydrocolloid is present in the dispersed or continuous phase depends on the relative concentrations. If either or both of the hydrocolloids can form gels independently then phase separation and gelation will occur simultaneously. The characteristics of the resultant gel will depend on the relative rates of these two

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Handbook of hydrocolloids

Fig. 1.10

Schematic representation of the interactions which occur in solutions containing mixtures of hydrocolloids.

processes. Careful selection of hydrocolloid type and concentration can, therefore, lead to the formation of a broad range of gel textures and this is currently an area receiving considerable attention.

1.6

Hydrocolloid fibres

Because there is a growing belief throughout the world that natural fibre foods are an integral part of a healthy lifestyle, food producers source an increasing proportion of their raw materials from nature itself. There is a growing demand from an increasingly healthconscious consumer for reduced fat and enhanced fibre foods of all types. If this can be achieved using materials that have low calorific value, further health benefits will result. Foods containing such ingredients will need to match the quality of the original product and without adverse dietary effects. This target cannot be achieved without the scientific use of thickeners, stabilisers and emulsifiers, particularly of the ‘natural type’. This calls for fibres, which can interact with water to form new textures and perform specific functions, which itself requires the use of hydrocolloids. In 1998 the world market for such hydrocolloids of the fibre type was 2.83 million US dollars and is set to grow significantly to meet the health aspirations of the consumer in the next millennium. It is the task of the food scientist to provide the hydrocolloids in the most appropriate form for inclusion in the food product. This requires an understanding of their structure and the way in which they act to produce the desired function in the food. Dietary fibre was first described as the skeletal remains of plant cell walls that are resistant to hydrolysis by the digestive enzymes of man. Since this excluded polysaccharide fibres in the diet, the definition was subsequently expanded to include all polysaccharides and lignin, which are not digested by the endogenous secretions of the human digestive tract. Dietary fibre thus mainly comprises non-starch polysaccharides, and indeed has been defined by Englyst and others as the ‘polysaccharides which are resistant to the endogenous enzymes of man’. Industrialised countries now generally

Introduction to food hydrocolloids

17

recognise the health-giving properties of increased consumption of fibre and reduced intakes of total and saturated fat. In this respect ‘fibre’ is used in a non-specific way, but is generally taken to mean structural components of cereals and vegetables. More recently the concept of ‘soluble fibre’ has emerged which assists plasma cholesterol reduction and large-bowel fermentation. The physical and fibre properties of such soluble and insoluble fibres allow them to perform both in a physical role and also to ferment through colonic microflora to give short-chain fatty acids (SCFA), mainly acetate, propionate and butyrate. These have a very beneficial effect on colon health through stimulating blood flow, enhancing electrolyte and fluid absorption, enhancing muscular activity and reducing cholesterol levels. The various hydrocolloids which are described in this handbook fall into this category.

1.6.1 Physical effect Dietary fibre to be effective must be resistant to the enzymes of the human and animal gastrointestinal tract. If physically suitable it can work effectively as a result of its bulking action. In the stomach and small intestine the fibre can increase digested mass, leading to faecal bulking, which readily explains the relief of constipation, which is one of fibre’s best-documented effects. It can increase stool mass and ease laxation very efficiently. This behaviour has considerable human and agricultural importance. The growth of the ruminant animal depends on the fermentable fibre content of the stockfeed. Soluble as well as non-soluble fibres exert their actions in the upper gut through their physical properties. Those which form gels or viscous solutions, can slow down the transit in the upper gut and delay glucose absorption, best explained in terms of ‘viscous drag’. Thus the reduction in glycemic response by soluble fibres can be explained.

1.6.2 Fermentation product effects Large bowel micro-organisms attack the soluble fibres, in fermentation resembling that in the rumen of obligate herbivores such as sheep and cattle. The products too are similar; short-chain fatty acids (SCFA), gases (hydrogen, carbon dioxide and methane) and an increased bacterial mass. The principal SCFA are the same in humans as in ruminants, and the concentrations are similar too, particularly for omnivorous animals with a similar digestive physiology (for example, the pig). The increased bacterial cell mass also has a positive effect on laxation. Faeces are approximately 25% water and 75% dry matter. The major components are undigested residuals plus bacteria and bacterial cell wall debris. These form a sponge-like, water-holding matrix which conditions faecal bulk and cell debris. The ability of different fibres to increase faecal bulk depends on a complex relationship between chemical and physical properties of the fibre and the bacterial population of the colon. The production of SCFA and their beneficial effects in humans and ruminant species has been well established for a considerable time, but the effect was not thought to be relevant to the carnivorous dog and cat. Now this too has been demonstrated.

1.6.3 Health benefits Whether by physical bulking action or through the production of SCFA, several health advantages are now established. Increasing fibre (20–30 grams per day in humans) can eliminate constipation through increased faecal bulking and water-holding. The

18

Handbook of hydrocolloids

fermentation to produce SCFA can also assist, since propionate stimulates colonic muscular activity and encourages stool expulsion. It was at one time thought that fibre lodged in the colon, leading to inflammation and herniation. This has now been disproved, and fibre can now relieve diverticular disease conditions, probably in the same way as it relieves constipation. Applying a solution of SCFA into the colon of ulcerative colitis patients or into the defunctioned portion of surgical patients has given rise to substantial remission in colitis. It could be that the condition arises due to a defect in the fermentation process in these patients or in the products. SCFA stimulate water and electrolyte absorption by the mucosa and enhance their transport through improving colonic bloodflow. Fibre fermentation also reduces the population of pathogenic bacteria such as Clostridia and can prevent diarrhoea due to bacterial toxins. Epidemiological studies have shown repeatedly that populations with high levels of fibre in their diet have reduced risk of colon cancer. Protection may be through the SCFA butyrate, which inhibits the growth of tumour cells in vitro. When applied to the companion animal, the increased production of SCFA increases gut acidity marginally, which reduces the activity of putrefaction and pathogenic bacteria and so lowers toxin and thus reduces bad odours and bad smelling faeces. The low level of toxin production reduces the load on the liver and results in better coat and skin quality. Therefore, the ageing animal can look better and produce less offensive faeces. The behaviour of SCFAs in the intestine can influence the immune system. Thus protection is possible against colonisation by opportunistic bacteria and the improved colonisation of beneficial indigenous bacteria in the gut, which gives greater resistance to infectious bacteria.

1.7

Future prospects

The introduction of totally new hydrocolloids for food use is restricted by the large financial investment required to obtain the necessary legislative approval. However, there is still a market for such materials in other industries. Welan gum, for example, another polysaccharide derived from bacteria, is finding increased use in oilfield applications. There are certain hydrocolloids, however, that have a long history of use in food in other parts of the world that may be introduced into the US and Europe as food additives. A notable example is konjac mannan which has been used for hundreds of years in Japan to produce noodles and is eaten as a food in own right. When dissolved in water this material has similar properties to locust bean gum but produces higher viscosity solutions and also has a stronger synergistic interaction with kappa carrageenan and xanthan gum. The search for new synergistic combinations continues and this is becoming more fruitful as our understanding of the interactions and phase behaviour of hydrocolloid mixtures increases at the molecular level. New processing procedures are also being introduced and an area of particular interest at present is the formation of sheared gels to give novel rheological characteristics. This involves applying shear as the hydrocolloid is undergoing gelation and usually results in the formation of micron-size hydrocolloid gel particles. At a sufficiently high concentration, the systems formed can have a very high low-shear viscosity and display strong shear thinning characteristics. As discussed above, although hydrocolloids have historically been used in foods to control the rheological properties and texture, consumers are being made increasingly aware of their nutritional benefits. Many hydrocolloids (e.g., locust bean gum, guar gum,

Introduction to food hydrocolloids

19

konjac mannan, gum arabic, xanthan gum, pectin) have been shown to reduce blood cholesterol levels. Others, (e.g., inulin, gum arabic) have been shown to have prebiotic effects. They are resistant to our digestive enzymes and pass through the stomach and small intestine without being metabolised. They are fermented in the large intestine to yield short chain fatty acids and stimulate the specific growth of beneficial intestinal bacteria, notably, bifidobacteria, and reduce the growth of harmful micro-organisms such as clostridia. All in all, the hydrocolloid market is currently very buoyant and the prospects for future growth are excellent.

1.8

Bibliography

DICKINSON, E. (1991) Food Polymers, Colloids, Gels DICKINSON, E. and WALSTRA, P. (1993) Food Colloids

and Colloids Royal Society of Chemistry. and Polymers. Stability and mechanical properties Royal

Society of Chemistry. and BERGENSTAHL, B. (1997) Food Colloids, Proteins, Lipids, and Polysaccharides Royal Society of Chemistry. DOMODARAN, S. (1997) Food Proteins and Their Applications Marcel Dekker. GLICKSMAN, M. (1982–6) Food Hydrocolloids Vols 1–3 CRC Press. HARDING, S. E., HILL, S. E. and MITCHELL, J. R. (1995) Biopolymer Mixtures Nottingham University Press. HARRIS, P. (1990) Food Gels Elsevier Science Publishers. IMESON, A. (1992) Thickeners and Gelling Agents for Food Blackie Academic and Professional. LAPASIN, R. and PRICL, S. (1995) Rheology of Industrial Polysaccharides; Theory and Applications Blackie Academic and Professional. MEUSER, F., MANNERS, D. J. and SEIBEL, W. (1993) Plant Polymeric Carbohydrates Royal Society of Chemistry. NISHINARI, K. and DOI, E. (1994) Food Hydrocolloids: Structure, Properties and Functions Plenum Press. NUSSINOVITCH, A. (1997) Hydrocolloid Applications; Gum technology in the food and other industries Blackie Academic and Professional. PHILLIPS, G. O., WEDLOCK, D. J. and WILLIAMS, P. A. (1984) Gums and Stabilisers for the Food Industry 2 IRL Press. PHILLIPS, G. O., WEDLOCK, D. J. and WILLIAMS, P. A. (1986) Gums and Stabilisers for the Food Industry 3 IRL Press. PHILLIPS, G. O., WEDLOCK, D. J. and WILLIAMS, P. A. (1988) Gums and Stabilisers for the Food Industry 4 IRL Press. PHILLIPS, G. O., WEDLOCK, D. J. and WILLIAMS, P. A. (1990) Gums and Stabilisers for the Food Industry 5 IRL Press. PHILLIPS, G. O., WEDLOCK, D. J. and WILLIAMS, P. A. (1992) Gums and Stabilisers for the Food Industry 6 IRL Press. PHILLIPS, G. O., WEDLOCK, D. J. and WILLIAMS, P. A. (1994) Gums and Stabilisers for the Food Industry 7 IRL Press. PHILLIPS, G. O., WILLIAMS, P. A. and WEDLOCK, D. J. (1996) Gums and Stabilisers for the Food Industry 8 IRL Press. ROSS MURPHY, S. B. (1994) Physical Techniques for the Study of Food Biopolymers Blackie Academic and Professional. STEPHEN, A. M. (1995) Food Polysaccharides and Their Application Marcel Dekker. WHISTLER, R. L. and BEMILLER, J. N. (1993) Industrial Gums: polysaccharides and their derivatives 3rd edn Academic Press. WILLIAMS, P. A. and PHILLIPS, G. O. (1998) Gums and Stabilisers for the Food Industry 9, Royal Society of Chemistry. WILLIAMS, P. A. and PHILLIPS, G. O. (in press) Gums and Stabilisers for the Food Industry 10, Royal Society of Chemistry. DICKINSON, E.

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2 Agar R. Armise´n and F. Galatas, Hispanagar S A, Madrid

2.1

Introduction

Agar-Agar, also called simply agar, was the first phycocolloid used as a food additive in our civilisation having been employed in the Far East over 300 years ago. Phycocolloids are those gelling products extracted from marine algae that are utilised in several ways solely because of their colloidal properties. The most important ones are agar, alginates and carrageenans that are produced in industrial quantities and presented in the form of clear coloured powders. In the Orient ‘natural agars’ in the old forms of strips and squares are still being used at home to prepare traditional dishes. Lately such types of agar have reached our dietetic and natural food stores while in Japan they are being substituted by powdered industrial agars prepared as tablets. Agar is defined as a strongly gelling hydrocolloid from marine algae. Its main structure is chemically characterised by repetitive units of D-galactose and 3–6,anhydroL-galactose, with few variations, and a low content of sulfate esters. We can also add that agar is also a mixture of polysaccharides made of dextro and levo galactoses united linearly.

2.1.1 Historical background In Japan, where agar has been used for several hundred years, Tarazaemon Minoya is generally considered its discoverer in 1658. In fact, this phycocolloid had certainly been utilised much earlier than any other phycocolloid such as alginates or carrageenans, which it predates by 200 years. From Japan its use extended to other oriental countries during the seventeenth and eighteenth centuries. Agar was introduced in the West by Payen (1859) as a Chinese foodstuff and its microbiological applications were presented by Koch (1882). Hence, it can be said that it was known in the Western world towards the end of the nineteenth century. Smith (1905) and Davidson (1906) contributed to its wider application by presenting very clear explanations of the raw materials and the manual processes involved in its production in Japan.

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Handbook of hydrocolloids

Being the first phycocolloid used by man, it is one of the food ingredients first approved as GRAS (Generally Recognized As Safe) by the FDA. This was in 1972, based on the positive as well as lengthy experience acquired by its usage in the Far East for more than three centuries. It also passed all other controls in its toxicological (FDA 1973a), teratological (FDA 1973b) and mutagenic (FDA 1973c) aspects.

2.1.2 Agarophyte seaweed used for production In the middle of the seventeenth century, agar was produced in Japan from Gelidium amansii exclusively, then China and Korea followed soon after. This ‘red’ (Rhodophyceae phyllum) seaweed type was the one most abundantly available along their respective coasts. As Gelidium amansii was in short supply, efforts were made to utilise other Rhodophyceaes as substitutes. When Gracilaria seaweeds started to be used, agars with very poor gelling power were obtained and these were called agaroids. Only in 1938, when Yanagawa discovered the alkaline hydrolysis of sulfates, was it possible to increase the gel strength and produce stronger agars from Gracilaria. Gracilaria contains in its cells an agar content of a very weak nature which Yanagawa learned to transform into a stronger final product by using this alkaline hydrolysis processing method. In Gelidium, Gelidiella and Pterocladia seaweed, there occurs a natural internal transformation through an enzymatic process that can be considered a maturing of the polysaccharide in the weed. In Gracilaria it is not converted in the needed amount during the weed’s lifetime so it becomes necessary to produce it industrially by means of a chemical method before extracting the agar from the weed. In Table 2.1 we can observe the different agarophytes used in the world for agar production. Due to some confusion in taxonomy because of frequent name changes, we have decided to use the classical names applied normally for agarophytes in worldwide commerce. Once DNA determinations are done on each weed, hopefully a permanent description basis will be established. There are several types of agars available with very different applications due to the distinct characteristics of each one. They have been developed to satisfy diverse applications and they originate from different agarophyte algae that are produced by diverse technologies. Mainly we should distinguish ‘natural agars’ from ‘industrial agars’. The first have been produced by artisans and lack technical controls but are still sanitarily clean and proper for its main use in home cooking. The latter types are manufactured in modern factories and are utilised as industrial food ingredients, so are subjected to all kinds of established controls. Agars utilised for microbiology and biotechnology are also included in this category even though they comprise only 10% of the total volume. Agar is a polysaccharide that accumulates in the cell walls of agarophyte algae. It is embedded in a structure of fibres of crystallised cellulose, constituting its polysaccharide reserve. For this reason, agar content in weed varies depending on the seasons. Initially an intermediate form of agar with low molecular weight and quite sulfated is secreted by the Golgi apparatus of the cell. Once deposited in the cellular wall it enzymatically polymerises and desulfates, being converted mostly into agarose that gives the agar its gelling power. The rest remains in the form of agaropectin. Matsuhashi (1990) suggested that agar could be linked to cellulose fibres by calcium ions. This would explain many phenomena that occur during the extraction process. In Fig. 2.1 we can observe a section of Gelidium sesquipedale, the agarophyte found in Western Europe and Morocco. The image shows the enormous thicknesses of the cellular

Agar Table 2.1

23

Taxonomic classification of agarophytes (Armise´n, R. 1995)

Phylum. Rodophyta. Class. Florideophyceae. Order. Gelidiales. Family. Gelidiaceae. Genus. Gelidium. Species. G. sesquipedale*, G. amansii*, G. robustum*, G. pristoides, G. canariense, G. rex, G. chilense, etc. Genus. Gelidiella. Species. G. acerosa. Genus. Pterocladia. Species. P. capillacea*, P. lucida* Order. Gracilariales. Family. Gracilariaceae. Genus. Gracilaria. Species. G. chilense*, G. gigas, G. edulis, G. gracilis, G. tenuistipitata*, Genus. Gracilariopsis Species. G. lamaneiformis, G. sjostedtii Order. Ahnfeltiales. Family. Ahnfeltiaceae. Genus. Ahnfeltia. Species. A. plycata. * The most used agarophytes in industry.

walls where agar is found. The smaller cells or rhizoids contain agaroses of higher molecular weight. It so happens that the number of rhizoidal cells is higher where the water movements are stronger. Therefore the hydrodynamic conditions of the environment where Gelidium grows play an important role in the agar content in the weed as well as its gel strength characteristic.

2.2

Agar manufacture

Agar is called Kanten in Japanese, which means ‘frozen sky’, referring to the way it was first manufactured by artisans based on freezing and thawing in the open fields of the extracted agar gel. It is derived from the technique recorded by Tarazaemon Minoya in 1658 that has its fundamentals in the insolubility of agar when cooled. The traditional technique adapted by Minoya, developed towards the middle of the seventeenth century, is still in use marginally to produce ‘natural agar’ in the oriental craft industry in the forms of strip agar (Ito-Kanten) or square agar (Kaku-Kanten). This technique started with careful washings of Gelidium amansii employing similar devices to those used to wash tea-leaves. The washed seaweed was selected by hand to eliminate any foreign body or extraneous seaweed. It was extracted in boiling water adjusting its pH. In olden days the

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Handbook of hydrocolloids

Fig. 2.1 Electronic scanner microphotograph of a section of Gelidium Sesquipedale. (Vignon, M. R., Rochas, C., Chanzy, H., Vuong, R. and Tekeley, P. (1994)).

adjustment was made with vinegar or sake but now diluted sulfuric acid is employed. The liquid extract was filtered while hot through cotton bags, poured into wooden trays and allowed to gel by cooling. The gels were cut into square bars (4 6 24 cm) or extruded to produce spaghetti-like strips 25–40 cm long. The gels prepared in this way were placed on bamboo grills and left to freeze all night in the open, usually in bluffs facing the northern winds. Once totally frozen during one or two nights, the agar was thawed during the daytime by sprinkling water over it. It was then sun dried and kept away from frosts. After the Second World War and up to the 1960s there were approximately 400 artisan plants in operation producing between 4 to 10 kg per day, climatic conditions allowing. It can be seen that this basic technique produces low standards and irregular qualities because the process is very dependent on climate. But it is reviewed here, as it is a simple way to comprehend the more involved processes that are performed now using more sophisticated mechanical means. What are known as industrial agars are produced in modern plants in which fully standardised agars are obtained, assuring qualities that comply with physicochemical and bacteriological specifications in accordance with sanitary codes. The best installed plants comply with ISO-9000 norms, that assure the market that all processes are controlled and are traceable from raw materials to shipped finished goods. In Fig. 2.2 the block diagram shows a general industrial process that can be utilised to produce agar. It is to be noted that two different paths can be followed to dehydrate the gellified agar extracts.

2.2.1 Freezing-thawing method This traditional method was first used for ‘natural agar’ production, and was not substantially changed until American Agar & Co. (San Diego, USA) started to manufacture agar industrially in 1939, in freezing tanks in the same way that ice bars are

Agar

25

Fig. 2.2 Agar fabrication diagram. (Armise´n, R. and Galatas, F. 1987)

made. Immediately after the Second World War the same technique was applied in Japan as well as in the new plants that were built in Spain, Portugal and Morocco. The seaweed extract, which normally contains between 1% and 1.2% of agar during the process, is concentrated after thawing and straining (normally by centrifugation) to contain 10% to 12% of agar which is a tenfold increase. The eluted water carries away oligomers, organic and inorganic salts as well as proteins from the algae including phycoerytrins that produce the red colour of the Rhodophyceae family. This process is one of purification. 2.2.2 Syneresis method This method is based on the property of gelling colloids by which the absorbed water can be eliminated by means of a properly applied force. The technique was utilised in Japan solely for Gracilaria agar on a semi-refined scale. Gelled extracts packed in cloths with a closed mesh were pressed under stone blocks to push the water out from the gel. Afterwards, it was pressed by means of small hydraulic presses to eliminate the residual water.

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Handbook of hydrocolloids

In 1964, Prona, a forerunner of Hispanagar, innovated a modern syneresis technique to concentrate gelled extracts for all agarophytes (not only Gracilaria) building new plants in France, Mexico, Chile and South Africa as well as modernising those in Spain, Portugal and Morocco. It gave the company worldwide leadership in agar production, only shared with Algas Marinas (Chile) in the late 1980s and with INA (Japan) in the last decade. Concurrently Okazaki wrote in 1971 that syneresis was possible only for Gracilaria. The method, which allowed a semi-automated process, was later further improved to a totally automated one which is in use nowadays. Vertical mechanical presses have been substituted by horizontal hydraulic ones. This syneresis technique has spread rapidly all over the world due to its reduced energy cost. The freezing method requires an ice production of 80/100 metric tons to produce one ton of agar while the consumption of energy by the syneresis method is very low. Both methods can be mechanised today with a slight advantage in costs for syneresis. On the other hand, some buyers still prefer the quality of freeze-thaw agar for which they are ready to pay a premium. Agar purity is increased in syneresis as the dry extract weight after pressing is as high as 20% compared to 11% in freezing which means that the latter has double the water content where the impurities stay while syneresis eliminates more soluble impurities. This is reflected in the lower ash content found in syneresis agars but again we state that many customers do not care about this. From those operations in Fig. 2.2 we can consider the most important ones in addition to dehydration to be: 1.

2.

3.

2.3

Treatment: usually an alkaline treatment is employed that allows a better extraction of the polysaccharide from the cell walls. For Gracilaria a stronger reaction is required to produce an alkaline hydrolysis of the sulfates in order to increase the agar gel strength. Extraction: the agar contained in the cell wall is detached and dissolved in boiling water, often under pressure. Careful control of the pH is needed to obtain the best yields. Filtration: this requires special care since clarity and purity will depend on this operation. Most standard filtering techniques can be applied.

Chemical structure of agar

Agar is a polysaccharide that formerly was considered to be formed by one unitary structure only having sulfate semiester groups linked to a few galactose oxydriles. Choji Araki (1937) showed that agar was formed by a mixture of at least two polysaccharides that he named agarose and agaropectin. Later in 1956 he assigned agarose the structure that can be seen in the upper part of Fig. 2.3. In 1938 Percival, Sommerville and Forbes, and independently Hands and Peat, discovered the existence of 3-6,anhydro-L-galactose as part of the agar molecule. When diverse types of agars were carefully studied, the presence of agarobioses which we can observe in the lower part of Fig. 2.3 (Lahaye and Rochas, 1991) was proven. These 11 agarobioses can be produced in many variable forms by the different agarophytes depending on gender and species which depend on their genetic characteristics. It is influenced by a series of ecological factors such as the nutrient availability, substrate composition on which they grow and the habitat hydrodynamic

Agar

27

Fig. 2.3 Agarose chemical structure. (Araki, Ch. (1956) Lahaye, M. and Rochas, C. (1991))

conditions. But of greater importance for the production of such agarobioses is the harvesting period as agar plants mature gradually through the summer season. Agarobioses are the fractions of agar that essentially gel. They have high molecular weights, above 100,000 Daltons and frequently surpass 150,000 Daltons, as well as a low

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Handbook of hydrocolloids

sulfate content usually below 0.15%. The rest of the fractions are known as agaropectins. They have a lower molecular weight, usually below 20,000 Daltons, with 14,000 Daltons being most usual. Sulfates are in much higher content registering sometimes 5% to 8%. This is far below carrageenans which range from 24% to 53% and even furcellaran which is the less sulfated carrageenans at about 17%. Agaropectins are fractions that have been studied less than agarose due to their lack of practical applications. Their properties do not match those of carrageenans for food applications and any process to produce them would be costly and complicated. As mentioned previously, some agaropectins are precursor polysaccharides for agaroses that are transformed internally by enzymatic polymerisation and desulfation processes. As will be shown later, the diverse forms of agarobioses determine the physicochemical characteristics of agar such as gelling and melting temperatures and reactivities or synergies of agars with other products.

2.4

Gelation of agar

Agar is a mixture of agarose and agaropectin fractions in variable proportions depending on the original raw material and the manufacturing process employed. Agar gelation occurs only by its agarose content that is produced exclusively by hydrogen bonds. Not needing any other substances to gel, it has an enormous potential in applications such as a foodstuff ingredient, for biotechnology uses, for cell and tissue culture or as support for electrophoresis or chromatography. Agarose produces ‘physical gels’ which means that these aqueous gels have all their structure formed only by the polymer molecules united solely by hydrogen bonds. Due to this unique gelling property, these gels hold in the interior network a great amount of water which can move more freely through the macroreticulum. Each molecule maintains its structure in complete independence so the process is not a polymerisation but a simple electrostatic attraction. On the contrary, ‘chemical gels’ have the polymer molecules united by covalent bonds to form large macromolecules so we can consider this polymerisation to be caused by a chemical reaction that forms the gel. The most remarkable property of ‘physical gels’ is their reversibility. They melt just by heating but gel again upon cooling. These transformations can be repeated indefinitely in the absence of aggressive substances that could hydrolyse their agarose molecules or destroy them by oxidation. ‘Chemical gels’ such as polyacrylamides that have their molecules joined by covalent bonds are irreversible. Another basic property is the gelling mesh size that in the case of agarose is identified by very high exclusion limits. Exclusion limit is defined by the greatest globular protein size that can traverse the gel in an aqueous solution. In the case of a 2% agarose gel the exclusion limit is 30,000,000 Daltons. Considering that there is no protein of such a size for calibration it has been necessary to resort to subcellular particles such as ribosomes or viruses. The agaropectin fractions present in agar narrow the reticulum reducing slightly its exclusion limit. Intermediate between the physical and chemical gels, we find those gels that require the presence of cations to form gel structures as in the case of carrageenans and alginates. In the case of gels formed by alginic acid with di- or tri-valent cations we face totally irreversible gels that will not melt by heating. These are gels that have formed ionic bonds that can be broken only by eliminating the bonding cation, which is normally calcium. It is done with the help of a complexing agent such as EDTA (ethylene diamine tetracetate). Hence, these gels can be considered as ‘ionic chemical gels’ as they form

Agar

29

Fig. 2.4 Gelling and melting temperatures of agar gels: gelation hysteresis. (Armise´n, R. 1997)

ionic bonds and are irreversible. An important property of agar gels derived from their agarose content is the very high gelling hysteresis, defined as the temperature difference between its gelling (around 38ºC) and melting temperatures (around 85ºC). In Fig. 2.4 we can observe the gelling and melting temperature graphs for regularly available agars. Concentrations are also typical (between 0.5% and 2%) being employed in agar applications. It shows a gelling hysteresis that in every case is above 45ºC. As a comparison, the most gelling carrageenans have a hysteresis of 12ºC that is 26% less than agar. These temperatures depend on the presence of agarobioses originally in the agarophyte seaweed from where the agar is extracted. Gel temperature is an indicator to identify the agarophyte used to produce an agar. Using Table 2.2 we can determine the origin of an agar by identifying its characteristic gelling temperature. It has been proven that the gelling temperature is influenced by the methoxylation degree of the C6 of the agarobioses present in the agar, in such a manner that the more methoxylated corresponds to Gelidiella agaroses and the least to the Pterocladia ones. This is the same as saying that a greater methoxylation in carbon 6 will correspond to a higher gelling temperature. Curiously, the methoxylation of the rest of the carbons reduces the gelling temperature and its gel strength at the same time. This is due to the inability to establish hydrogen bonds by the hydroxyl group located in C6 because of their

30

Handbook of hydrocolloids Table 2.2 Typical gel temperatures of agars extracted from several agarophytes (Armise´n, R. 1993) Genus

1.5% solution, gel temperature

Gelidiella Gracilaria Gracilariopsis Gelidium Pterocladia

42–45ºC 40–42ºC 38–39ºC 36–38ºC 33–35ºC

Fig. 2.5 Agarose gelation. (Medin, A. S. 1995)

position in the gelling helices while the rest of the hydroxyl group are bonding points where hydrogen bridges can be formed. The gelling process of agarose can be seen in Fig. 2.5, obtained from Medin (1995). This is an exothermic process which develops when agarose molecules are dissolved in water. It can be seen on the left that these molecules are real ‘statistical random coils’ subject to Brownian movements. When cooling down close to the gelling temperature, the next structures start to form. According to Rees and Welsh (1997) we observe in the upper part of Fig. 2.5 how antisymmetric double helices (B1) are formed in their aggregation to form a macroreticulum as pictured in the upper side of C and D. In the lower part of Fig. 2.5 and according to Foord and Atkins (1989) we can see simple helices B2 that are joined by hydrogen bridges that produce folded structures (symmetric double helices) that will form the macroreticulum as can be seen in the lower side of C and D. It seems that both gelling processes can coexist and one or the other dominates depending on the cooling speed. A faster rate favours the first process. Both are based in the formation of hydrogen bridges and produce a macroreticular structure. In Fig. 2.6 we can observe the spongy structure of a 2% agarose gel. The mesh cavities of the gel which can be seen are filled with solvent water that circulates freely through the mesh capillaries. The very high exclusion limit of agarose gels allow the passage of soluble macromolecules up to 30,000,000 Daltons of molecular weight. A characteristic property of an agarose gel is its syneresis capacity which relates to the capacity to eliminate water contained in its gel mesh. The ejection of aqueous solvents is speeded by

Agar

Fig. 2.6

31

Electronic scanner microphotograph of 2% agarose gel. (Medin, A. S. 1995)

pressure conveniently applied on a properly confined gel. In these conditions a 1% agar or agarose gel can eject a great proportion of the water soaked in the capillaries ending the process with a 20–25% dried extract which means that 95% of the water used to dissolve the agar/agarose has been eliminated. If the undried synergised gel is submerged in water it will recover its original size. The gel structure has been maintained during syneresis and upon rehydration it will recover exactly to the previous form. This is known as gelling memory.

2.4.1 Synergies and antagonisms of agar gels We shall consider in this section the most important cases of blended products which modify agar by increasing its gel strength, modifying its texture or elasticity and the antagonist products that reduce the gel strength or block in any way the gelling process. Traditionally food agar gel strengths are controlled following the Japanese Nikan Sui method that measures the charge in grams that ruptures a gel by means of a cylindrical piston with a surface of 1 cm2 after applying force for 20 seconds. This traditional method is used universally even though there are other more precise ones that operate with a growing load that also permits valuation of the elasticity module of the gel. No doubt these controls will be improved in the coming years but nowadays Nikan values are the basis for commercial transactions worldwide. Agar-locust bean gum (LBG) synergies Synergies with this gum are possible only with Gelidium and Pterocladia agars. It has practical applications because gel strength is increased and the gel texture is modified in a

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Handbook of hydrocolloids

Fig. 2.7 Agar-locust bean gum synergies. (Armise´n, R. and Galatas, F. 1987)

way that rigidity is decreased and elasticity is enhanced becoming less brittle. As we can see in Fig. 2.7 a mixture of LBG and Gelidium agar dissolved at 1.5% gives Nikan gel strengths (g/cm2) which increase to a 9:1 ratio when maximum hardness is achieved, returning to the corresponding agar strength when the agar-LBG proportion is 4:1. Contrary to this, Gracilaria agars do not show such synergy and upon substitution hardness falls in the same proportion as if the agar concentrations were reduced. The synergism agar-LBG is caused solely by the agaropectins contained in Gelidium and Pterocladia but not by those in Gracilaria. Sugar reactivity Reactivity with sugar (saccharose) is basically present in Gracilaria agars when dissolved in aqueous solutions with a high sugar concentration (around 60%). In agars with high gel strengths and low sulfate content the synergy is caused by the thread pitch of the gelling helices. It always requires the presence of agarose of high molecular weight (around 140,000 Daltons) and is a consequence of the 3-6,anhydro bridge in the L-galactose which is able to assist in the building of hydrogen bridges. Gelling blockade by tannic acid (TA) The presence of TA (pentadigaloil glucose) may inhibit agar gelation if the TA quantity is large enough. This acid is found in some fruits such as squash, apple and prune in variable concentrations. Adding glycerol in small amounts is usually enough to avoid this reaction. Gelling blockade by chaotropic agents Chaotropic agents which capture protons can also block agar gelation by avoiding the hydrogen bridges which form between the agarose molecules present. In any case, there is no problem associated with chaotropics for the use of agar in the food industry, as foodstuffs do not contain significant quantities of agents such as urea, guanidine, sodium thiocyanate or potassium iodide.

Agar

33

Acid and alkaline hydrolysis As with all polysaccharides, agar can suffer hydrolysis reducing its molecular weight and consequently losing its gelling power. Acid hydrolysis in agar appears more readily, as a result of lowered pH and the longer time the agar stays in dissolution at a high temperature. In general, hydrolysis is not an important problem unless the agar undergoes extensive heating at pH below 5.5. Alkaline hydrolysis is not important at pH below 8. Enzymatic hydrolysis is not relevant as there are few agarases (enzymes that break down agaroses) which are found only in marine bacteria, in a few bacilli and Esquizosaccharomycetes that are not normally found in food products.

2.5

Applications of agar

Agar applications are fundamentally based on the enormous gelling power, high hysteresis and perfect gel reversibility which are unique properties conferred by its special ‘physical gel’ structure. Although agar has multiple applications, the traditional one is as a food ingredient accounting for 80% of its consumption. The remaining 20% is accounted for by biotechnological applications. A list of different uses and the corresponding type of algae required can be found in Table 2.3.

2.5.1 Agar in food applications Agar is a food additive of universal use considered in the US as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration). In Europe it is considered an E406 additive. In the Register Service of the Chemical Abstracts it is registered as 9002-18-0. The synergies that were described when dealing with agar gelation are important. The synergy between agar-LBG shown by Gelidium and Pterocladia agars improves hardness as well as texture, making the mixture more palatable due to the elasticity conferred and the elimination of the brittleness period. The

Table 2.3 Agar grades depending on their final uses and agarophytes used for their production (Armise´n, R. 1995)

Natural agar

Industrial agar

Agar type applications

Agarophytes used

‘Strip’ ‘Square’ Accustomed only on Far East traditional kitchen

Produced mostly with Gelidium by traditional methods

Food grade agar used for industrial food production

Gelidium, Gracilaria, Pterocladia, Ahnfeltia, Gelidiella

Pharmachological agar

Gelidium

Clonic plants production grade

Gelidium or Pterocladia

Bacteriological grade used for bacteriological media formulation

Gelidium or Pterocladia

Purified agar used in biochemistry and in media for very difficult bacteria

Gelidium

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Handbook of hydrocolloids

mixture of LBG-agar reduces the syneresis of the gels which makes them exude less liquid during handling, transportation and storage. In the same way, Gracilaria agars that show reactivity with sugar, experience an increase in gelling power when used with sugars with a high sugar content (60% or more) such as jams and jellies. Adding glycerin or sorbitol to aqueous gels prepared with agar, reduces dehydration to such an extent that with sufficient quantities of these humectant products, the drying of gels exposed to air can be avoided. Obviously, the higher the relative humidity of the atmosphere, the added quantity of humectants should be lower. The ambient temperature changes during the expiration life of the product also affect this behaviour. Agar is tasteless and cannot be detected in foodstuffs with delicate flavours. In contrast, those gelling agents that need the presence of cations (calcium or potassium) to gel should be blended with foodstuffs with strong flavours to mask the characteristic flavour of said cations. Some agars with an elevated melting point such as those produced in Portugal by Iberagar are employed to prepare Mitsumame. This popular Japanese fruit salad mixes fruits and coloured gel cubes properly flavoured and, after canning, is subjected to heat sterilisation which the agar gel cubes withstand without melting. Normally agar is dissolved by adding it slowly to water with good stirring. In sweet products agar is usually premixed with a part of sugar, then adding it slowly later to avoid clumps which can form during dispersion. If acidification is required in a process, it has to take place once all the agar has been dissolved and whenever possible at reduced temperatures to minimise hydrolysis risks. Nevertheless, agar has enough resistance to hydrolysis to the degree that, in meat preserves which require sterilisation in an autoclave over 121ºC, it undergoes the treatment successfully withstanding hydrolysis. Aromas, especially volatile ones, should be administered at the final stage of cooling, just prior to moulding and packing of the product. In this way evaporation losses are avoided. In some industries it is customary also to add at this stage the colour ingredients. Given that agar is utilised in food as a minor ingredient (0.5% to 1.5%) relative to the total weight of the finished product, its nutritional contribution is low because the human digestive system hardly absorbs it. Agar has been consigned a digestibility below 10% of what is ingested. For this reason agar is used to prepare dietetic formulas and foods for diabetics since it does not depend on sugar to gel as with pectins. Formulations can be made employing edulcorators as substitutes for sugar together with agar which will create very low-calorie food products. For many years agar has been included in the US Pharmacopoeia as a laxative as it has a convenient effect as a soft voluminator. In fact it is contained in several formulas for this purpose. It behaves just like natural fibre with the great quality of being totally soluble and resistant to hydrolysis. In addition the human digestive system produces no agarases, which contributes to the extremely low digestibility of agar. Today agar is included in the US National Formulary to be used as a slow-release ingredient for the slow absorption of pharmacological agents. It is very interesting to realise that the alimentary applications of industrial agar differ depending on the cultural area that is considered. In Table 2.4 we can observe how they vary among geographical areas. We shall follow with some formulations used to prepare these products from our experience in this field and the bibliographical data available. In necessary cases indications will be given to prepare these products. We wish to state that in no circumstances can this information be considered as an aid to infringe patents or protected processes.

Agar Table 2.4 1990)

35

Agar applications in several cultural world regions (from a lecture by Modliszewsky. J.

Applications

Regions

Ice cream Milk shake Sherbet Custard pudding Cakes Pie filling Flat icing Meringue Cookies Candy (agar jelly) Fruit jelly dessert Jams, Jellies Processed cheese Ferm. dairy products Wine clarification Gelled meats Dulce de batata Mitsumame Red bean jelly Agar jelly beverages

Asia

US/Europe

Latin America

+ + + + + ÿ + ÿ + + ++ + ÿ + ? ++ ÿ ++ ++ ++

+ + + + + + + + + + + + + ++ + ++ ÿ ÿ ÿ +

+ + + + + + + ÿ + + + + ? + + ? ++ ÿ ÿ +?

Some formulations for agar in human diet Yokan Matsuhashi, T. (1990) Yokan, a traditional agar hard gelatin consumed usually in the tea ceremony but also on different occasions. Its ingredients are sugar, azuki bean pure´e, agar and water. Sometimes chestnuts are used instead of beans for higher priced Yokan. An approximate formula would be: Agar Water Sugar Inverted sugar Citric acid Flavour and colour Azuki beans

50 gm 2000 to 3000 gm 1000 to 2000 gm 1500 to 3000 gm 2 gm If desired Variable according to each case but enough to produce a hard gel.

Agar is dissolved in boiling water with sugar and inverted sugar and maintained at 106ºC for a few hours to reduce the volume. After brief cooling, the fruit pure´e previously prepared and the acid are added together with flavours and colourings. It is left to cool overnight at room temperature. This gel has a dried weight of 70–75%. It is placed in an oven at 55ºC as long as needed to reach a dry weight of 84–86% and is cut in small pieces that are first folded in an oblate and later in plastic. This oblate is an edible paper made of starch and agar. Inverted sugar is hydrolysed saccharose that avoids crystallisation.

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Handbook of hydrocolloids

Sweet potato sweet (An Argentinian traditional dessert) Fiszman, B. (personal communication towards 1975). This is a very popular sweet in Argentina and Uruguay and also in some parts of Brazil, being part of its culture. Components: Sugar 37% Glucose 21%, usually a syrup of 76º Brix Agar 0.181% Locust bean gum (LBG) 0.29% Fresh sweet potato with skin 80%. As it is an elaborate process, it is recommended to: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Peel the potato. Boil it in water. Sieve it to a state of pure´e. Mix the pure´e with sugar and glucose. Concentrate by boiling until 60º Brix. Add the agar that has previously been dissolved at 25%. Bring the mixture to 58/60º Brix. Add to this mixture the LBG that previously has been dry mixed at four times its weight in sugar and dissolved in cold water at 6–7 times the total weight. Carry all of it to a 60.5º Brix, flavour and can while hot.

Flat cans of 25 cm in diameter and 5 cm in height are usually employed. Sugar icings Meer, W. (1980) Components: Agar Salt (NaCl) Emulsifier Granulated sugar Coating sugar (Glasee) Water

0.35% 0.3% 0.4% 15.0% 73.0% Enough to complete 100%

Different coatings are prepared with agar using several formulations depending on the solidification speed that is expected for each coating. These icings are prepared by boiling the agar, used as stabiliser, in a sugar solution followed by the addition of coating sugar. The icing heated up to 50–60ºC is applied over the products to be coated in 60 seconds. Gelation times can be regulated, increasing or reducing the agar amounts.

2.5.2 Agar in insect culture media formulations Agar is used for the breeding of larvae and other smaller sized animal species. It is applied for fattening silkworms year round in order to lengthen the season that was previously a limited one. The tiny worms could feed only on tender mulberry leaves that were produced at early budding. Agar for this purpose is dissolved and the feed, composed of carbohydrates and proteins, is suspended in the solution. Left to cool, the

Agar

37

mixture is extruded in the form of thin spaghettis with adequate sizes for the different worm constitutions from the egg eclosion period until the formation of the silk buds. Only agar could be employed for its composition because all other gelling agents have tastes that are rejected by silkworms. Another classic use of agar is a similar application for feeding larval phases of flies such as Drosophila melanogaster used for genetic research. Obviously when technologies for biological insect plague contentions were developed, analogous methods for larvae feeding were implemented using agar as a base. It dealt with insects that damaged intensive crops and modern methods were required for its control. In this way the Mediterranean fly that damages orchards or Pectinophora glosipeii caterpillars that destroy cotton farms are artificially grown in order to be subsequently sexually sterilised by c (Gamma) radiation. The sterile individuals are kept hibernated and set free at the proper mating period for intercourse. Because these insects are able to copulate only once in their lifetime, their copulation capacity is frustrated rendering void their intercourse with a fertile mate. Without the need of organochlorated insecticides the species is thus controlled but not destroyed as some individuals can mate with fertile partners.

2.5.3 Vegetable tissue culture media formulations The gelling properties of agar enhance the formulation of solid media for tissue culture growth originated in techniques developed to obtain orchid clones. Media has been formulated to reproduce plant specimens to grow identical plants free of viruses from each one. Usually vegetable meristems of plants to be cultivated are cultured in media of the adequate composition, enriched with vegetable hormones such as auxines or cytokines that are applied depending on the rooting desired for the plant and/or its growth velocity. Once the proper plant development is achieved, they are transferred to vegetable ground to continue its growth.

2.5.4 Culture media for microorganisms Microbial cultivation began with R. Koch in 1882 and since then its use has been related to the development of microbiology in such a way that it has never been substituted, repeated efforts to the contrary notwithstanding. The special properties of the physical gels that agars form, together with their gelling and melting temperatures and especially due to their enormous gelling hysteresis and reversibility, all contribute to and offer characteristics which are unique for these applications and have found no substitute so far. Likewise their huge resistance to degradation by enzymes that damage other gelling agents and their ability to gel in the absence of cations, enable agars to support culture media with very well adjusted osmotic pressures to cell requirements whether red blood cells, bacteria, yeasts or moulds are to be grown.

2.5.5

Industrial agar application formula

Dental moulds Meer, W. 1980 A formula for an industrial use of agar is offered as an example of a general application that is quite common in very diverse uses where high-precision moulding is sought. This composition is employed to prepare dental moulds in the US but in other countries it is

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Handbook of hydrocolloids

utilised to prepare plasters of archaeological pieces or moulds for sculptures. Police use it to preserve footprints or similar clues for criminal cases. The enormous reversibility of agar gels that change from solution to gel states by simply cooling them, is an incentive due to its convenient use. Gels can be kept for long periods in tubes similar to toothpaste tubes. Before use, they are melted in boiling water baths and the solution is placed in a tray prepared for the dental imprint. Once the adequate temperature (39ºC) is reached, the imprint is performed immersing the tooth (or the object to be copied) until the Gelidium agar gels at 36ºC. Other agars that gel at higher temperatures are not recommended as they can annoy patients. This is not the case for inanimate objects that may employ other agars. A common formulation is: Gelidium agar Borates Sulfates Hard wax Thixotropic materials (Bentonite type products) Water

13–17% 0.2–0.5% 1.0–2.0% 0.5–1.0% 0.3–0.5% Sufficient to arrive to 100%.

This formulation can be modified to achieve gels that will not dry in practice in normal atmospheric conditions. For this purpose sorbitol glycerin should be added or any other humidifier agent that will absorb the ambient dampness to compensate for the evaporation losses. In normal weather conditions 8–10% of glycerin is required but this percentage must be adjusted for practical uses depending on the maximum and minimum temperatures the product can withstand and on the relative humidities of the environment.

2.6

New developments

It has been a continuous goal to obtain an agar with good gelling properties but with a greater ease for solution. Specifically, efforts have been made to try to obtain agars that can be dissolved without the need to reach boiling temperatures of 100ºC. It would enable the preparation of foods with ingredients that do not resist temperatures above 85ºC. In parallel, industrial agars have increased in gel strength to be formulated in lesser proportions saving costs, as agar is one of the most expensive ingredients. In general there has been a tendency to increase the gel strength by increasing the average molecular weights and reducing the sulfate contents in industrial agars. It is necessary to spend longer periods at boiling temperature to dissolve agars of this type. Practically, when dealing with high sugar content products (saccharose), solution always occurs above 100ºC due to the boiling phenomena that sugar produces. New agars intend to improve the solution by enabling the agar to dissolve at lower temperatures and in shorter periods. They have been developed lately to achieve these seemingly opposed targets of a lower dissolving temperature with a minimum sacrifice in gel strength. One of the methods employed for some of these agars is based on the addition by means of mechanical pressure of substances that unite strongly to the agar in a dry state and upon dissolution accelerate the binding of the agar and water molecules. Agars obtained in this way present the inconvenience of lower gel strength (caused by the increased proportion of a non-gelling agent) as a trade-off for lowering the temperature of

Agar Table 2.5

39

Some easily soluble agars: comparative analysis

Product Produced by Moisture Ash Solution clarity at 1.5% pH in 1.5% solution pH in 1.5% gel Viscosity 1.5% 60ºC Gel temperature at 1.0% Melting temperature at 1.0% Gel strength (Nikan) at 1.5% Gel strength at 1.5% (dissolved 5 minutes at 85ºC) Gel strength at 1.5% (dissolved 5 minutes at 90ºC)

Grand agar

Speed agar-80

Hispanagar (Spain)

Taito (Japan)

7.16% 1.53% 26 Nephelos 6.80 6.48 6.5 cps 31.9ºC 87.5ºC 1,270 gr/cm2

7.08% 1.47% 510 Nephelos 6.18 6.56 4 cps 34.9ºC 75.6ºC 590 gr/cm2

680 gr/cm2

430 gr/cm2

820 gr/cm2

440 gr/cm2

solution. Hence, it loses some yield when used as an ingredient and has limitations for biotechnology uses such as microbiology culture media due to the foreign substance added that can interfere with the growth of organisms or even inhibit then. In other cases, production processes have been refined to optimise the binding of agar and water molecules at lower temperatures without additives and without affecting the gel strength that is kept at normal agar levels. Therefore its performance as a gelling ingredient has not changed and its uses in biotechnology has not been disturbed. The utilisation of these agars in industry is dependent on the balance of its higher price and the convenience it can bring to foodstuff manufacturers. Being in the market only since the late 1990s, the novelty has not yet been fully introduced into each of the traditional areas of application. In Table 2.5 we can observe a comparison of the analysis performed on two of these types of agars that are easy to solubilise, samples which were available to us in sufficient quantities and have been tested in our laboratories. As can be seen in all cases, gel strengths of agars that have been dissolved at 80ºC are specified. Ina Food Industry and Setexam also produce agars which are easily soluble. However, at the time of writing we have not been able to obtain samples to carry out comparative analysis.

2.7

References and further reading

ARAKI, CH. (1937) ‘Acetylation of agar like substance of Gelidium amansii’ J. Chem. Soc. Japan, 58; pp. 1338–50. ARAKI, CH. (1956) ‘Structure of agarose constituent of Agar-Agar’ Bull. Chem. Soc. Japan, 29; pp. 43–4. ARMISE´N, R. (1991) ‘Agar and Agarose biotechnological applications’ Hydrobiology, 221; pp. 159–66. ARMISE´N, R. (1993) ‘Agar-Agar’. Lecture in Training Course T004 ‘Gels Thickeners and Stabilizing Agents’

held in Leatherhead, Surrey, UK on 25–8 May 1993 organised by Leatherhead Food Research Association (a copy of the paper was delivered to the thirty delegates present). ARMISE´N, R. (1995) ‘Worldwide use and importance of Gracilaria’ Communication presented in the Workshop ‘Gracilaria and its Cultivation’. Organised in the University of Trieste (Italy) 10–12 April 1994, under the auspices of COST 48 of the CCEE. Journal of Applied Phycology, 7; pp. 231–43. ARMISE´N, R. (1999a) ‘Agar’ Thickening and Gelling Agents for Food. 2nd edn. pp. 1–21. Ed. Alan Imeson. Blackie Academic and Professional, London. ARMISE´N, R. (1999b) ‘Applications of agar-agar and agaroses in Microbiology, Electrophoresis and Chromatography’. Proceedings of Workshop COST 49 ‘Technology and and Biotechnology of Algal

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Polysaccharides; Future Trends’. Edited by Crescenzi, V., Rizzo, R. and Skaak-Braek, G. pp. 3–16. Published by European Commission EUR 18951 Luxembourg 1999. ARMISE´N, R. and GALATAS, F. (1987) Production Properties and Uses of Agar: pp. 1–57. Production and Utilization of Products From Commercial Seaweed; Ed. McHugh, D. J. FAO Fisheries Technical Paper No. 288; Rome. DAVIDSON, C. J. (1906) ‘The seaweed industry of Japan’ Bull. Imp. Inst. Japan, 4; pp. 125–49. FDA (1972) Agar-agar, GRAS (generally recognized as safe) Food Ingredients Food and Drug Administration, PB-221225 NTIS, US Department of Commerce. Washington DC. FDA (1973a) Evaluation of Health Aspects of Agar-agar as a Food Ingredient Food and Drug Administration, PB-265502, Federation of American Societies for Experimental Biology. Bethesda MD, USA. FDA (1973b) Tetratologic Evaluation of FDA 71-53 (agar-agar). PB-223820 US Department of Commerce, Washington DC. FDA (1973c) Mutagenic Evaluation of FDA 71-53 (agar-agar). PB-245-443 US Department of Commerce, Washington DC. FOORD, S. A. and ATKINS, E. D. T. (1989) ‘New X-ray diffraction result from agarose: extended single helix structure and implications for gelation mechanism’ Biopolymers, 28; pp. 1345–65. GLICKSMAN, M. (1969) Seaweed Extracts pp. 199–266. Academic Press, New York. GLICKSMAN, M. (1983) Food Hydrocolloids vol. II pp. 74–83 ed. Martin Glicksman, CRC Press Inc. Boca Raton, Florida. HANDS, S. and PEAT, S. (1938) ‘Isolation of an Anhidro-L-Galactose derivate from agar’ Nature 142, p. 797. KOCH, R. (1882) ‘Die Aetiologie der tuberculose’ Berl. Klin. Wochensch. 15, pp. 221–30. LAHAYE, M. and ROCHAS, C. (1991) ‘Chemical structure and physico-chemical properties of agar’ Hydrobiologia 126; pp. 137–48. LAWRENCE, A. A. (1973) Edible Gums and Related Substances pp. 165–74. Noyes Data Corporation. Park Ridge NJ. LAWRENCE, A. A. (1976) Natural Gums for Edible Purposes pp. 238–49. Noyes Data Corporation. Park Ridge NJ. MANTELL, C. L. (1965) The Water-Soluble Gums Haefner Publishing Company, New York. MATSUHASHI, T. (1990) Food Gels. Ed. Peter Harris. pp. 1–51. Elsevier Applied Science, London. MEDIN, A. (1995) Studies of Structure and Properties of Agarose Ph. D. Thesis. Acta Universitatis Upsaliensis, 126. MEER, W. (1980) Handbook of Water-Soluble Gums and Resins Ed. Robert L. Davidson. pp. 1–19. McGraw-Hill Book Company, New York. MODLISZEWSKI, J. (1990) ‘Food uses of Gelidium Extracted Agars’ International Workshop on Gelidium. Santander (Spain) 3–8 Sept. 1990. OKAZAKI, A. (1971) Seaweeds and their uses in Japan. Tokai University Press. PAYEN, M. (1859) ‘Sur la gelose et les nids de salangane’ C.R. Acad. Sci. Paris 1856: pp. 521–32. PERCIVAL, E. G. V., SOMERVILLE, J. C. and FORBES, L. A. (1938) ‘Isolation of an Anhydro-L-Galactose derivative from agar’ Nature, 142, pp. 797–8. REES, D. A. and WELSH, E. J. (1977) ‘Secondary and tertiary structure of polysaccharides in solution and gels’ Angew. Chem. Int. De. Engl. 15, pp. 214–24. SELBY, H. H. and WYNNE, W. H. (1973) Industrial Gums; Polysaccharides and Their Derivatives pp. 29–48. Eds Whistler, R. L. and BeMiller, J. N. Academic Press, New York. SMITH, H. M. (1905) ‘The seaweed industries of Japan’ Bulletin of the US Bureau of Fisheries vol. 24; pp. 135– 65. STANLEY, N. F. (1995) Food Polysaccharides and their applications ed. Alistair M. Stephen. pp. 187–204. Marcel Dekker, New York. VIGNON, M. R., ROCHAS, C., CHAMZY, H., VUONG, R. and TEKELEY, P. (1994) ‘Gelidium sesquipedale. Gelidiales, Rhodophyta). II. An ultrastructural and morphological study’ Botanica Marina 37, pp. 331–40. YANAGAWA, T. (1938) ‘The influence of sodium hidroxide on mucilagenous extracts of red algae’ Bull. Jap. Soc. Sci. 6; pp. 274–6.

3 Starch P. Murphy, National Starch and Chemical, Manchester

3.1

Introduction

No other single food ingredient compares with starch in terms of sheer versatility of application in the food industry. Second only to cellulose in natural abundance, this polymeric carbohydrate was designed by nature as a plant energy reserve. Man, however, has extended the use of starch far beyond this original design. Modifications – physical, chemical or biochemical – mean that numerous highly functional derivatives have enabled the evolution of new processing technologies and market trends. To this end, speciality starches have been tailored to: create competitive advantage in a new product; enhance product aesthetics; simplify a label declaration; reduce recipe/production costs; increase product throughput; eradicate batch rejects; ensure product consistency; and extend shelf life. Clearly, understanding the capabilities of starch and how to exploit its potential is of relevance to all stages of a food product’s lifecycle from development, through production and marketing, to retail.

3.2

Manufacture

3.2.1 Sources To ‘plug’ into the sun is to trap the cleanest and ultimate source of energy for the planet. Photosynthesis does precisely this. Leaves – nature’s ‘solar panels’ – trap light energy and, through a cascade of physico-chemical processes, involving carbon dioxide and water, translate this into sugar molecules, such as glucose. In itself, glucose is too mobile to act as a long-term energy storage system. Nature’s solution to this is to immobilise the glucose by forming a polymer; more precisely, a condensation polymer in which glucose chains are linked together by the elimination (condensation) of water. This is starch, an anhydroglucose polymer. Starch is a member of the ‘polysaccharide’ group of polymers.

The author would like to thank M. Croghan, J. Scott and K. Hughes, National Starch and Chemical and Dr B. Murphy, MMU for the information and support provided.

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It is laid down as insoluble, compact and microscopic semi-crystalline granules of size 1– 100m. In the human dimension, this means that 1g of starch typically contains something of the order of 1,000,000,000 granules with each granule, in turn, containing about 10,000,000,000,000 starch molecules. Seeds, tubers, roots and stem piths are the repository sites for the starch. When digested, the trapped energy is released as the starch is broken down by hydrolysis back to its constituent glucose molecules and thence further back to the original carbon dioxide and water. Despite the ubiquity of starch in nature, the number of commercially viable sources is small. The most important origins of starch are maize, potato, wheat, tapioca and rice. ‘Corn’ and ‘maize’ starches are American and European terms that actually refer to the same range of starches. The cereal crops maize, waxy maize and wheat are grown in America and Europe whilst potato is largely derived from the cooler climes of northern Europe. Most cassava (the base crop for tapioca starch) is sourced from Brazil, Thailand and Indonesia, while rice originates mainly from Asia.

3.2.2 Extraction The manufacture of starch employs a variety of processes which isolate purified starch from the other constituents of the raw material. For example, the extraction of maize starch from the maize kernel is a process known as wet milling in which the starch is separated from the fibre, oil and tightly bound protein. Regardless of the extraction processes involved, the objective is to recover the insoluble starch as undamaged or intact granules. In this form it is known as native starch. It can be washed and dried or left as a slurry for future processing into modified starches.

3.2.3 Processing Unprocessed native starches are structurally too weak and functionally too restricted for application in today’s advanced food technologies. Processing is necessary to engender a range of functionality. Processing can be chemical, biochemical or physical. Table 3.1 serves as a ready reference to the type of modification, its effect on the native starch (the objective) and the functionality produced (the benefit). The effects of modification processes on starch structure are explored in Section 3.4. Chemically modified starches are, in most cases, labelled as ‘modified starch’. Physically treated starches (without any additional chemical modification) carry a simple ‘starch’ label. This is elaborated in Section 3.7.

3.3

Structure

3.3.1 Starch molecule The glucose polymers that make up starch come in two molecular forms, linear and branched. The former is referred to as amylose and the latter as amylopectin. The basic glucose building block is a ring-shaped molecule with six atoms in the ring, see Fig. 3.1. Although for simplicity often drawn as a flat structure, the ring is, in fact, mobile and can take many shapes through ‘puckering’. Of these many shapes or conformations one – known as the chair form – is favoured. Moreover, the chair form comes in two varieties (stereoisomers): -D-glucose and -D-glucose. The two are interconvertible using heat and one is transformed into the other by a series of twists around the six-membered ring

Starch Table 3.1

43

Modifications of starch

Modification*

Objective

Benefit to user

Cross-linking

Strengthen starch granule

Improved process tolerance to heat, acid and shear

Ambient stable products

Delay viscosity development by retarding granule swelling

Production efficiency: increased heat penetration allowing shorter process time

Bottled sauces

Stabilisation

Dextrinisation

Typical uses

Sterilised soups and sauces

Prevent shrinkage of starch Excellent chill and freeze/thaw granule and provide stability to extend shelf life stability at low temperatures

Chilled and frozen processed foods

Lower gelatinisation temperature

Easy to cook in high solids systems

High brix fillings and toppings

Break down and rearrange starch molecule providing lower viscosity, increased solubility and a range of viscosity stability from liquid to gel

Easily handled or applied at higher dosage than parent native starch for desired effect

Fat replacers

Create film-forming properties

Protective coatings in confectionery

Bakery glazes

Blistered, crispy coatings in fried snacks Enzyme conversion (Biochemical modification)

Produce varied viscosity, gel strength, with thermoreversibility and sweetness

Contributes texture and rheology

Fat mimetics

Economic dispersant

Flavour carriers Dry mix fillers

Acid thinning

Lower viscosity and increase gel strength

Enhances textural properties at higher usage concentrations of starch

Gums, pastilles, jellies

Oxidation

Introduce carbonyl and carboxyl groups which increases clarity and reduces retrogradation of cooked starch pastes

Improves adhesion of coatings

Battered meat, poultry and fish

Creates soft stable gels at higher dosage than parent native starch

Confectionery

Provide lower viscosity and low temperature stability Lipophilic substitution

Introduce lipophilic groups Emulsion stabiliser which improves quality of any fat/oilcontaining product Reduces rancidity by preventing oxidation

Beverage, salad dressings Flavourencapsulating agents

Pregelatinisation

Pre-cook starch to give cold water thickening properties

Cold water thickening eliminates need to cook, offers convenience and energy savings

Instant soups, sauces, dressings, desserts, bakery mixes

Thermal treatment

Strengthen starch granule

Unique functional native starch with ‘Starch’ on label declaration

Ambient stable products

Improved process tolerance to heat, acid and shear

Bottled sauces

Delay viscosity development by retarding granule swelling

Production efficiency: increased heat penetration allowing shorter process time

Sterilised soups and sauces

* Combination treatments are also commonly used to achieve the desired objective. The most popular are crosslinking/stabilisation or cross-linking/stabilisation/pregelatinisation.

Fig. 3.1

Linear and branched starch polymers.

Starch Table 3.2

Starch granule characteristics

Starch

Type

Maize (b)

Cereal 5–30

Waxy Maize Tapioca

Cereal 5–30 Root

4–35

Potato

Tuber

5–100

Wheat

Cereal 1–45

Rice

Cereal 3–8

Sago

Pith

High Amylose Maize

Cereal 5–30

Diameter Morphology microns (m)

15–65

Round Polygonal Round Polygonal Oval Truncated ‘kettle drum’ Oval Spherical Round Lenticular Polygonal Spherical Compound granules Oval Truncated Polygonal Irregular Elongated

45

Gelatinisation Pasting temp. ºC temp. ºC (a)

Amylose Cooked content properties

62–72

80

25

Opaque gel

63–72

74

<1

62–73

63

17

Clear cohesive Clear cohesive, tendency to gel

59–68

64

20

58–64

77

25

Clear cohesive, tendency to gel Opaque gel

68–78

81

19

Opaque gel

69–74

74

26

Opaque gel

63–92 (c)

> 90

50–90

Very opaque, very strong gel

(a) Measured for 5% starch suspension. (b) Maize is also often referred to as ‘corn’, ‘dent corn’ or ‘regular maize’. (c) High amylose maize starches are not completely gelatinised in boiling water.

which cause the groups to ‘wag’ sequentially in a molecular-scale ‘Mexican wave’ around the ‘arena’ of the glucose ring. It is the -D-glucose that is used in nature to form the starch polymers. Once polymerised into starch, -D-glucose is locked into this chair form. When the ring atoms are numbered as in Fig. 3.1, it is clear that the links (glucosidic links) between carbons 1 and 4 of neighbouring units give rise to amylose, while occasional branches from this linear chain between carbons 1 and 6 give rise to the larger, more highly branched amylopectin. In fact, the ‘linear’ amylose has a small degree of branching but it is predominantly regarded as a single chain. The chain length can vary with the botanical origin of the starch but will be of the order of 500 to 6000 glucose units. Because of its more simple polymeric structure, amylose has a greater propensity to deposit in a regular manner forming crystals. In nature, three crystalline forms of amylose, A, B and C exist, depending upon the source: cereals (A), tuber (B) and certain pea and bean varieties (C). Precipitated starch complexes (with iodine, long-chain alcohols and fatty acids) are found in the V form. The so-called linearity of the amylose is further complicated by a twisting of the polymer into a helix. It is different degrees of hydration of the helix that gives rise to the A, B and C forms. In contrast to amylose, each branched chain of amylopectin contains only up to 30 glucose units. However, the multitude of branching in amylopectin gives it a molecular weight that is 1000 times that of amylose. Indeed, amylopectin is a titan of a molecule from nature: one of the largest with a molecular weight of 400 million. The ratio of amylose and amylopectin in any native starch is dependent not only on its source, Table 3.2, but also on selective crop breeding, a process known as hybridisation. Waxy and high amylose starches are widely used examples of this.

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3.3.2 Starch granule Figure 3.1 also shows that the starch backbone has numerous ‘OH’ (hydroxyl) groups projecting into the surrounding space. Hydroxyl groups have a particular affinity for other hydroxyl groups and can serve as a driving force in bringing starch chains together in an ordered manner through hydrogen bonding (Section 3.3.3). Where such ordering occurs, crystalline regions are deposited in the granule. The remaining regions of unordered starch are referred to as amorphous. It is the crystalline regions that give the granule its structure and facilitate identification of a raw (uncooked) starch. Under a microscope, starch granules illuminated with polarised light, show a characteristic ‘Maltese Cross’ pattern. This phenomenon is also known as birefringence. The microscopic appearance of each granule is diagnostic of its source: Table 3.2 – morphology. Microscopic identification can be enhanced using iodine to stain the amylose to a characteristic blue-black colour. This is the result of an association between the two to form a complex in which the amylose forms a helical coil around iodine molecules. Waxy maize starch, which contains negligible amounts of amylose, does not form a complex and as a result takes on the red/brown solution colour of iodine.

3.3.3 Hydrogen bonding Hydroxyl groups are the very same groups that make up water – the usual medium for starch in the food industry. There is consequently a strong interaction (hydration) and affinity, through hydrogen bonding, between the colossal starch and the diminutive water molecules. Hydration, when brought about by cooking, produces an irreversible change in the structure of the starch granule whereby the starch–starch interactions are ‘unzipped’ and replaced by starch–water interactions. This forces the chains apart and the granule swells. Eventually the granule ruptures and starch polymers are dispersed in solution producing a viscous colloidal state. This is a form of water management that controls structure and texture in food products. ‘Gelatinisation’ and ‘pasting’ are, respectively, the technical descriptions of the hydration within the granule and the irreversible granule swelling that builds viscosity. Dispersal of the simpler and more linear form of starch, amylose, allows for greater mobility which can result in the molecules self-assembling into a more ordered structure. Aligning themselves parallel to each other, the hydrogen bonding that previously may have involved water, can be reduced and replaced by hydrogen bonding between the aligned chains. Ordering in this manner produces a three-dimensional network that constitutes an opaque gel. Otherwise referred to as ‘retrogradation’ or ‘set-back’, only cooking and cooling can bring about this phenomenon.

3.4

Modifications

Starch modifications are a means of altering the structure and affecting the hydrogen bonding in a controllable manner to enhance and extend their application. The alterations take place at the molecular level, with little or no change taking place in the superficial appearance of the granule. Therefore, the botanical origin of the starch may still be identified microscopically. Each chemical and biochemical modification described below is represented schematically in Fig. 3.2.

Starch

Fig. 3.2

47

Chemical and biochemical modifications of starch.

3.4.1 Cross-linking Cross-linking is the most important chemical modification in the starch industry. It involves replacement of the hydrogen bonding between starch chains by stronger, more permanent, covalent bonds. In this manner, the swelling of the starch granule is inhibited, pre-empting disintegration either by chemical attack, mechanical attrition (shear) or cooking. Simply put, the starch granule is, in the molecular dimension, ‘spot welded’ at random locations to reinforce it. Distarch phosphates and distarch adipates are the most commonly encountered cross-linked starches wherein a phosphate or adipate bridge is present; the latter containing a longer bridge than the former. Because of the more permanent nature of the covalently bonded bridge or cross-link, only a small degree is necessary to produce beneficial effects: typically one cross-link per 100–3000 anhydroglucose units of the starch. As the number of cross-links increases, the starch becomes more resistant to gelatinisation. Consequently, cross-linked starches offer acid, heat and shear stability over their parent native starches.

3.4.2 Stabilisation Stabilisation, the second most important modification, is usually used in conjunction with cross-linking. The primary objective of stabilisation is to prevent retrogradation and thereby enhance shelf-life through tolerance to temperature fluctuations such as freezethaw cycles. In this modification, bulky groups are substituted onto the starch in order to take up space and hinder (steric hindrance) any tendency for dispersed (cooked), linear fragments to re-align and retrograde.

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Handbook of hydrocolloids

The effectiveness of stabilisation depends upon the number and nature of the substituted group, of which there are primarily two food-approved types: acetylated and hydroxypropylated. The Degree of Substitution (DS) is a measure of the number of substituents per 100 anhydroglucose units. Low DS starches, those with a DS below 0.2 (i.e. 2 substituents per 10 anhydroglucose units), are the most important commercially. As the DS level is raised, the starch–starch interactions in the granule are weakened and, consequently, hydration and gelatinisation by cooking is achievable at lower temperatures. Such starches benefit from easy cooking and are particularly useful in low-moisture environments and where the moisture level is restricted by competition from co-ingredients.

3.4.3 Conversions Conversion is a collective term for a range of chain–cleavage reactions of starch. Acid hydrolysis Acid-thinned, thin-boiling and fluidity starches are all terms which refer to starches which have been subjected to acid hydrolysis. This form of hydrolysis differs from dextrinisation in that it is conducted in aqueous conditions. The acid predominantly attacks and depolymerises the amorphous regions of the granule such that when the starch is heated beyond its gelatinisation temperature the granules rupture quickly. The effect on cooking is a lower hot viscosity and, due to the increase in the ratio of smaller, linear molecules, a stronger gel develops on cooling (set-back) compared to the native parent starch. Oxidation The production of oxidised starches employs alkaline hypochlorite as a reagent. Two important modifications occur: the relatively bulky carboxyl (COOH) and carbonyl (CO) groups are introduced together with partial depolymerisation of the starch chains. Oxidised starches, like acid-thinned starches, exhibit a significantly reduced hot viscosity due to breakdown of the starch beyond its gelatinisation temperature. Unlike acid-thinned starch however, the steric hindrance of the bulky groups disrupts any tendency towards re-association (set back) of the shorter chains thereby significantly reducing the gel strength. This is an advantage of oxidised over acid-thinned starches. ‘Bleaching’ is, in fact, a milder form of oxidation with less than 0.1% of carboxyl groups. Dextrinisation Dextrinisation, also known as pyroconversion, refers to two aspects of the structural modification of starch. The first is a partial depolymerisation achieved through hydrolysis. Hydrolysis is the reverse of condensation. It is the addition of water across a bond resulting in cleavage of that bond. It is usually brought about by dry roasting the starch either alone, making use of its natural 10–20% moisture content, or in the presence of catalytic quantities of acid. This gives rise to a range of polymer fractions of varying chain length (low conversion). The second aspect involves a recombination of these fragments (repolymerisation) but this time in a branched manner (high conversion). The starches so produced are called dextrins or pyrodextrins. They are typically classified as white dextrins, yellow dextrins or British gums depending on their range of viscosity, cold-water solubility, colour, reducing sugar content and stability.

Starch

49

Enzyme hydrolysis Selective enzyme hydrolysis is a form of biochemical modification. This reaction can result in a wide range of functionalities. Depending on the extent of enzyme hydrolysis, a range of chain lengths corresponding to glucose (dextrose), maltose, oligosaccharides and polysaccharides can be produced. The exact composition can be determined by measuring the dextrose equivalent (DE), where a DE of 100 corresponds to pure dextrose and a DE of 0 to the native starch. Hydrolysates with a DE below 20 are referred to as maltodextrins whilst those with a DE between 20 and 100 are referred to as glucose syrups. A range of enzymes is employed to produce such a spectrum of hydrolysates. These commonly include amylases for breaking up the straight chains as well as other enzymes to break off the branched segments of starch. -Amylase selectively and randomly attacks the 1,4-linkages of the starch to produce maltodextrins and low DE syrups. -Amylase is more thorough and attacks every other 1, 4-linkage to give lower molecular fragments and higher DE syrups, e.g., maltose which has a DE of 50. Other commonly used enzymes include iso-amylase and pullulanase which also give high DE syrups through hydrolytic attack at other specific sites on the starch, such as de-branching reactions at the 1, 6-linkages.

3.4.4 Lipophilic substitution The hydrophilicity of starch, its propensity to interact with water, can be transformed into a schizophrenic hydrophilic-hydrophobic duality. This is particularly useful for stabilising interactions between materials such as oil and water. To achieve this the already hydrophilic starch must be given a characteristic that mirrors that of an oil, namely, a long hydrophobic, i.e. lipophilic, hydrocarbon chain. Octenylsuccinate groups containing an eight-carbon chain provide the lipid-mimicking characteristic. Starch octenylsuccinates are attracted to, and stabilise, the oil–water interface of an emulsion. The glucose part of starch binds the water while the lipophilic, octenyl part binds the oil. In this way complete separation of the oil and water phases is prevented.

3.4.5 Pregelatinisation Pregelatinisation is a physical rather than a chemical modification. Certain starches require cooking to develop their function. These are referred to as ‘cook-up’ starches and the process of pregelatinisation is designed to remove the necessity for cooking. Pregelatinisation may be applied to native or modified cook-up starches to achieve a versatile range of cold thickening starches. The starch is pre-cooked or ‘instantised’ by simultaneously cooking and drying using one of the following processes: • drum drying (starch suspensions or starch pastes) – very widely used • extrusion (semi-dry starch) – rarely used • spray-drying (starch suspensions) – increasing in use Drum-dried starches give a slightly lower viscosity than their base starch because of the damage sustained by the granules when the drum-dried flakes are ground to the desired particle size, of which there are two, fine and coarse. Further, the finer the particle size required, the greater the damage incurred. Consequently drum drying, albeit a traditional and economic method of processing, is not without its drawbacks. Moreover, the fine and coarse grades perform differently in respect of dispersion, smoothness and

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Handbook of hydrocolloids

rate of viscosity development. As a result, spray-dried starches are finding popularity. They offer properties that are closer to their base cook-up starch.

3.4.6 Thermal treatment As recently as 1997 it was claimed that: ‘the search for physical modification techniques that are ‘‘greener’’ and ‘‘more natural’’ alternatives to chemical modification continues . . . but today there is no economically viable alternative to chemical modification to meet the many requirements of food processing in the late 1990s and beyond’ (Imeson, 1997). This market deficit was remedied while the above article was in press in 1996. This is a true revolution in starch technology and offers manufacturers ‘thermally-treated’ or ‘functional native starches’ – new terms in the field – with all the process tolerance of chemically modified starches whilst, crucially, remaining classified as ‘native’. Processing involves physical rather than chemical modification under proprietary technology. These functional native starches are simply labelled as ‘starch’, thus avoiding the need for the somewhat confusing ‘modified starch’ label.

3.5

Technical data

3.5.1 Structure–function relationship Native starches The technical attributes of a native or modified starch are brought to the fore through cooking. Depending upon the origin or modification, phases such as gelatinisation (1), pasting (2) and retrogradation (3) can be achieved within temperatures and times that are suited to the food product in which the starch is an ingredient. These three phases, characterised by the viscosity profiles of Fig. 3.3, are captured using a viscometer such as the Brabender Viscoamylograph, and the light microscope. The viscoamylograph is the industry-standard instrument used to control mixing, heating and cooling during viscosity measurement. The unit of viscosity measured in this way is the arbitrary Brabender Unit and measurements are always made relative to an internal standard. It must be stressed that Brabender measurements are not comparable with viscosity measured by rheometers such as, the Brookfield or Bostwick instruments. The viscosity changes of the native starches are related to the structural transformations of swelling, rupture, dispersion and set-back (all of which have been detailed previously in Section 3.3.3). These transformations can be observed using the light microscope with iodine-staining for further enhancement. The Brabender profiles of Fig. 3.3 illustrate the limitations of native starches. In the figure, the viscosity changes are related schematically to changes in granular morphology, or shape. During the initial stages of heating, no viscosity change is noted as hydration occurs at the molecular level within the granule. This is the gelatinisation stage which, in the absence of a detectable viscosity change, is better gauged using the microscope. The first indication of gelatinisation is the loss of the diagnostic ‘Maltese Cross’ or birefringence as viewed under plane polarised light. At this stage the hydration and swelling are reversible. If cooled and dried at this juncture, sufficient molecular ‘memory’ remains in the starch interactions for the granule to revert to its original state. However, once the hydration has reached the critical pasting stage, a rapid onset in the development of viscosity is seen. At this point the structural changes in the granule are irreversible. Pasting temperatures differ with starch origin

Fig. 3.3

Changes in traditional native starch during processing.

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Handbook of hydrocolloids

due to the varying accessibility of the granule to hydration. The accessibility is determined by: the ratio of amylose to amylopectin; the concentration of lipid material; and other factors such as the detrimental effect of phosphate groups. The cereal starches, maize and wheat, in contrast to waxy maize, tapioca and potato, reach their pasting temperatures at a slower rate due to an amylose-lipid complex that reinforces the granular structure. With continued heating the paste viscosity climbs to a peak and then decreases as the granules rupture. This is over-cooking. On cooling, retrogradation and gel formation in cereal starches sets in quickly and strongly due to the reassociation of a high proportion of small and mobile (dispersed) amylose polymers. Significantly, waxy maize contains next to no amylose thereby allowing the granules to swell more freely to a peak viscosity and ultimately to produce, not a gel, but a thick, colloidal amylopectin dispersion. Potato starch exhibits the greatest swelling due its larger granule size. In this case, the very rapid viscosity development at low temperatures is due to naturally occurring phosphate groups which are responsible for starch-starch repulsions that weaken the granule and accelerate its rupture. The phosphate groups also impart a strong salt/electrolyte sensitivity. For native starches, the process tolerance is extremely fragile and, in general, native starches suffer from over-cooking. Process-tolerant starches In modified starches that are cross-linked, the peak viscosity region is drawn out into an extended viscosity plateau. In this region, the modified starch is tolerant of over-cooking and retains its high viscosity-building potential (Fig. 3.4). Thermally treated starches can now achieve the same profiles.

Fig. 3.4

Effect of cross-linking and stabilisation.

Starch

53

Stabilisation is used in conjunction with cross-linking to produce process tolerant starches with freeze-thaw stability. In addition to enhanced process tolerance, stabilisation also lowers the gelatinisation temperature.

3.5.2 Nutrition Fat mimetics Marketing initiatives in the ‘diet’ and ‘health’ sector of the food industry presents new challenges for the food industry. The thrust of starch technology in this direction has been to support the development of new wholesome and balanced foods without sacrificing the desirable characteristics associated with the traditional high-calorie fat-containing food products. In fat-reduction programmes, the key functions of starches are in achieving a significant calorie reduction coupled to the rebuilding of features which hitherto would have been provided by the fats in the product. These are viscosity, body and mouthcoating. The traditional approach is the partial replacement of fat using starches which, when dissolved in water, create stable thermoreversible gels. Soft, fat-like gels can be created by conversion modifications to the degree necessary to produce thermoreversible, spreadable gels. Typically, 25–30% solids, i.e. starch in water, form an optimal stable structure for fat replacement. New generation fat replacers are tailored to mimic more closely the many and complex properties of fats or oils in a particular application. These are referred to as fat mimetics. Whilst normal, viscosifying starches require intact, swollen starch granules for maximum viscosity and stability, fat mimetics generally require granule rupture and solubilisation for their functionality. Therefore, the lubricity they are designed to contribute is not generally affected by extended heating, shear or acid. Maximising the synergies of functional ingredients such as hydrocolloids generally in combination with specific starch fat mimetics can mean that 100% fat reduction is achievable. Resistant starch In modern societies, great emphasis is frequently placed on the relationship between health, lifestyle and diet. With major infectious disease under control, focus has shifted from reactive cure to proactive prevention. Given the nutritional connotation that ‘you are what you eat’, there is much debate surrounding food fortification. Resistant starch (RS), be it natural or commercial, has a role to play with regard to the nutritional benefits of fibre fortification. RS goes under many definitions but, in essence, it is starch that is resistant to digestion in the stomach and small intestine. RS offers advantages over cellulosic sources of fibre such as bran. It provides low water-holding capacity thereby aiding processing; it enhances the organoleptic qualities of food as a replacement for, or complement to, natural fibre and it can be labelled as ‘dietary fibre’. Although research in this area is still in its infancy, there are potential physiological benefits in relation to the biochemistry of the colon and glucose/insulin metabolism. In the former, it is said that ‘desirable’ microflora are encouraged in the gut from the prebiotic action of RS leading to improved colonic health. In the latter, it offers a controlled rate of digestion to glucose without the peaks and troughs produced by less complex carbohydrates. There are four types of resistant starch. RS II, III and IV are commercially available. RS I is the physically inaccessible starch found in grain, seeds and legumes. In general, it is not suited as a food ingredient since processing can destroy it. RS II is a granular starch which, in an uncooked state, is naturally resistant to enzyme attack. It is found in green

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bananas, potatoes and very high amylose starch. RS III and IV are formed through: thermal modification, e.g., as in bread crusts, cornflakes or retrograded high amylose starch; or chemical modification, e.g., as in repolymerisation to alter the glycosidic linkages such that they are no longer recognised by -amylose. Commercial sources of RS assay at 30–60% dietary fibre (as measured by the Prosky AOAC method). Assuming 50% of the RS product is indigestible (i.e. reaches the colon) then for labelling purposes it can be considered as non-calorific and has a theoretical calorific value of only 1.9 kCal/g. This makes resistant starch products ideal ingredients for the fibre fortification of lowcalorie products. Note: Given that material reaching the colon does provide energy to the body the actual physiological calorific value will be approximately 2.8 kCal/g. Processing of high-fibre products has traditionally been fraught with problems related to the high water-binding capacity of cellulosic fibres. RS offers processing advantages, not only due to its low water-binding capacity, but also due to a negligible impact on dough viscosity and rheology, since it does not compete for water. There is even a textural benefit observed with RS in low moisture systems where, for example, cereals and snacks containing RS are more expanded and retain a light, crispy texture. This is in contrast to the texture attributed to oat bran-substituted snacks and cereals which are dense and hard, and accordingly have a reputation of limited palatability. However, a synergistic action of RS complimenting traditional cellulosic fibres can dispel this reputation and open up opportunities for dietary enhancement with texturally appetising and nutritionally balanced foods.

3.6

Uses and applications

3.6.1 Starch selection As a multifunctional and user-friendly ingredient, starches are found widely applied across the food and beverage industry. Matching the right starch within any of these applications requires a multitude of criteria to be considered. The process of starch selection can be rationalised through a knowledge of the range of features in a food product or process that starch can control or facilitate. These include sensory properties, method of manufacture, co-ingredients and shelf-life expectations. All are dealt with in greater detail in the following sections. Sensory properties Figure 3.5 displays some key sensory descriptions for appearance, structure, taste and mouthfeel together with examples of how such attributes can be achieved. Ingredient factors It is not uncommon, in complex food-product formulations, to encounter effects arising from ingredient interactions. Indeed, on occasions when synergistic interactions are observed, manufacturers will seek to optimise them and, where the opportunity arises, protect their invention through patents. In cooking a starch-based formulation, the effects of three critical co-ingredients should be considered. Acid Many foods contain acids either as a means of preservation or for flavour. In such cases, the acidity or pH of the food product is very important to the selection of a suitable starch. Viscosity can vary significantly as a function of pH, not only for native starch but also for

Starch

55

Fig. 3.5 Starch sensory attributes.

modified starch. Acids disrupt naturally occurring hydrogen bonding causing the starch granule to swell more easily and, in extreme pH conditions (e.g. pH 2.5), cooking can lead to premature rupture of the granules with an accompanying breakdown in viscosity. To overcome the effects of acid and provide maximum viscosity at minimum starch usage levels, the starch should be inhibited, i.e., strengthened by chemical cross-linking or thermal treatment, to the degree necessary. This will usually include consideration of the manufacturing process involved for the food product. Sugars In ‘high-solids’ or ‘high-Brix’ systems, the presence of water-soluble solids, notably sugars, can have a detrimental effect on starch hydration. By competing for, and preferentially tying up, the water necessary for hydration, sugars can cause an increase in the starch gelatinisation temperature which makes it more difficult to cook-out the starch and achieve the desired functionality. A formulation containing 60% sugar raises the starch gelatinisation temperature to above 100ºC. To achieve such temperatures in water, pressurised cooking would be needed to cook-out the starch. As an alternative in simple operations, the solution may be to add the sugar after the starch has been cooked; or, to limit the sugar addition to 20% whilst cooking the starch, the remainder of the sugar being added at the end. Complex manufacturing, however, may not be amenable to such alteration. In these circumstances, it is advisable to use a light to moderately inhibited starch which is also highly stabilised or, indeed, a pregelatinised starch. Hydroxypropylated, stabilised starches offer processing advantages in this respect. Their lower gelatinisation temperature allows easy hydration and cooking, even in high-solids formulations. Their low hot viscosity allows excellent heat penetration and affords a

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shorter process time. This, in combination with their better freeze-thaw stability, provides for a premium quality product with good shelf-life. Fats and oils The rate of viscosity development is often lowered in high-fat or oil products. This occurs due to the fat or oil coating the starch and delaying hydration. This feature can be taken advantage of to achieve easier dispersion and rehydration of pregelatinised starches. In food emulsions, lipophilic starches are used as emulsion stabilisers to ensure product quality and shelf-life stability. Process factors There are essentially three parameters to consider here, namely, time, temperature and shear during manufacturing and reconstitution. These parameters should not be viewed in isolation since, in most processed foods, at least two of the parameters are involved. Table 3.3 lists examples of processing equipment together with the conditions associated with them. In general, higher temperature, longer hold time and greater shear forces will promote granule swelling and, consequently, the starch will be more susceptible to rupture and breakdown. Tolerance to such processing factors is achieved by strengthening the granule through cross-linking or thermal treatment. Shelf stability The conditions that a finished food product has to withstand between production and consumption will also influence the selection of an appropriate starch. Ensuring a processed food reaches the consumer in the same ideal state as it left the factory, is the consideration of shelf stability. The most critical factor is temperature during storage, especially if the temperature fluctuates. Assuming the relevant modified starch in the product is optimally cooked, and the level of cross-linking is matched to the required degree of process tolerance, then a combination modification is required for temperature-storage tolerance. The key combination with cross-linking is stabilisation which prevents freeze-thaw intolerance, i.e., retrogradation, water loss or any textural changes. In this context, the more bulky blocking groups of hydroxypropylated starches, as opposed to those of acetylated starches, offer better freeze-thaw stability and longer term storage stability. Table 3.3

Effects of food processing*

Equipment Steam jacketed kettle Plate heat exchanger Scraped-surface heat exchanger Jet cookers Direct steam injection cookers Retorts Extruder cookers Flash cooling Piston pump Mono pump Centrifugal pump Colloid mill, homogenisers Microwave

Time

Temperature

Shear

Pressure

Long Short Short Short Short Very short Long Short

Low–medium Medium–high Medium–high Medium–high High High High High

Low High High Medium–high Very high High Low High High Low–moderate Moderate Moderate–high Very high

Low–medium High High

Low–High

* High temperature, shear and pressure are often referred to as ‘high stress’ processes. For such environments, specific modified starches will be required.

Starch

57

End use The end use of a product defines how it will be reconstituted or prepared for consumption. The meticulous consideration of factors affecting starch selection during manufacture should be extended to include those factors which will influence the starch cook during reconstitution, and which will consequently affect product quality. If a product is to be reheated, then the method used is important since, not only time and temperature, but shear may also need consideration. Flexibility in reconstitution is a selling point. For example, in a three-way cook using either hob, casserole or microwave, temperature and time are involved in two of them whilst the microwave additionally includes shear. Microwaves have a different mechanism for heating than the conventional oven cooker. Microwave radiation produces heat by ‘activating’ friction between dipoles such as the hydroxyl groups of molecules (water, starch and sugar have these in abundance). Consequently, microwave processing is a high shear environment and the assessment of the degree of cross-linking needed should therefore include consideration of the means of reconstitution. Fats, compared to water, exhibit little dipolar charge but their much lower specific heat means that they warm considerably faster than water in microwave cooking.

3.6.2 Applications Baked goods Wheat flour is the basis of many baked goods. It is an economic commodity but is overstretched in respect of modern products such as frozen, chilled, low-fat and gluten-free foods, where aesthetics and processing can be limiting without recourse to starch coingredients. Whilst formulations will vary significantly depending on the desired final product, typical ingredients, apart from starch, could include wheat flour (8–16% protein, 71–79% carbohydrate), fats, sugars, eggs, emulsifiers, milk and/or water. Processing conditions will also vary with the formulations. The effect of these ingredients and processes on the use of starch or modified starch can be exacerbated since baked goods have a limited amount of moisture. Gelatinisation of the starch (in the wheat flour as well as in the added starch) is critical to building structure and texture in bakery products. Since wheat starch will only thicken during baking, pregelatinised starches can be used to bind the limited water available early on. This creates a host of benefits: suspension of particulates (as in muffin mixes); reduction in the stickiness of doughs; improved handling and machinability; increased cake volume; improved water binding for increased moistness; and softer textures. A common problem in bakery products is staling caused by retrogradation. Stabilised starches, in particular hydroxypropylated, pregelatinised starches bind water more effectively thereby providing baked goods with enhanced shelf-life through extension of the perception of freshness. Native maize, waxy maize or tapioca starches are alternatives to wheat flour for gluten-free products for coeliac sufferers who are intolerant to wheat gluten. Glazes and icings are used to enhance aesthetics and add value to baked goods. A glaze is typically a thin wash containing sugar, water and/or milk, whilst an icing may be applied as a thickened layer and usually contains fats. Pregelatinised starches and, especially dextrins, find application here where the main functions are viscosity control, colouring, softening and texturing. Batters and breadings The concept of a breaded or battered coating is to create additional value for meat, poultry, seafood and vegetables. Flours are a major component in batters and breadings

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but a common problem for food manufacturers is fluctuation of the quality of commodity native flours. Speciality starches are used to solve this and extend the features of the native base flour. The wide array of substrates for coatings means that the accompanying wide range of storage and reconstitution methods become key considerations, in addition to textural attributes, in the selection of a starch. Starches in batters provide viscosity control which, in turn, controls the quantity and thickness of the batter layer; the adhesion efficiency; visual effects (smooth to blistered); texture; storage; and reconstitution stability. Pregelatinised starches are used to control the cold viscosity. These can be cross-linked and stabilised to provide shear and freezethaw stability for batter slurries which are to be recycled or used in chilled or frozen products. The textural attributes can be enhanced by high amylose starches for fried or oven-baked poultry and meat products. High amylose starches need higher temperatures, as in frying, to functionalise them. Stabilisation lowers the gelatinisation temperature and therefore makes the starches suitable for applications with lower cooking temperatures. Dextrins are used for vegetable coatings where they will enhance colour and, at high dosage rates (~30%), will create blistered effects to transform the surface of a coating. Very high levels of dextrins or incompletely gelatinised, high amylose starches, may cause stickiness. This problem is overcome by reducing the dextrin level or increasing the amylose content to reach the desired crispiness. To achieve a smooth, uniform coating at lower cooking temperatures, a modified high amylose starch is recommended. Since amylose has excellent film-forming properties, the incorporation of high amylose starches in batters can reduce oil pick-up in fried, battered products. Beverage emulsions and flavour encapsulation Lipophilic starches have replaced gum arabic, the traditional emulsion stabiliser, in concentrated flavour emulsions for soft drinks and in encapsulated ingredients, e.g., spray-dried flavours and creamers. Indeed, lipophilic starches can be extended beyond gum arabic applications to high-load encapsulation of flavour oils. Generally these starches provide improved oxidation resistance and low temperature emulsion stability. Confectionery Starches are found in a wide range of confectionery products, contributing from soft through to hard gels, and from brittle through to chewy textures. Starch is also active as the structure builder in coatings and even as the moulding medium to support the shaping of confections. Starches are selected primarily on their ease of cooking in high-sugar environments and their ease of handling during production. The majority of confections are high in sugar, sugar syrups or polyols, with solids in the region of 68–72%. In these products there will be considerable competition for water, with the starch suffering in that gelatinisation temperature is increased and the product more difficult to functionalise under 100ºC. There are several methods used in confectionery processing to functionalise ingredients: the traditional steam-jacketed kettle (low-to-medium temperature, low shear); heat exchangers (medium-to-high temperature, high shear); or direct steam injection, as in jet cooking (high temperature and very high shear). Converting amylose and high amylose starches to different degrees of hydrolysis creates a suitable range of acid-thinned or oxidised starches for confections. These starches have a range of low hot viscosities and consequently they allow rapid and efficient cooking of starch solutions in the presence of concentrated sugar syrups. A

Starch

59

further useful modification is stabilisation where the reduced starch gelatinisation temperature allows easier cooking, especially of high amylose starches, so that stronger gels can be achieved with increased clarity and extended shelf life for the confections. Converted starches are also used in confections on the exterior of products, as in pancoating. Dextrins, with their good film-forming properties are used with high-sugar solutions to create stable, flexible coatings as in jelly beans or shell-coated chocolates. Texture can be further modified by using starches in combination with other hydrocolloids. Starches also serve as a process aid rather than an ingredient in confections. The shaping of confectionery pastes normally occurs in starch moulds where the desired shape or design has been imprinted into trays of moulding starch. The moulding starch is also treated with a small percentage of mineral oil which enables it to hold the mould imprint and to minimise dusting during the manufacture process. The moulding starch has two key functions, its shape and the absorption of moisture. The moisture content of the moulding starch is critical in obtaining a high-quality confection. Above 9% moisture, the drying time is extended – this reduces production rates – whilst below 6% moisture, the confection has a crust of hardened exterior as the rate of moisture loss is too fast. Dairy products Dairy products have flourished as they are convenient at most daily eating occasions. The latest developments are primarily focused on chilled and frozen products, but there remains a substantial market for ambient and dry mix products. Chilled, frozen and ambient dairy products are processed in order to deliver the required shelf-life. Processing factors which influence the selection of starch are heat and shear. Heating is achieved through direct or indirect processing, and shear is predominantly achieved by the design of the heating equipment or by homogenisation of the product (in this instance before or after heating). Direct processing involves heating the product by steam injection (where steam is forced into product through a nozzle) or by steam infusion (where the steam and product are mixed in a chamber). These systems involve very high temperatures and high shear but short process times. Indirect processing relies on heat exchangers such as the ‘scraped surface’ or ‘tubular’ varieties. More viscous products are best handled through scraped surface heat exchangers where, despite their higher shear intensity, the shear profiles are still lower than those experienced in direct processing. Heat penetration is critical to the production of a high-quality, safe and stable dairy product. Low initial viscosity during processing contributes to a better heat penetration which, in turn, means increased product throughput with less fouling and downtime, together with improved plant efficiency through shorter process times. Hydroxypropylated and cross-linked, waxy maize or tapioca starches offer lower hot viscosities than their acetylated counterparts and yet produce up to 50% greater cold viscosity after 24 hours storage. Hydroxypropylated starches also provide better freeze-thaw stability and produce richer, creamier textures. They are more effective thickening and stabilising agents in low-fat or low-calorie dairy products due to their greater compatibility with milk protein. Retorted (canned) dairy-based products can bring out the advantages of using hydroxypropylated starches. By comparison, acetylated starches may de-acetylate, creating dramatic localised changes in pH which cause the product to curdle or precipitate. Achieving the desired texture of desserts is dependent upon an optimal cooking of the starch. Understanding the impact of shear on the product is necessary to select a crosslinked or thermally treated starch which provides the level of process tolerance necessary

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for production. Upstream homogenisation of products at temperatures below the gelatinisation temperature of starch, 60ºC, will not adversely affect starch functionality. Downstream homogenisation is where the starch is subjected to high shear well above its gelatinisation temperature. In this instance, very highly cross-linked or thermally treated starches are recommended in order to deliver an optimum viscosity. Starch selection will be influenced not only by the process requirements but also by the product characteristics. Blandly flavoured starches, such as modified tapioca starches or certain thermally treated starch, are particularly important in dairy products due to the high dosage levels required. Starch produces a higher viscosity when cooked in whole milk (3.5% fat) than in either skimmed milk (0.5% fat) or water. Typically, 1–3% starch is used for pouring consistencies, 3–4% for medium viscosity and 4–6% for thick, spoonable textures. Gelled textures, as in cheese analogues, are achievable with some converted starches or high amylose starches. Gelled dairy desserts will also use other hydrocolloids, notably carrageenans, in combination with starch to create the desired textures. Fruit preparations Regardless of season, there is now a wide range of fruit bases available throughout the year. For the food manufacturer, this presents a range of conditions, such as pH, to be contended with. This is compounded further by the fact that, even within season, there will be a further natural variation in pH (3–4.5) and pectin within and between fruit types. To achieve consistent fruit preparations, a thickener or stabiliser is required to even out these variations. But the thickener and stabiliser must also be able to tolerate the processing involved to provide the desired texture, freeze-thaw or bake stability. Fruit preparations find application in: yoghurts, layered dairy desserts and ice-creams; as fillings for baked goods, e.g. doughnut; and, as toppings for pastries such as cheesecakes. Formulations will include 20–90% fruit, and sugar solids from 7% (no added sugar) to 70%. Sugar solids (measured as percentage Brix) above 40% increase the gelatinisation temperature beyond 100ºC. In these circumstances a jet cooker is preferred over the kettle variety to functionalise the starch. Alternatively, adding the sugar at the end of the process, allows the starch to fully gelatinise. In systems where a ‘one-shot’ addition of dry ingredients is only possible, then cross-linked and stabilised, pregelatinised starches or highly stabilised and light-to-moderately cross-linked cook-up starches are suitable as thickeners. The starches must be capable of developing sufficient viscosity to suspend the fruit, yet be pumpable and mixable with, for example, a yoghurt base. Fruit pieces are particularly delicate during cooking. Therefore, reducing the process time through an increased heat penetration will protect fruit integrity. This is achieved by selecting a cross-linked and stabilised starch which will thicken enough to suspend the fruit pieces; provide a low viscosity for effective heat penetration; provide acid stability; and excellent chill and freeze-thaw stability. Flavour release in fruit preparations is particularly enhanced with process tolerant, thermally treated starches. Gravies, soups and sauces This sector includes an enormous range of food products that span the spectrum of shelf stability from immediate consumption to very long ambient shelf life. Starch selection in this instance depends upon the production process which, in turn, is usually influenced by the pH of the product. Acidic products (especially pH < 4.5) will require a higher degree of cross-linking than neutral products, provided the heat process is the same. Normally acidic products will have a shorter process time or temperature to reach their target

Starch

61

sterilisation factor or F0 value. Cross-linking or, more recently, thermal treatment introduces process tolerance by strengthening the starch granule. In ‘wet’ sauces, processing factors to consider are preheat temperature and hold time; pasteurisation/ sterilisation temperature and time; method and rate of cooling; mechanism of pumping; and, indeed any other forms of shear used during cooking or in the introduction of difficult-to-disperse powders. Reference has already been made to the contribution of these factors during food processing and their effect on starch gelatinisation in Section 3.1.3. The shelf-life requirements of this group of products are achieved with stabilised starches which imply freeze-thaw stability. This feature is particularly important in the development of chilled, frozen and ambient-stable products which are recommended for refrigerated storage after opening. Hydroxypropylated starches are the best to use here, especially since their excellent compatibility with milk or milk proteins leads to an enhanced texture and mouthfeel of cream-based sauces. Besides processing and shelf-life considerations, there are two other important criteria, fill-viscosity and heat penetration. The function of starch as a fill-viscosity aid is to provide sufficient high viscosity during the initial stages of cooking, typically during preheat, that particulates are homogeneously suspended and then evenly distributed into the container. The high initial viscosity is also designed to prevent splashing and reduce unhygienic conditions around the production filling line. Fill-viscosity starches are commonly native starches, primarily waxy maize, which thicken easily and quickly to achieve their peak viscosity. They then breakdown during the sterilisation stage with little or no residual viscosity. The final desired viscosity is achieved by cross-linked and, usually, stabilised starches. This combined, modified starch facilitates heat penetration which supports a higher quality of product with shorter, optimal heat processing. Increasing the level of cross-linking delays viscosity development which is desirable for better heat penetration. However, there will be an optimum economic level for process tolerance. In general, viscosity-building starches are used at dosages of 2–6%, whilst fillviscosity starches may be used at 1–3%. Dry mixes are often required by consumers, food caterers and manufacturers for reasons of convenience, ease of handling and storage stability. Starch selection will depend on the specific requirements of the product and its use. In the case of food manufacturers, time, temperature, shear, pH and storage conditions apply as for wet sauces. Dry mixes for the consumer will demand dispersibility in hot or cold liquid as the critical factor. Whisk-and-serve applications require easy dispersion with fast hydration of the starch to achieve the target viscosity within a convenient time. Pregelatinised or easy-to-cook, ‘cook-up’ starches are recommended. However, pregelatinised starches may ‘lump’ as they try to wet-out too quickly, particularly in hot liquids. Lumping restricts viscosity development thereby compounding the problem. Agglomerated, pregelatinised starches can disperse more easily than the traditional drum-dried starches as they are more granulated. These easily dispersed starches are particularly desirable in low-sugar/salt formulations as there is less granulated material to blend. Alternatively, a highly stabilised, hydroxypropylated starch with a lower gelatinisation temperature will overcome the lumping to hydrate quickly and produce a rich, thick and creamy product. In some applications, e.g., microwave reconstitution or in cold pies requiring additional bake stability, a combination of pregelatinised and cook-up starch is used. The pregelatinised starch builds viscosity immediately to suspend the other ingredients, including the cook-up starch. This is essential in microwaveable dry mixes since stirring during cooking is impossible. The cook-up starch will contribute to the final viscosity and stability.

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Potato starch has the highest swelling capacity combined with a low gelatinisation temperature. Native or lightly modified potato starches find application in economy-style dry mixes. When products require greater flexibility, e.g., three-way cooking on the hob, casserole or microwave, followed by chilled or frozen storage, as in catering, then crosslinked or stabilised waxy maize or tapioca starches are preferred. Tapioca starches are more suited to delicately flavoured sauces as in fish dishes. Often dry mixes will require low-moisture starches (2–8%) depending upon the other ingredients and the required water activity for long-term shelf stability. Native starches such as maize or wheat, may be used to increase opacity and change mouthfeel or as bulking agents to aid dispersion. For all varieties of gravy, soup and sauce, when traditional native starches are used, the recommendation for superior product quality is one part native to two parts modified starch. This ratio ensures the beneficial effects of the modified starch compensates for the limitations of the traditional native starches. Thermally treated starch in total replacement of the modified starch, can retain the attributes of the modified whilst dispensing with the ‘modified’ label declaration (Section 3.7). Mayonnaise and salad dressings The main function of starches in this category is to thicken and stabilise. Cross-linked and stabilised starches are commonly found here. More recently, thermally treated starches are finding increased popularity. Two further starch types find specialised application here: lipophilic starches to stabilise emulsions, particularly in egg-free dressings and fat mimetics in low-calorie or fat-free formulations. The processing conditions are very rigorous for mayonnaise and salad dressings. Starches need to be able to tolerate the acidity, where pH can be as low as pH 2.8; a heat process, which generally involves ‘hotfill’ as a key sterilisation factor; shear for the emulsification, which will depend on the fat content; and the type of emulsification unit used. Products which are hot-filled and directly stacked onto pallets have a tendency to suffer from ‘stack-burn’. This occurs as the core temperature of the stack of product on the pallet remains relatively high for prolonged periods and product viscosity begins to break down as the starch is degraded. A highly cross-linked starch will provide additional heat and acid tolerance to overcome stack-burn. Starches are also used to ensure product quality over a long shelf life, which could be up to 12 months. While moderate-to-high cross-linked starches are essential for the heat, acid and shear tolerance stabilised, and in particular, hydroxypropylated starches, bring added benefits by introducing freeze-thaw stability. This is a benefit for catering and consumer products which require refrigeration after opening. Low-calorie or low-fat dressings also benefit from hydroxypropylated starches as the main thickeners since they develop rich, creamy textures. Mouthfeel can be further enhanced using starchbased fat mimetics. These are available as replacements for vegetable oils. They bring a unique rheology suitable for pourable dressings, or as thermoreversible gels they are suited to spoonable dressings. A gelled structure can be further enhanced using amylosecontaining starches but typically, they are used in combination with cross-linked and stabilised waxy maize starches since these contribute to the process and storage stability. Salad dressings and mayonnaise will vary in oil content. Here the processing equipment is designed to create the emulsion by dispersing the oil as fine droplets with a diameter of less than 1 m throughout the aqueous phase. Lecithin, the natural emulsifier in egg yolk and/or any added emulsifier, will stabilise the emulsion by reducing the surface tension of the oil droplets. This makes large droplets unstable and prevents ‘creaming’ (where oil migrates to the surface). Strictly, starches act as emulsion

Starch

63

stabilisers rather than as true emulsifiers. Viscosity-building starches achieve this by structuring the aqueous phase and restricting the movement of the oil droplets. Lipophilic starches are introduced under agitation for optimal dispersion so that they interact with both water and oil to produce a stabilising effect at the oil–water interface. Another advantage of using lipophilic starches is that, as modified starches, they may already be covered on the label declaration if a modified starch has been used as the viscosity builder – this simplifies the list of ingredients on the label. Meat products In processed meats, starches are used as water binders to increase yields, reduce cooking losses, improve texture, sliceability and succulence, and extend shelf life. Reformed or comminuted meat products require cooking to a minimum internal temperature of 72– 75ºC for product safety. In order to function at such low temperatures with little or no shear or acid, potato starches, which swell easily at low gelatinisation temperatures, are recommended. Waxy maize and tapioca starches, with a high degree of stabilisation to reduce their gelatinisation temperatures, have the additional benefit of excellent water binding during chilled or frozen storage. Lipophilic starches, too, find application in comminuted meats and pate´s as stabilisers of the emulsion and in the strengthening of the fat- and water-binding properties of the meat protein. Coarse grind, pregelatinised starches bind water quickly and increase the viscosity which assists the moulding of reformed or comminuted meats whilst enhancing a more open, coarse texture. Savoury snacks Savoury snacks are low-moisture products (15–30% water used in the dough, less than 2% moisture in the final product) and the choice of starch is critical since manipulating the formulation and process equipment will affect gelatinisation and, consequently, will influence the final product characteristics. ‘Expansion’ is a very important process for snacks. Several processes are available for expanding snacks: extrusion (puffed corn snacks); baking (low fat, sheeted baked snacks); frying (traditional crisps) and microwave (popcorn). Pregelatinised starches are recommended in snacks that are manufactured under low-to-medium shear and medium temperatures (75–120ºC). Cross-linked or thermally treated ‘cook-up’ starches are required to withstand the rigours of higher shear, pressure and temperature (160–180ºC) found in the extrusion process for directly expanded products. In snacks, texture and appearance are of high priority. The starch polymers of amylose and amylopectin exhibit a very obvious effect on these. In general, the highly branched amylopectin polymer increases dough viscosity and expansion, and a waxy maize starch therefore is the preferred base starch in the production of light, crispy expanded products. Amylose, in contrast, is responsible for strengthening the dough which improves forming, cutting and a harder, more crunchy final texture. In this instance, the linear chains can align very closely. Amylose also exhibits excellent film-forming properties which can be advantageous in the reduction of oil pick-up in fried snacks.

3.6.3 Viscosity-troubleshooting When a starch containing product fails to meet its viscosity specification, it is a common and expensive mistake to add further starch to correct the viscosity. This leads, at worst, to a scenario in which the initial product is overcooked and the additional starch is undercooked. The result will be an unstable product which will taste ‘starchy’. Rather

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Table 3.4

Clarity Viscosity/ texture Taste Stability

Viscosity troubleshooting – evaluation of starch cook*

Cloudy

Cloudy

Clear

Clear

Clear

Starch will sediment unless agitated

Thin

Heavy-bodied, short texture

Reduced, cohesive

Reduced, cohesive

Starchy

Starchy

Neutral

Neutral

Neutral

Poor

Poor

Good

Poor

Poor

* Light microscope @ 200 magnitude

than compensating for viscosity it is better to investigate the root cause. Ensuring that the starch is optimally cooked is a good place to start. Table 3.4 illustrates the diagnoses of the various stages of a starch cook. For example, in the case of undercooking, either further cooking to achieve a ‘good cook’ or the adoption of a less inhibited starch is required. The converse will apply for overcooking. Sliced and diced vegetables in a product can present viscosity problems due to natural enzyme activity from the high surface area of the vegetable. -Amylases are the source of the problem. The viscosity loss from enzyme hydrolysis of the starch can be so dramatic as to leave either ‘ghost granules’ or no granules at all under microscopic examination. The most problematic sources of -amylase are cabbage, onions and kidney beans. Unheat-treated wheatflour is an important and often over-looked cereal source of amylase. Heat treatment provides a straightforward means of inactivating the enzyme.

3.7

Regulatory status: European label declarations

Food ingredient suppliers are obliged to label their products with information which assists the food manufacturer when declaring the ingredient list of the final food product. Table 3.5 outlines the current status of native and modified starches; it highlights those that are classified as ingredients and those as additives. The majority of chemically modified food starches are approved as permitted food additives within the European Union and, as such, have been assigned E-number classifications. Food additives include artificial sweeteners, colours, preservatives, flavour enhancers, emulsifiers, stabilisers, thickeners and most modified starches. Modified starches which are classed as additives are defined as ‘substances obtained by one or more chemical treatments of edible starch which may have undergone a physical or enzymatic treatment and may be acid- or alkalithinned, or bleached’. Thermally treated starches, native starches, dextrins, starch modified by acid or alkali treatment, bleached starch and starch treated by enzymes are classed as food ingredients and have no E-number classification. Starch food additives are regulated under the terms of the EC Miscellaneous Additives Directive, 95/2/EC (and amendment 98/72/EC), on food additives other than colours and sweeteners. As a broad class of additive, modified starches (E1404–E1451) have ‘horizontal’ approval throughout the European Union for food use. They may be added to foodstuffs following the quantum satis principle (no maximum level indicated) unless otherwise

Starch Table 3.5

65

List of food starches permitted under European Food Law

Starch

E Number classification

Physically modified Enzymatically modified Dextrinised Acid treated Alkali treated Bleached Oxidised starch Monostarch phosphate Distarch phosphate Phosphated distarch phosphate Acetylated distarch phosphate Starch acetate Acetylated distarch adipate Hydroxypropyl starch Hydroxypropyl distarch phosphate Starch sodium octenyl succinate Acetylated oxidised starch

– – – – – – E1404 E1410 E1412 E1413 E1414 E1420 E1422 E1440 E1442 E1450 E1451

Classified as

Food product label

Ingredient Ingredient Ingredient Ingredient Ingredient Ingredient Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive

Starch Starch Dextrin or modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch Modified starch

specified or their use is restricted in food for which there is existing ‘vertical’, or specific, legislation, e.g., as in chocolate, fruit juices, jams, jellies, etc. For labelling purposes, manufacturers of food products sold to the consumer need only list the generic term ‘modified starch’ within the ingredient list. This must be accompanied by an indication of its specific vegetable origin, when the modified starch may contain gluten.

3.8

Bibliography

CROGHAN, M. (1998) ‘The search for a high fibre snack’ Kennedy’s Ready Meals & Snacks, vol. 1, no. 1, 58–60. CROGHAN, M. and MASON, W. (1998) ‘100 years of starch innovation’ Food Science & Technology Today, March,

17–24. EMSLEY, J. (1994) The Consumer’s Good Chemical Guide, London, W. H. Freeman & Co. Ltd. IMESON, A. (ed.) (1997) Thickening and Gelling Agents for Food, London, Chapman & Hall. LIGHT, J. M. (1990) ‘Modified food starches: why, what, where and how?’ Cereal Food World, 35 (11), 1081–92. MACDOUGALL, A. (1999) ‘Native vs Modified starch’, Food Manufacture, February, 16–18. THOMAS, D. J. and ATWELL, W. A. (1997) Starches, Eagan Press, USA. WHISTLER, R. L., BEMILLER, J. N. and PASCHALL, E. F. (eds) (1984) Starch: Chemistry and Technology, 2nd edn,

Academic Press, New York. (1986) Modified Starches: Properties and Uses, CRC Press, Florida. Food Starch Technology, National Starch and Chemical Brochure. ‘Stability and flavour in fruit preparations’, (1998) Milk Industry International, Technical and Research Supplement, MII, 11/98, 4–5.

WURZBURG, O. B.

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4 Gelatin D. A. Ledward, University of Reading

4.1

Introduction

Gelatins do not exist in nature but are derived from the parent protein collagen by processes that destroy the secondary and higher structures with varying degrees of hydrolysis of the polypeptide backbone. Its name is derived from the Latin gelata which describes its most characteristic quality, i.e. gel formation in water.

4.1.1 Manufacture The major international suppliers of gelatin are given in Table 4.1. Collagen, the basic raw material for gelatin production, is the major constituent of all white fibrous connective tissue occurring in animal bodies such as cartilage, sinews, the transparent sheaths surrounding muscles and muscle fibres, skin and ossein (the protein matrix of bone). Although the relative proportions and sequences of the constituent amino acids in collagen and gelatin are substantially the same, the physical and chemical properties of the two proteins differ markedly. For example, in dilute acid or alkali collagen will swell (hydrate) but not dissolve, whereas gelatin will dissolve. On mild heating (less than 50ºC) gelatin will readily dissolve to form a viscous solution at all pHs, collagen merely shrinks and loses its ability to hold water. For gelatin production the raw material may be any collagenous-containing material, but hides, skins or bones are the preferred sources. The raw material is washed to remove surface soil and other impurities. Bones are processed somewhat differently in that, after washing, crushing and rewashing they are subjected to counter current treatment with acid (usually 4–7% hydrochloric) over five to 14 days to decalcify or leach out the calcium phosphates and leave a residue of ‘bone collagen’ or ossein. The concentrated raw material, from all sources, may be processed directly or it may be dried and stored. Following the preliminary treatment described above the raw material is subjected to either acid or alkaline treatment depending on the ultimate use to I would like to thank Bernd Eggersglu¨ss and Peter Findlater of DGF Stoess and Mrs Joan Meakin for much helpful information.

Table 4.1

International suppliers of gelatin

Nordisk Gelatine, Denmark

Fredrich Naumann Gelatine Und, Germany

Sunny South Canners (PTY) Ltd, South Africa

COPIAA, France

CE Roeper GmbH & Co, Germany

Miquel Junca SA, Spain

Croda France SA

Thew Arnott & Co Ltd, UK

Protein SA, Spain

Gelatine Products Ltd, UK

Leiner Davies Gelatines, France

BM Burke & Co Ltd, Ireland

Univar Food Ingredients Ltd, UK

P Leiner (GB) Ltd, UK

Protilact, France

Figli di Guido Lapi, Italy

Biogel AG, Switzerland

Lion Foods Ltd, UK

SBI Systems Bio Industries, France

Italgelatine, Italy

AMPC Inc, US

Leiner Davis Gelatin, Argentina

SBI Systems Bio Industries Ltd, UK

Lapi Gelatines, Italy

Atlantic Gelatin, US

Germantown (Europe) NV, Belgium

SYNERCHIM SA, France

GI Lim Foods Ltd, Korea

Dynagel Incorporated, US

PB Gelatins, Belgium

Weishardt International, France

Gelatine Delft BV, Netherlands

Eastman Gelatine, US

PB Gelatins UK Ltd

G Fiske & Co Ltd, UK

Hormel Foods Corporation, US

Leiner Davis Gelatin Brazil

Dena GmbH

Leiner Davis Gelatin, NZ Ltd, New Zealand

Cangel Inc, Canada

DGF Stoess AG

Rieber & Son ASA, Norway

Nitta Gelatin NA Inc, US

Zhejiang Yiwu Natural Pigment, China

Gelatine Products Ltd (UK)

Rieber & Son plc, Food Division, UK

Norland Products Inc, US

Leiner Davis Gelatine, Inds. Pty, South Africa

Vyse Gelatine Company, US

ACP Ingredients, UK

Danisco Ingredients, UK

Arthur Branwell & Co Ltd, UK Croda Colloids Ltd, UK Geistlich Sons Ltd, UK

Danisco Ingredients, Denmark

Mitros GmbH Germany

Div of Knox Gelatine Inc, US

Gelatin

69

which the gelatin is to be put and the source of the collagen. Acid treatment is particularly useful for the preparation of gelatins from pigskins and ossein and is widely used in America. In Europe the major sources are hides and bones and alkaline extraction is the more common extraction process. 4.1.2 Acid treatment This gives rise to Type A gelatins and in this process the washed hydrated stock is immersed in cold dilute mineral acid (pH 1.5 to 3.0) for eight to 30 hours (usually 18 to 24 hours) depending on the thickness and/or degree of comminution of the raw material. During this period the material swells to two or three times its original volume. After treatment the material is washed in running water to remove excess acid so that the material is approximately neutral. 4.1.3 Alkaline treatment This gives rise to Type B gelatins and although a range of alkaline agents may be used it is usual practice to use saturated lime water (Ca(OH)2, pH 12.0) as the curing liquor. The washed stock is placed in pits or vats along with the liquor and sufficient hydrated lime to maintain saturation. The temperature is kept below 24ºC and the mixture is agitated at intervals using poles or other mechanical means. The process lasts for one to six months (usually two to three months) depending on the thickness and type of the raw material. When treatment is completed the limed material is washed with water to remove excess lime, then with dilute acid (HCl) until the external areas are acidic. Washing with water is resumed until the whole is approximately neutral. 4.1.4 Extraction To extract gelatin the treated raw material is placed in extraction kettles and covered with hot water. A series of extractions are made with consecutive lots of hot water (usually three to five), each extraction is somewhat hotter than the preceding one in the range 55 to 100ºC. Each extraction usually lasts from four to eight hours. Highest quality gelatins, as judged by gelling ability, are made by the lower temperature extraction since, at these temperatures, less hydrolysis of the polypeptide chains occurs. Each subsequent extraction gives a poorer gelling, more highly coloured product. At the end of each run or extraction all possible grease is removed by skimming and after allowing the liquor to cook or settle for a short time further fat may be removed. All the liquor, containing 2– 4% gelatin, is removed prior to the addition of further hot water. The various extractions may be combined to yield gelatins of the required quality prior to filtration to remove further fat and other suspended impurities. Active charcoal may be used to minimise high colour. The ash content of the gelatins is at this stage 2–3% and if lower values are required ion-exchange can be used. Many pharmaceutical and photographic users will specify lower ash contents. These light liquors, which are crystal clear and transparent, are then continuously evaporated until the increased viscosity makes further concentration by evaporation impractical. This occurs at a concentration of 8–12% in high-quality gelatins and 15–20% in low-quality material. Beyond certain limits most film type evaporators cause further degradation of the gelatin. If desired the concentrated liquors from the evaporators may be filtered and bleached again to improve quality. Following the evaporation stage further drying is usually by air, the liquor being run onto a wide endless conveyor belt to produce a strong gel. This thin gel is placed on metal

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nets and blown with cold air until a skin develops after which hot air is used to complete the drying. The resultant transparent sheets are ground as necessary. Other methods of drying include spray and roller drying. For food grade gelatins all equipment must be manufactured to avoid heavy metal contamination and aluminium may be used for acid processed gelatin and nickel, or more commonly, stainless steel for both acid and alkaline processed gelatins. Food grade gelatins, although varying markedly in their functional and rheological properties have similar empirical compositions, typically containing 8–12% moisture and less than 2% ash, the remainder being protein (gelatin).

4.2

Structure

4.2.1 In solution Since all gelatins are derived from collagen it is pertinent to describe the structure of these macromolecules before discussing the gelatins. The collagen monomer (tropocollagen) is a triple helix or rod about 300nm long and 1.5nm in diameter of molecular weight about 300,000. On mild heating (40ºC) the helix unfolds to yield a mixture of chains (molecular weight about 100,000), chains which consist of two covalently bound chains and units consisting of three chains. The amino acid composition of these chains (Table 4.2) is quite unusual, since glycine accounts for about one-third of all the residues and the polypeptides are also very rich in both proline and hydroxyproline. Sulphur-containing amino acids are virtually absent and the crosslinks between the chains do not involve these types of residue. However, in most tissues only a small amount (< 5%) of collagen is soluble in dilute acid or salt solution and with increasing maturity Table 4.2

Amino acid composition of collagen and gelatin – residues per 1000 residues

Amino acid Alanine Arginine Aspargine Aspartic acid Glutamine Glutamic acid Glycine Histidine 4-Hydroxyproline "-Hydroxylysine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

Type I collagen (bovine)

Type A gelatin [a]

Type B gelatin [b]

114 51 16 29 48 25 332 4 104 5 11 24 28 6 13 115 35 17 4 22

112 49 16 29 48 25 330 4 91 6 10 24 27 4 14 132 35 18 3 26

117 48 – 46 – 72 335 4 93 4 11 24 28 4 14 124 33 18 1 22

[a] Type A gelatin: acid-pre-treated pigskin gelatin; [b] Type B gelatin: alkali-pre-treated bone gelatin.

Gelatin

Fig. 4.1

71

Formation of the two reducible aldimine and keto-imine crosslinks found in collagen.

the amount of soluble collagen decreases. This is because as well as intramolecular crosslinks collagens usually contain a number of intermolecular bonds which give the tissue its high tensile strength, the number of such linkages increasing with age. Bailey and co-workers have elucidated the nature of these unique linkages. It would appear that their formation involves a condensation reaction between the amino group of lysine and an aldehyde group formed by the enzymic oxidation of a lysine or hydroxylysine residue. If the aldehyde is produced from lysine then a heat labile aldimine is formed (Fig. 4.1), but if it is formed from hydroxylysine then a heat stable keto-imine crosslink forms via an Amadori rearrangement (Fig. 4.1). With increasing age these intermediate crosslinks undergo further reactions to produce stable trivalent, rather than divalent linkages (Fig. 4.2). These linkages can link three rather than two collagen molecules and account for the

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Fig. 4.2 Formation of two proposed trivalent ‘mature’ collagen crosslinks formed from the divalent aldimine and keto-imine crosslinks.

increase in tensile strength with age. It is these stable crosslinks which mean rather severe processing is necessary to yield soluble gelatins and why commercial gelatins are so heterogeneous in size; individual molecules may be single chains of molecular weight less than 50,000 or multistrand polymers with molecular weights of over one million. Although gelatins do have almost identical amino acid compositions to their parent collagen if they are prepared by alkaline pre-treatment many of the non-ionisable glutamine and asparagine residues are converted to carboxyl groups, with the evolution of ammonia, and the gelatins become more acidic (Table 4.2). Thus the isoelectric points of acid processed gelatins tend to be in the range 7–9.4 whilst alkali processed ones have isoelectric points in the range 4.8–5.5. The lowest isoelectric point observed in an alkali processed gelatin is 4.6 which presumably corresponds to a molecule virtually free of amide; however, most commercial alkali processed samples contain individual molecules with isoelectric points differing by 0.5–0.7 pH units. Gelatins with average isoelectric points in the range 7–9.4 usually exhibit a larger range of individual values since at these

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73

pH values proteins are poor buffers. Apart from the differences in isoelectric points acid precursor gelatins usually have lower intrinsic viscosities for a given molecular weight than alkali processed ones. As acid treatment is usually used for material containing a low concentration of intra and inter chain crosslinks gelatins prepared by this method are usually less branched than commercial alkali pre-treated samples. Although gelatins vary widely in size and charge distribution all collagens (and thus gelatins) have a characteristic primary structure and with the exception of the telopeptide regions, i.e., a small region at the end of the -chain, glycine is found as the third residue in all chains whilst proline, but not hydroxyproline, is frequently found to follow glycine in the sequence. To a large extent proline, hydroxyproline (which together account for about onefifth of the total amino acids in mammalian collagens) and alanine (which accounts for about one-ninth of the total) are found in ‘non-polar’ regions where tripeptides of the type glycineproline-R, R is a non-polar amino acid, including hydroxyproline, predominate. These ‘nonpolar’ regions are interspersed with polar regions, which are relatively deficient in both proline and hydroxyproline. An interesting difference is that the total proline plus hydroxyproline content of collagens vary quite markedly between species. In vertebrates it may vary from about 155 residues per thousand for a cold-water fish, such as cod, to about 221 residues per thousand for most warm-blooded mammals, such as sheep and cattle. Invertebrates display an even greater variation ranging from about 112 residues per thousand for the collagen of the body wall of the Metriculum to about 304 residues per thousand in the Ascaris worm. Several workers have shown quite clearly that the thermal stability of a collagen is directly related to its pyrolidine content and there is evidence that, although proline may be important, hydroxyproline located in the third position of the triplet is the major determinant of stability due to its hydrogen bonding ability. Just as the proline plus hydroxyproline content dictates, to a large extent, the thermal stability of a collagen so the content and distribution of these imino acids are major factors in dictating the gelling ability of a gelatin. Fish skin gelatins of low pyrolidine content are far poorer gelling agents than gelatins of similar molecular weight derived from warm-blooded mammals. Although at low temperatures gelatin molecules readily aggregate to form gels at temperatures of 40ºC and above gelatin is assumed to exist as a random coil and most physical measurements suggest this is so. However, the high imino acid content (proline and hydroproline) means the chains do have limited flexibility and it has been suggested that even at these temperatures the chains possess some helical structure. If the polypeptides consisted purely of proline residues the chains would adopt a rigid poly-Lproline II helix in solution (Fig. 4.3). In a native tropocollagen molecule the whole chain is stabilised by three such chains aggregating via hydrogen bonding to form the collagen triple helix (page 70). It is believed that the trans form of the helix (Fig. 4.3) is found in tropocollagen, but to accommodate all the side chains on the polypeptide chains in tropocollagen the three chains are not parallel but wind about each other in a gentle spiral, i.e., go anticlockwise around one another on ‘climbing’ the common axis, a rotation of ÿ324º taking one back to the original chain (cf. 360º) and a rotation of ÿ108º taking one to a similar residue on the next chain (cf. 90º).

4.2.2 In the gel Although at temperatures above 35–40ºC gelatins in solutions behave as random coils, which can take up an infinite number of transient configurations, on cooling the solution aggregation occurs and at concentrations above about 1%, depending on the quality of the gelatin and pH, a clear, transparent gel will form. Unlike most protein and polysaccharide

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Fig. 4.3 The two forms of the poly-L-proline II helix.

gels, gelatin gels are thermoreversible since on warming to 35–40ºC the gel will dissolve. It is this property which makes gelatin such a useful and unique food ingredient since such gels will ‘melt in the mouth’. It is generally accepted that the pyrolidine-rich regions of the gelatin chains act as nucleation sites for the formation of potential junction zones in that, as the temperature is lowered these regions, especially those with the sequence glycine-proline-proline (or hydroxyproline) tend to take up the proline-L-proline II helix (Fig. 4.3) and aggregation of three such helices form a collagen-like triple helix which act as the gel junction points or zones. These junction zones are stabilised by inter chain hydrogen bonds, which being temperature sensitive break at 35–40ºC causing the gel to melt.

Gelatin

Fig. 4.4

75

Rigidity modulus, G, as a function of time for 2% gels of a commercial limed hide gelatin matured at 20ºC and pH7.

In a normal gelatin gel matured at high temperatures, only a few collagen-like junctions will form and the remainder of each polypeptide chain will be disordered and weak gels are formed. On further cooling of this gel additional parts of each chain become ordered, either by the growth of existing junctions or the formation of new, but less stable junctions from the regions containing lower contents of pyrolidine residues; most workers believe the growth of existing junctions is the major contributor to the increased rigidity or strength seen at lower maturing temperatures. Several studies have shown that at relatively high temperatures the gel network, once formed, is continually being reorganised to include junctions of increasing thermal stability. At all temperatures the strength of a gelatin gel increases with time, albeit only slowly and rarely achieves an ‘equilibrium’ value since the junctions are continually reorganising themselves and perhaps some new junctions also slowly form with time (Fig. 4.4). More concentrated gels, matured at lower temperatures reach almost constant gel strengths more quickly than shown for the dilute gel matured at 20ºC in Fig. 4.4. Since the poly-proline II helix in gelatin has to be stabilised by three such regions coming together it is readily apparent that at low gelatin concentrations it is likely that these three regions may be derived from one chain to give an intramolecular collagen-like structure which will make no contribution to a gel network. However, as the concentration is increased the likelihood of two or even three different chains being involved also increases. Thus not all reformed collagen-like structures will form ‘useful’ junction zones. It would be expected that the ratio of useful to non-useful junctions increases with gelatin concentration so that there is no simple relationship between gel strength and concentration though over the range 2–6% the strength varies according to the (concentration)n where n approaches two for many gelatins but can be as low as 1.3. As

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well as the concentration, the ratio of useful to non-useful junctions will also depend on the chain size and whether the individual molecules are multistranded or not. Since the strength of a gelatin gel is primarily dependent on the concentration and distribution of the pyrolidine residues and the overall size and shape of the molecules, gel strength in the pH range 4–10 is virtually independent of pH, at least for concentrated systems. Outside this pH range gelation is markedly inhibited and this probably reflects the fact that at these extreme pHs the chains carry a high net positive or negative charge and electrostatic forces inhibit the ability of the chains to come into suitable juxtaposition for the formation of junction zones.

4.3

Technical data

Although industrial gelatin production processes have existed for at least two centuries, methods for the comparative testing of gelatins have evolved only in the last 70–80 years. Initially, interest was focused primarily on the strength of gels formed under standard conditions. Later, other physical properties were recognised and included in gelatin specifications. Today, the monograph specification for gelatin covers physical, chemical and microbiological tests besides the standard gel strength test (BP, 1993, USP NF, 1995). In spite of this, gel strength remains the property on which the commercial value of gelatins is based and this is indicated by the Bloom strength.

4.3.1 Bloom strength Bloom strength is essentially the rigidity of a gelatin gel formed under standard conditions, commercial value increases with increasing Bloom. The Bloom gelometer was developed and patented by O. T. Bloom in 1925 to measure gelatin gel rigidity by determination of the weight (force in grams) required to depress the surface of a gelatin gel 4mm using a 12.7mm diameter flat-bottomed cylindrical plunger. The gel must contain 6.67% w/w of air-dried protein and is prepared by placing 7.5g of gelatin into a Bloom jar and mixing well with 105g of distilled water. The mixture is allowed to stand until the gelatin has completely swollen, then heated in a water bath at 60ºC with gentle swirling to form a homogeneous solution. Samples are then left at room temperature for about 15 minutes prior to being placed in a bath at 10ºC for 16–18 hours before testing with a Bloom gelometer, or its modern equivalent the Stevens LFRA texture analyser, which is believed to provide more reliable results. The gelatin Bloom test has a poor record for repeatability and reproducibility unless strict control of all experimental parameters is maintained. Hence the method for the determination of gelatin gel strength has been standardised (BSI, 1975, GMIA, 1986). To achieve reproducible results it is important that • The temperature of the water bath is 10.00ºC0.02ºC. • The capacity of the bath is sufficient to reduce the water temperature to 10.00ºC within 1 hour of the gelatin samples being placed in it. • The platform that the jars are placed on is perfectly flat and horizontal. • The water level in the bath is at least 1cm above the surface of the gel. • The texture analyser is thoroughly warmed up before use and calibrated using standardised weights. The plunger is of BSI or AOAC standard without chipped edges or face. The AOAC plunger has the same dimensions as the BSI equivalent but has

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77

a sharp edge giving a larger surface area and subsequently slightly higher Bloom values. • The test jars are of standard shape, surface area, depth and construction material, fitted with rubber bungs, which have small-diameter holes through them to allow air expansion during heating and to minimise evaporative losses. • Standard gelatins of known Bloom are included in the test sequence to account for any other influences on the test procedure such as preparation errors and instrumental faults. As discussed earlier (page 75) the exact relationship between concentration and gel strength depends on the type and origin of the gelatin itself. For a 100–250g Bloom gelatin the relationship becomes: C1 n B2 C2 n B1

where C = gel concentration, B = Bloom value of gel, n = 1.7 for high-Bloom gels and 1.8 to 1.9 for low-Bloom gels (150–100g). Therefore, for Bloom determinations, the weight of the gelatin must be 7.50.01g and the volume of the water 1050.1g in sample preparation. Additionally, small changes in the moisture content of gelatin samples can appreciably affect their Bloom strength. The moisture content of commercial gelatins can range from 7–15% therefore determination of the water content at the time of gel strength testing is important. This would normally be done just prior to use. The gel strengths of all types of gelatins decrease below pH5 and above pH9 however, in the range pH5–9 gel strength remains almost constant, although differences have been noted between acid and alkali processed gelatins. Bloom determination is normally made at the pH of the sample which can vary over the range 4.6–7+. It has been suggested that control of pH is essential for the comparison of the protein component of gelatins, but this is not normally done. Generally, the influence of pH on gel strength is greater for dilute gels (< 2% w/w).

4.3.2 Chemical and microbiological properties Although Bloom strength is commercially important it is not essential information for all gelatins; however, all suppliers of gelatin must supply details of their product, including a certificate of analysis to show the sample meets British Pharmacopoeia (BP) specification. Specifically gelatin should meet the following criteria. Characteristics Be light amber to faintly yellow translucent sheets, shreds, powder or granules with little odour. It should swell when immersed in water which on heating gives a colloidal solution, and on cooling forms a more or less firm gel. It should be practically insoluble in most organic solvents. Identification 1. A 1% solution held at 55ºC (S) should give a violet colour when mixed with copper sulphate at alkaline pH demonstrating the presence of protein. 2. pH of S should be in the range 3.8–7.6. 3. A 5% solution, prepared by standing for ten minutes and then heating to 60ºC for 15 minutes, when cooled at 0ºC for six hours should not flow out of the test-tube on inversion.

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Clarity and colour of solution S The solution should not be more opalescent than a standard reference suspension and should have a less intense colour than a given standard reference solution. Purity The gelatin sample should also contain less than 1 ppm arsenic, less than 50 ppm heavy metals, less than 200 ppm sulphur dioxide, less than 100 ppm peroxides (as H2O2) and be essentially free of phenolic preservatives. The total ash content should be less than 2% and the moisture content less than 15% as judged by drying. Standard well-documented analyses are described in the BP for all these factors. Microbiological criteria are that 1g of the gelatin must be free of Escherichia Coli and 10g be free of salmonella. It is also usual in most parts of the world that the gelatin must have a total viable aerobic count of less than 103 micro-organisms per gram, determined by plate count.

4.3.3 Viscosity Viscosity data for a gelatin may be supplied as this property is of critical importance in many uses. Routine commercial determination of gelatin viscosity is usually done at 60ºC0.1 using a gelatin concentration of 6.67% (as in Bloom testing) and is carried out using a BSI standard U-tube viscometer made of borosilicate glass and the International standards quote the size of viscometer to be used for given viscosities. Capillary methods for determining viscosity rely on the pressure under which the liquid flows to provide the shearing stress. In the U-tube viscometer liquid is introduced into the viscometer and drawn up into the right-hand limb until the liquid level is about 5mm above a given mark (A). After releasing pressure or suction the time taken for the bottom of the meniscus to fall from the top edge of mark A to the top edge of a further mark (B) is recorded. The use of a precalibrated viscometer enables comparison and measurement of relative viscosity directly, water being the usual reference liquid. Accurate temperature control is necessary due to the dependence of viscosity on temperature; also the capillary must be clean and dry. Since gelatin viscosity is very dependent on concentration the accuracy limits required for the Bloom test are also required for viscosity determination.

4.3.4 Molecular weight distribution Although it would be very unusual for a supplier routinely to supply any more technical data than outlined above, some users may carry out additional tests to ensure the sample is appropriate for its intended use. For example, for certain uses manufacturers may require a specific size distribution within a sample. This would normally be achieved by the use of gel permeation chromatography (GPC) or size exclusion chromatography (SEC) using a column that has been calibrated against polymer standards, polystyrene standards being most commonly used for gelatin characterisation. The gelatin molecules are subdivided into several molecular weight ranges corresponding to the most commonly occurring sizes. These are 340,000 to 540,000 which correspond to hepta and penta derivatives; it would be unusual to find many samples with a high concentration of such derivatives unless specially prepared. The more commonly found ranges correspond to ‘ chains’ of molecular weight 230–340,000, ‘ chains’ of molecular weight 123–230,000, ‘ chains’ of molecular weight 80,000–125,000 and sub units: unit 1, 49,999–80,000; unit 2, 35,000–49,000; unit 3, 25,000–35,000; and unit 4,

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79

Table 4.3 Examples of molecular weight distribution (10ÿ3) of collagen and collagen hydrolysates Molecular mass distribution

> 360 300 200 100 55 38 25 18 13.5 7.5 3.5 1.5 < 0.5 MW average

Native collagen

Type A gelatin Bloom 310 visc. 5.0 MPas

Type B gelatin Bloom 270 visc. 11.9 MPas

Collagen hydrolysate Gelita–Collagel A (Pig-skin based)

Collagen hydrolysate Gelita–Sol C (Bovine-hide based)

MW-fract

MW-fract

MW-fract

MW-fract

MW-fract

12.9 13.9 25.2 18.6 13.0 7.2 6.5 2.7 0 0 0 0 0 174

11.1 13.6 18.2 40.2 5.8 6.2 2.1 2.8 0 0 0 0 0 168

0 0 0 0 3.5 8.3 18.4 14.4 21.5 15.9 7.0 6.6 4.5 16.7

0 0 0 0 0 0 0.4 0.5 2.9 16.8 24.2 39.8 15.3 3.4

100 0 0 0 0 0 0 0 0 0 0 0 0 > 360

corresponding to molecular weights in the range 10,000–25,000. Obviously each of these fractions will be heterogeneous with regard to size and shape and the , , definitions merely reflect that these are the ranges in which , or gelatins would be found. It is apparent that the amount of each of these fractions will, to some extent, be reflected in the Bloom gel strength and viscosity but their determination will permit far better quality assurance for certain uses. For example, in the manufacture of gelatin capsules too high a content of chains gives a very fast setting, viscous solution which gives rise to misshapen capsules. Conversely if the chains content is too low the gel will set too slowly and fail to peel adequately from the gel-spreading drum. The and fractions contribute to the gel strength and viscosity and if the sample is rich in sub- chains it has a relatively low viscosity and a slow-setting, sticky gel results which is not suitable for encapsulation. In general a higher content of chains give a high-setting gelatin while a sample rich in sub- particles gives a low-setting sample. A typical 150 Bloom lime bone gelatin (alkaline pre-treatment) yielded a chain content of 16.5%, a chain content of 12.2%, an chain content of 32.4% and a total sub- content of about 38.5%. A lower Bloom lime bone gelatin would usually have correspondingly lower concentrations of the higher molecular weight fractions. The higher molecular weight fractions are the major determinants of viscosity and the gel strength correlates quite well with the sum of , and higher molecular weight components and is inversely related to the concentration of sub- particles. In Table 4.3 the molecular weight ranges of a range of commercially available collagen and gelatin derivatives is given.

4.4

Uses

Gelatin is usually used at relatively low concentrations in water or polyhydric alcohols in the manufacture of sweets, marshmallows and a whole range of dessert products. The major reasons it is so widely used in the food industry are because

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• it gives high-quality gels in dilute solution with a clean ‘melt in the mouth’ texture • at higher concentrations it gives elastic gum-like textures which slowly dissolve in the mouth • it is an effective emulsifying and foaming agent • as a polyelectrolyte it is able to flocculate suspended particles. In the pharmaceutical industry it is widely used to bind tablets and it aids the slow release of the active ingredient(s). It is also very widely used in the manufacture of hard and soft gel capsules to contain many drugs and nutritional supplements. It also has other medical uses. Another major user of gelatin is the photographic industry, which utilises both its gel-forming ability and its surface activity to suspend particles of silver chloride and light sensitives dyes without agglomerisation.

4.4.1 Food uses The food industry is still a large user of gelatin although in some cases it has been replaced by other hydrocolloids and the recent concerns about the possibility, however slight, of products derived from beef carcasses being transmission agents for the bovine spongiform encephalitis (BSE) prion has caused some concern about its use in food products. In addition, followers of some religions will not consume foods derived from pork. The major users today include manufacturers of • Frozen cream products who would use 0.25% of a 250 Bloom gelatin to inhibit the crystallisation of ice and sugar. • Ice cream who would use 0.5% of a similar gelatin (250 Bloom) to prevent crystallisation. Its use in ice cream manufacture is though diminishing. • Marshmallows who may incorporate up to 1.5% gelatin (of good Bloom strength) to prevent crystallisation. It is also useful during the manufacture of light aerated confectionery such as marshmallows and foam wafers as it increases the viscosity of the system and stabilises the foam during processing, transport and storage. • Lozenges, wafers and sweet coatings where up to 1% gelatin may be added to limit dissolution. • Low-calorie sweets and spreads where its excellent water-holding capacity is utilised. Gelatin itself has an energy value of 14.7kJ/g. Thus a 2% gelatin gel will contain less than 30kJ per 100g, i.e. (less than 8k calories per 100g). The development of low-fat and low-calorie foods has been intensively researched in recent years and the possibility of using mixed gels of, for example, egg white and gelatin in fat-free products so that the water is partitioned between the two gel phases, one of which is dispersed within the other to give the appropriate mouthfeel has been researched. In full fat products there are two distinct phases (lipid and aqueous) one of which is dispersed in the other. Thus if the phase volumes of the gels are appropriate and the gelatin ‘melts in the mouth’ then a similar mouthfeel may result. Such technology is in its infancy. • Meat products such as corned beef and luncheon meats where it is primarily used to hold water (retain the juices). In the manufacture of pasteurised canned hams and other such products gelatin (in the form of granules or portions of sheets) may be introduced into the product just prior to cooking so that the hot water released on heating the meat matrix dissolves the gelatin so that on cooling, a gel forms around the product to fill the space left as the meat shrinks. In pork pie manufacture a similar approach is

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81

adopted except that a hot gelatin solution, at over 45ºC to reduce microbial contamination, is injected into the cooled pies after baking. Pies are best ‘jellied’ at a core temperature of 65–70ºC, when the pastry is sufficiently cool to minimise the absorption of the gelatin solution. Usually a 6% solution of Bloom strength 160–200g is used. Lower core temperatures than 65–70ºC may be used and this will help minimise any softening of the pastry due to migration of water from the gelatin solution/gel. However, this is not usually feasible because of the possibility of microbial contamination. • Novel dairy products where small amounts can produce creamier stir yoghurts or firmer, gelled products with good organoleptic properties. It is helpful if the gelatin can be added directly to the cold fresh milk to enable it to swell and subsequently dissolve during processing (heating). • Drinks industry as a flocculating agent. There is no uniform quality characteristic for the best fining gelatin as it depends on the nature of the drink and even then the results may be somewhat variable. Both extremely high Bloom gelatins, mostly in the form of the leaf gelatin and low Bloom powdered gelatins (ca. 80 Bloom grams) are used. Clarification of beer using gelatin is widely used in North and South America and is being increasingly used in Europe, outside Germany. Unlike the clarification of wine, juices and beer where the gelatin is reflocculated, in lemonades it remains in the drink to stabilise the essential oils.

4.4.2 Medical uses Gelatin is an ingredient of pastilles, pastes, pessaries, beugies and glycerol suppositories and isotopic solutions containing 0.5–0.77 gelatin and a suitable bactericide may be used as artificial tears. However, uses unique to the medical field are as follows. Capsules. One of its major uses in the health/medical field is as the main constituent of hard and soft (flexible) capsule shells. The hard gelatin capsule is a unit solid dosage form. It consists of two pieces, a cap and a body, which have the form of open-ended cylinders and which fit one over the other. They are produced by dipping a cold metal forming mould into a hot gelatin solution. The gelatin gels around the mould as it is withdrawn, so forming a continuous film. This is then dried, removed from the mould, cut to the correct length and the two pieces joined together. They are manufactured by a small number of specialist companies who supply them to the pharmaceutical industry where they are filled with active compounds to produce the final dosage form. Their use has increased significantly in Europe in the last 20 years. It is estimated that the current annual world-wide consumption is in excess of 100,000 million. For hard gelatin capsules the walls need to be strong and fairly rigid and a high Bloom gelatin is essential and the viscosity is used to control the wall thickness. In a typical process a 30–40% gelatin solution is prepared in hot (60ºC–70ºC) demineralised water in stainless-steel pressure vessels. The solution time is two to three hours. After this time it is subjected to vacuum to remove entrapped air bubbles. Gelatin in hot solution hydrolyses and loses its desired physical properties; thus the quantity prepared at any one time is governed closely by its consumption rate to ensure that it is not held at these temperatures for too long. The other main raw material is the colourant. Capsules are made in a vast range of different colours and to achieve this two sorts of colourants are used; soluble dyes and pigments. All of these are synthetic in origin. Natural dyes have been tried but they

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present problems of low tinctorial power and high cost. The colourants which can be used are governed by legislation which, unfortunately, varies widely from country to country. This means that sometimes the same colour has to be prepared with different dyes, depending upon the area of the world in which the finished product is to be sold. The capsule manufacturing machine is usually about 10m long and 2m wide. It is divided longitudinally into two halves, which are mirror images of each other and hold over 20,000 stainless-steel mould pins. On one side the caps are made and on the other the bodies. The pins, in groups of five at room temperature, are lowered into the solution at 45– 55ºC and the gelatin gels on their surfaces. The temperature must be carefully regulated to maintain a constant viscosity. The moulds are slowly withdrawn and the excess solution allowed to run off. The quantity of gelatin picked up by the mould is dependent upon the viscosity of the solution; the higher the viscosity the thicker the resulting capsule shell wall. The gelling of the gelatin is assisted by the use of cool air currents and the moulds then travel through a series of drying kilns. At the start of the process the capsules contain about 70% moisture and at the finish about 15–18%, which is higher than required in the finished capsule, which is 12.5–16%. This is because if they were dried to this level they would be difficult to remove from the moulds and might split in the process. However, they need to be dried down to a level where they are firm enough to be handled. The capsules are automatically removed from the moulds and the matching halves transferred to a central joining block and the complete capsules are ejected in groups. The machine cycle is in the order of 45–55 minutes. Each machine is capable of producing between 750,000 and 1,000,000 capsules per 24-hour day depending on capsule size. Soft gelatin capsules use a lower Bloom gelatin than that used for hard capsules, typically 150–200 Bloom, and in addition a plasticiser is incorporated into the formulation. Generally a good gelatin for manufacturing soft gelatin capsules has a chain content of 11–13% and less than 43% sub- units. The amount of plasticiser can be varied to produce capsules for different applications (Table 4.4). The plasticiser most frequently used is glycerol although sorbitol, propylene glycol, sucrose and acacia have also been used. The manufacture of soft capsules is very different from that used for hard ones since it is manufactured and filled in one operation. In 1932, R. P. Scherer developed the first continuous method of encapsulation, the Rotary Die Method. This was later refined to give a fully automated process for the production of soft gelatin capsules. Today this process may be described as follows.

Table 4.4 Control of the platiciser content of shells for soft gelatin capsules in conjunction with their intended use. Hard gelatin capsules rarely, if ever, contain added plasticiser. Glycerol:gelatin ratio (parts of dry glycerol to one part of dry gelatin) 0.35 0.46 0.55–0.65 0.76

Application Oral capsules with oil fills where final capsule should be hard Oral capsule with oil fills where shell requires to be more elastic Capsules containing oils with added surfactant or products with hydrophilic liquid fills Oral capsules where a chewable shell is required

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83

Liquid material is fed from two tanks into the encapsulation machine, one contains molten gel material at 60–65ºC and the other contains the medicinal fill material usually at 25ºC. Molten gel (approximately 40% gelatin plus the plasticiser) flows down two heated pipes into two heated spreader boxes and then onto two large cool-casting drums where flat gelatin ribbons are cast. These ribbons are fed into rollers, lubricated with oil and then fed into the encapsulating die mechanism. Here liquid fill flows under gravity to a pump that injects the fill from a heated wedge at 37–40ºC into the space between the gelatin ribbons as they pass between the die rolls, forcing the gelatin to expand and form the shape of the die, which determines the size and shape of the capsules. The ribbon continues to flow past the heated wedge and is pressed between the die rolls, where the capsule halves are sealed together by the application of heat and pressure, the capsules being cut from the gel ribbon by the dies. Thus the material to be encapsulated is injected between two sheets of plasticised gelatin at the exact instant when the capsule is formed between two revolving dies. Most of the moisture is then removed by passing the capsules through a series of tumble driers followed by spreading onto trays and stacking in a tunnel drier at ambient temperature and air at approximately 20% relative humidity. The rotary die method has the advantage of producing a capsule which has an accurate dosage – an essential requirement for pharmaceutical products. Capsules are hygienically filled without the inclusion of air, which is essential for prevention of oxidation of capsule fill components, enabling the encapsulation of vitamins and actives sensitive to oxidative degradation which can remain stable for several years when produced in soft gel capsule form. Here a role is thought to be played by the gelatin shell itself, acting as a physical barrier between atmospheric oxygen and the capsule contents. Gelatin sponges. Gelatin is often used as the base for the foam cubes used by dentists to absorb blood during treatment and help stop bleeding. The aerated gelatin gels have a high absorption capacity and the good compatibility of gelatin with human tissue rules out allergic reactions to a high degree. Blood substitutes. To counteract high blood losses blood substitutes, which have optimum dwelling periods in the blood stream and thus allow blood volume to be regulated, are used. Here infusion therapy using suitable gelatin solutions is important, the solutions are so designed to have similar viscosities to blood and gelatin has among other advantages the fact that it is not stored in the body but is completely decomposed. Obviously high purity is essential for such gelatins and DGF Stoess are the major suppliers.

4.4.3 Photographic uses Modern silver bromide photographic materials are mainly composed of emulsions containing gelatin on a backing material (paper or film). Here gelatin has three functions. 1. 2.

3.

It acts as a bonding agent for the photosensitive silver bromide. For the fabrication of the emulsion, it is essential the gelatin swells and forms a solution when heated, which turns into a gel on cooling and, after the water has been extracted, changes into a durable state. The swelling capacity of gelatin guarantees that the photographic baths, that are necessary for the chemical reactions during the processing of the exposed

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Handbook of hydrocolloids photographic materials, penetrate into the emulsion and can be easily removed by rinsing.

With the introduction of gelatin more than one hundred years ago, films became about 1,000 times more sensitive than their predecessors. However, the spectrum of gelatin applications includes a lot more than just prints, slides, movie and cine films. Industry processes photographic gelatin to various types of repro-films for the printing trade (intermediate stage for the multi-colour prints of today). To scientific and technical photographic emulsions, such as nuclear trace emulsions for localising radio isotopes in nuclear medicine, to infrared sensitive emulsions for taking pictures in the ‘dark’, in astronomy, and in geology and photogrammetry for pictures taken from great heights. Nowadays the highest demands are made on photographic gelatin for manufacturing Xray films.

4.5

New gelatin derivatives

4.5.1 Cold water soluble gelatins Commercial gelatins usually possess both amorphous and crystalline character. However, if the drying process is very carefully controlled it is possible to produce a very finely powdered gelatin which, in contrast to the coarsely granulated conventional gelatin possesses no crystalline character. The amorphous structure of instant gelatin enables it to swell very rapidly and intensively. Its three-dimensional molecular network is weakly linked; the molecular arrangement is purely coincidental and the physical inter and intra-molecular binding forces are weak. Water can readily be taken up by the structure therefore swelling never actually ceases and all the water that is available is absorbed to a gel-like texture. In rheological terms, the instant gels can be compared with those formed by dissolution in hot water. However, the gel-forming kinetics are different; whilst instant gelatin has achieved 90% of its firmness after ca. 30 minutes, normal gelatin gels require much more time. In addition, normal gels are much firmer, even with similar concentrations and gelatin quality. The strengths are often about four times less than one might expect. To avoid ‘clumping’ and ensure homogeneity it is advisable to mix the cold water soluble gelatin with other fine particle ingredients in any food formulation at a ratio of 1:5–1:7. Such gelatins are used extensively in formulating easy to prepare dessert products, especially creams and mousses (normally at 0.1–3% concentration) where the powdered ingredients can be stirred in cream, milk or other liquid and sometimes whipped. After a short time in the refrigerator they can be eaten. As with conventional gelatins these cold water soluble gelatins can be supplied with a wide range of gelling and viscous properties.

4.5.2 Gelatin hydrolysates Although gelatin loses its ability to gel when hydrolysed to small peptides there is an expanding market for such products. Such hydrolysates are prepared by the controlled enzymic hydrolysis of gelatin followed by sterilisation, concentration and finally spraydrying and thus their gross composition is very similar to that of the native protein (89– 93% protein, 2% ash and 5–9% water). Unlike many other protein hydrolysates gelatin

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85

hydrolysates do not possess a bitter taste and thus can be used in a whole range of products; they typically possess a viscosity of 20–50mPas in 35% solution at 25ºC. They are supplied as light-cream coloured water soluble powders for use as nutritional supplements, binding agents, foaming and emulsifying agents and carriers. A wide range of gelatin hydrolysates are available with molecular weights in the range 3,000–20,000. DGF Stoess market Gelita Sols of molecular weight 3,000 and Gelita Collagels with molecular weights in the range 7,000–11,000 (BS-GA), 12,000–18,000 (BS) and 10,000– 20,000 (A). These zero Bloom gelatins do not gel but are used in confectionery as substitutes for carbohydrates, a protein source, whipping agent and a binding agent for cereal bars. A typical sugar-free gum may contain about 20% gelatin hydrolysate and 7% conventional gelatin while a muesli bar may contain 23% hydrolysed gelatin. In the dairy industry such hydrolysates are usually used as whipping agent, 1–3% of the higher molecular weight hydrolysates enable creamy and soft textures to be obtained and provide a final product of high whipping volume. In the meat industry they have been used in finely homogenised canned meats where addition of 1.5–2% can reduce jelly and fat deposits by two-thirds, in cooked sausage at about 2% to reduce cooking losses and improve sliceablility, as edible films on frozen meat to prevent oxidative changes and freezer burn (in conjunction with conventional gelatin). Higher molecular weight hydrolysates have been used in the manufacture of soups, sauces and prepared meals to impart a creamy smooth consistency to the product and in low-fat meat spreads where they act as a binding agent.

4.5.3 Chemically modified gelatin Gelatin contains a number of amino acids which possess side chains with amino-, carboxyl- and hydroxyl groups. These groups can be reacted with numerous monoand bifunctional reagents, hence altering the chemical and physical properties of gelatin and its derivatives. Chemically modified gelatin is mainly used in the photographic and cosmetic industries; use in the food and medical industries is restricted by law.

4.6

Regulations

Until recently there were no real limitations on the use of gelatin for food and pharmaceutical uses as, according to EU and US legislation, gelatin is classed as a food and not a food additive. Its use with other food ingredients is therefore generally permitted where good manufacturing practices are in place. Only in special cases might its use be limited to avoid deceiving the consumer about the nutritional value of a product, for example, injecting gelatin into ham to enhance water binding is forbidden in Germany. At present, purity requirements are still regulated on a national basis by individual European countries. However, the EC is currently working on a proposal to standardise quality requirements for food grade gelatin on a pan-European basis. The advent of BSE has complicated the situation since, as with other beef products, the EC in a series of directives banned the UK from selling gelatin derived from UK-bred animals over 30 months of age and although Northern Ireland has now been exempted; the ban still applies to the rest of the UK. The situation is continually being reviewed and gelatin from all sources may be given the ‘all clear’ in the near future.

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Further reading

(1985) ‘The biological diversity of collagen: A family of molecules’ in Advances in Meat Research Vol. 4, (eds) A. G. Ward and A. Courts. BLOOM, O. T. (1925) Penetrometer for testing jelly strength of glues, gelatins, etc. US Patent, 1, 540–979. BORSTEIN, P. and TRAUB, W. (1979) ‘The Chemistry and Biology of collagen’ in The Proteins, Vol. IV, 3rd edn pp. 412–605. British Pharmacopoeia, Vol. 1 (1993). Monograph for Gelatin. BRITISH STANDARDS INSTITUTE (BSI), (1975) Methods for sampling and testing gelatin (physical and chemical methods) BS 757. GELATIN MANUFACTURERS INSTITUTE OF AMERICA (GMIA), (1986). Standard methods for the sampling and testing of gelatins. HARRIS, P. (1990) ‘Gelatin’ in Food Gels, Elsevier, London, pp. 233–89. HUTCHINSON, K. (1992) ‘Encapsulation of soft gels for pharmaceutical advantage’ in Encapsulation and Controlled Release. Proceedings of a Symposium, Royal Society of Chemistry (eds) D. R. Karsa and R. A. Stephenson, pp. 86–97. JONES, B. E. (1982) ‘The Manufacture of Hard Gelatin Capsules’, Chem. Eng. London, 380, 174–7. JONES, B. E. and TURNER, T. D. (1974) ‘A Century of Commercial Hard Gelatin Capsules’, Pharm. J., 213, 614– 17. LEDWARD, D. A. (1986) ‘Gelation of Gelatin’ in Functional Properties of Food Macromolecules (eds) J. R. Mitchell and D. A. Ledward. Elsevier Applied Science, pp. 171–201. PARRY, D. A. D. and CRAEMER, L. K. (1979) ‘The Primary Structure of Collagen’ in Properties of Fibrous Proteins. Scientific, Industrial and Medical Aspects, Vol. 1, Academic Press, London, pp. 133–50. SIMS, T. J. and BAILEY, A. J. (1992) ‘Structural Aspects of Cooked Meat’ in The Chemistry of Muscle-based Foods (eds) D. A. Ledward, D. E. Johnston and M. K. Knight, Royal Society of Chemistry, Cambridge, pp. 106– 27. United States Pharmacopoeia National Formulary (1995) 23, p. 2247 & 781, p. 1812. VEIS, A. (1964) Macromolecular Chemistry of Gelatin, Academic Press, London. WARD, A. G. and COURTS, A. (1977) The Science and Technology of Gelatin, Academic Press, London. BAILEY, A. J.

5 Carrageenan A. P. Imeson, FMC Corporation (UK) Ltd

5.1

Introduction

For many centuries, red seaweeds have been used for foods in the Far East and Europe. Different species of Rhodophycae contain naturally occurring polysaccharides which fill the voids within the cellulose structure of the plant. This family of polysaccharides include carrageenan, furcellaran and agar. These polymers have a backbone of galactose but differ in the proportion and location of ester sulfate groups and the proportion of 3,6anhydrogalactose. The differences in composition and conformation produce a wide range of rheological properties which are utilised in a large number of foods. Different carrageenans cover a wide spectrum of rheological behaviour going from a viscous thickener to thermally reversible gels which range in texture from soft and elastic to firm and brittle. Kappa carrageenan is able to interact synergistically with other gums, such as locust bean gum and konjac mannan, to modify further the gel texture. A specific interaction between kappa carrageenan and kappa casein is widely used to stabilise dairy products.

5.2

Manufacture

5.2.1 Raw materials The main species of Rhodophycae used in the commercial production of carrageenan include Euchema cottonii and E. spinosum. These are spiny bushy plants, about 50cm high, which grow on reefs and in shallow lagoons around the Philippines and Indonesia and other island coasts in the Far East. E. cottonii yields kappa carrageenan and E. spinosum contains iota carrageenan. Chondrus crispus is the most familiar of the red seaweeds and is found as a small bushy plant, only about 10cm in height, widely distributed around the coasts of the North Atlantic. Carrageenan extracted from this species comprises both kappa and lambda types although it has been shown that these do not occur within the same plant but in individual plants which grow together (McCandless et al., 1973). Gigartina species are large plants up to 5m in length which

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are collected from the cold deep coastal waters off Chile and Peru to give kappa and lambda carrageenans. Furcellaria species are found in the cold waters around Northern Europe and Asia and yield kappa and lambda carrageenans.

5.2.2 Manufacturing process The manufacturing processes for extracting carrageenan are shown in Fig. 5.1. The processes commence with the selection of seaweed to ensure it is harvested at the right time. After the seaweed is collected it is washed to remove sand and stones and then dried quickly to prevent microbial degradation and thus preserve the quality of carrageenan. The weed is then baled and shipped to the processing plants and warehoused before use. Manufacturing plants located near the harvesting site may utilise wet seaweed to avoid the costly drying and subsequent rehydration stages. At the manufacturing site, the baled seaweed is tested and various lots are selected to produce the desired extract. Proper selection of the raw materials and an understanding of the influence of the process on the properties of the final carrageenan are vital to the production of a high quality and consistent end-product. After the seaweeds are identified and chosen to make a particular extract, they are washed to remove sand and stones before treating with appropriate amounts of various alkalis to swell the seaweed and extract the carrageenan. The alkali may be selected to determine the particular salt type of carrageenan produced by the process which, as discussed later, has important consequences for the properties of the resultant extract, including dispersion, hydration, thickening and gel formation. Prolonged treatment with

Fig. 5.1

Manufacturing processes for carrageenan and processed Euchema seaweed.

Carrageenan

89

alkali promotes an internal rearrangement which modifies the polysaccharide backbone. With kappa carrageenan the anhydride bridge formed by this rearrangement allows adjacent chains to form helical structures which, after neutralisation of the exposed sulfate groups by appropriate cations, aggregate to form junction zones. Hence, the alkali modification produces carrageenans that form firm, brittle gels. Iota carrageenans are modified by alkali so that they form weak, elastic gels. After extraction and modification, the dilute carrageenan solutions are filtered and clarified by high-speed centrifugation and concentrated by a range of methods. The solutions are then precipitated with isopropyl alcohol to give a fibrous mass which is pressed to remove impurities and then dried. An alternative recovery process utilises the specific selectivity of kappa carrageenan for potassium salts to form a gel. As kappa carrageenan solution is extruded into a concentrated solution of potassium chloride a fibrous mass is formed. The precipitated mass synerises or exudes free water and is dewatered under pressure to make ‘gel press’ carrageenan. The precipitated carrageenan may be frozen and thawed to assist this dewatering step. The pressed fibres are then dried and ground to the appropriate particle size. Each manufacturer carefully controls the raw materials and process parameters to produce a large number of extracts with well-defined properties. Individual extracts are characterised by their thickening and gelling properties and finished products are made by blending various extracts in order to maintain consistent quality from lot to lot and to provide the specific properties needed to meet particular customer requirements and to suit particular applications. Processed Euchema seaweed (PES), Philippines Natural Grade (PNG), semi-refined carrageenan (SRC), alternatively refined carrageenan (ARC) and alkali-modified flour (AMF) are the various terms used to describe Euchema seaweeds which are harvested around the Philippines and Indonesia and directly treated with alkali to modify the carrageenan within the seaweed. This is a more economic process as it avoids extracting carrageenan into dilute solutions which require expensive concentration and drying steps to make the finished carrageenan powder. The process by which processed Euchema seaweed is converted into commercial gelling grade products is compared to the traditional extraction process in Fig. 5.1. The process commences with selection and washing. Thereafter the process diverges from the manufacture of extract carrageenans. The Euchema seaweed is soaked in potassium hydroxide solution in situ before chopping and bleaching to reduce the colour of the finished powder. After washing, drying and grinding, the powder is sterilised to control the microbiological levels of the finished product, which are now tightly controlled by legislation.

5.3

Structure

Carrageenan is a high molecular weight linear polysaccharide comprising repeating galactose units and 3,6-anhydrogalactose (3,6 AG), both sulfated and non-sulfated, joined by alternating -(1,3) and -(1,4) glycosidic links. An almost continuous spectrum of carrageenans exist but the work of Rees and co-workers (Rees, 1963; Anderson, Dolan and Rees, 1965) was able to distinguish and attribute definite chemical structures to a small number of idealised polysaccharides. The main carrageenan types, lambda, kappa and iota, can be prepared in pure form by selective extraction techniques. Mu and nu

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Fig. 5.2

Carrageenan structures (alkali conversion of mu > kappa, nu > iota and lambda > theta).

carrageenans are postulated as the precursor structures which, as a result of the internal rearrangement by alkali treatment, form kappa and iota carrageenans. The basic disaccharide units that make up the various carrageenans are shown in Fig. 5.2. The distinct carrageenan structures differ in 3,6-anhydrogalactose and ester sulfate content. Variations in these components influence hydration, gel strength and texture, melting and setting temperatures, syneresis and synergism. These differences are controlled and created by seaweed selection, processing and blending of different extracts. The ester sulfate and 3,6-anhydrogalactose content of carrageenans is approximately 25% and 34% respectively for kappa carrageenan and 32% and 30% respectively for iota carrageenan. Lambda carrageenan contains 35% ester sulfate with little or no 3,6anhydrogalactose content. Even furcellaran, which in the past has been rather misleadingly called ‘Danish agar’, contains 16–20% sulfate content. These high sulfate levels contrast with agar-agar which has a very low sulfate content, always below 4.5% and typically it is from 1.5–2.5%. For food applications, carrageenan is best described as ‘extracts from Rhodophyceae which contain an ester sulfate content of 20% and above and are alternately -(1,3) and -(1,4) glycosidically linked’ (Anon., 1988). Processed Euchema seaweed differs from the traditional carrageenan extracts in that it contains 8–15% acid insoluble matter compared to 2% maximum for an extract (Anon., 1998). The acid insoluble matter mainly consists of a network of cellulose which modifies the hydration, appearance and gel characteristics of the gels which are described in more detail later. The heavy metal content of processed Euchema seaweed is higher than extract carrageenan but still well below limits set for food additives (Phillips, 1996). Carrageenan is a high molecular weight, polydisperse material. Commercial kappa carrageenan extracts are between 400–560kDa and processed Euchema seaweed has a slightly higher molecular weight of 615kDa (Hoffmann, Russell and Gidley, 1996). All carrageenans contain a fraction (< 5%) of material below 100kDa and this low molecular weight material is believed to be inherent in native algal weed.

Carrageenan

5.4

91

Physical properties

The thickening and gelling properties of the different types of carrageenan are quite different. For example, kappa carrageenan forms a firm gel with potassium ions while iota and lambda are only slightly affected. Iota carrageenan interacts with calcium ions to give soft elastic gels but salts have no effect on the properties of lambda carrageenan. Application of these combinations requires experience and understanding of carrageenans but this expertise is available from the major suppliers of this material. 5.4.1 Solution properties All carrageenans are soluble in hot water. However, only lambda and the sodium salts of kappa and iota are soluble in cold water. Lambda carrageenan gives viscous solutions which show pseudoplasticity or shear-thinning when pumped or stirred. These solutions are used for thickening, particularly in dairy products, to give a full body with a nongummy, creamy texture. The influence of temperature is an important factor in deciding which carrageenan should be used in a food system. All carrageenans hydrate at high temperatures and kappa and iota carrageenans in particular exhibit a low fluid viscosity. On cooling, these carrageenans set between 40–60ºC, depending on the cations present, to form a range of gel textures. 5.4.2 Acid stability Carrageenan solutions will lose viscosity and gel strength in systems below pH values of about 4.3. This effect is due to autohydrolysis which occurs at low pH values as carrageenan in the acid form cleaves the molecule at the 3,6-anhydrogalactose linkage (Hoffmann, Russell and Gidley, 1996). The rate of autohydrolysis increases at elevated temperatures and at low cation levels. However, once it has been cooled below the gelling temperature, carrageenan retains the sulfate-bound potassium ions and this prevents autohydrolysis proceeding. Consequently, in acidic products, the carrageenan should be added at the last moment to avoid excessive acid degradation and, if possible, acid should be added to the food immediately before depositing and filling to minimise polymer breakdown. Table 5.1 shows approximate processing times at various pH values for a gel produced with 0.5% kappa carrageenan and 0.2% potassium chloride, such that no more than 20– Table 5.1

Gel processing times

Temperature

Final pH

(ºC)

3

3.5

4

4.5

5

5.5

6

120 110 100 90 80 70 60 50 40

2s 6s 20s 1min 3min 10min 30min 1.5h 5.0h

6s 20s 1min 3min 10min 30min 1.5h 5.0h 15.0h

20s 1min 3min 10min 30min 1.5h 5.0h 15.0h 2.0 days

1min 3min 10min 30min 1.5h 5.0h 15.0h 2.0 days 6.0 days

3min 10min 30min 1.5h 5.0h 15.0h 2.0 days 6.0 days 20.0 days

10min 30min 1.5h 5.0h 15.0h 2.0 days 6.0 days 20.0 days 60.0 days

30min 1.5h 5.0h 15.0h 2.0 days 6.0 days 20.0 days 60.0 days 200.0 days

* Gel process times are the times at various pH and temperatures to reduce the gel strength by about 25%.

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25% of the original gel strength is lost when the solution is cooled. In general, each 0.5 pH unit reduction will decrease the potential processing time by a factor of three. Times will vary somewhat depending on carrageenan concentrations or system ingredients such as salts and sugars. In a continuous process the processing time should be kept to a minimum. In systems above about pH 4.5 the process conditions become irrelevant as the carrageenan solution is stable to most food processing times.

5.4.3 Gel properties Hot solutions of kappa and iota carrageenans set to form a range of gel textures when cooled to between 40 and 60ºC depending on the cations present. Carrageenan gels are thermally reversible and exhibit hysteresis or a difference between setting and melting temperatures. These gels are stable at room temperature but can be remelted by heating to 5–20ºC above the gelling temperature. On cooling the system will re-gel. The ionic composition of a food system is important for effective utilisation of the carrageenan. For example, kappa carrageenan selects for potassium ions to stabilise the junction zones within the characteristically firm, brittle gel as shown in Fig. 5.3(a). Iota carrageenan selects for calcium ions to bridge between adjacent chains to give typically soft elastic gels in Fig. 5.3(b). The presence of these ions also has a dramatic effect on the hydration temperature of the carrageenan and on its subsequent setting and remelting temperatures. For example, iota carrageenan will hydrate at ambient temperature in water but the addition of salt raises the gel point so that the solution is converted into a gel with distinct yield point which is used for cold-prepared salad dressings. Sodium salts of kappa carrageenan will hydrate at 40ºC but the same carrageenan in a meat brine will only show full hydration at 55ºC or above. These effects are shown in Fig. 5.4.

Fig. 5.3

Gelation of kappa and iota carrageenans with cations.

Carrageenan

Fig. 5.4

93

Hydration and setting temperatures for dispersions of kappa and iota carrageenan in different salt solutions.

As a carrageenan dispersion is heated there is no significant particle swelling or hydration until the temperature exceeds about 40–60ºC (Fig. 5.4). As the particles hydrate the viscosity rises as the swollen particles offer more resistance to flow. Further heating to 75–80ºC produces a drop in viscosity. On cooling, the solution shows a marked increase in viscosity followed by gelation below temperatures of 40–50ºC. The hydration and gelation temperatures are strongly dependent on the salts associated with the carrageenan or added separately to the solution. For example, levels above about 4% sodium chloride can prevent full hydration of carrageenan in meat products. In contrast, the very dilute levels of around 200ppm of carrageenan used to stabilise chocolate milks and other dairy beverages may not form a stabilising gel network until the temperature drops below 20ºC. The presence of high solids, such as in confectionery, effectively concentrates the carrageenan and cations on the aqueous phase so the gelation may occur at 80–85ºC or higher placing limitations on the levels and types of carrageenan suitable to such food applications. As mentioned above, kappa carrageenan makes a firm brittle gel which has very poor freeze-thaw stability whereas iota carrageenan forms a thixotropic solution or very elastic gel which has good freeze-thaw stability. Kappa and iota systems may be blended to obtain a range of gel textures with intermediate properties of freeze-thaw stability and moisture binding as illustrated in Fig. 5.5. Processed Euchema seaweeds yield a limited range of products suited to medium gel strength applications such as cooked meats and some dairy products. The solution and gel properties of processed Euchema seaweed and kappa carrageenan obtained by traditional extraction processes are similar with some specific differences. The cellulose network in processed Euchema seaweed reduces the rate of hydration so that solutions develop viscosity after longer heating periods or after heating to higher temperatures. The presence of cellulose in the finished gel gives a lower rupture strength with a more brittle and fragile gel. The cellulose particles make the product cloudy and therefore unsuited to clear gel applications such as water dessert gels and cake glazes. The less refined material may also have slight odour and colour differences compared to carrageenan extracts.

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Fig. 5.5

Gel properties of mixtures of kappa and iota carrageenans.

5.4.4 Synergism with other gums Hot solutions of kappa carrageenan-locust bean gum form strong elastic gels with low syneresis when cooled below 50–60ºC. Locust bean gum is a galactomannan with a level of substitution of one part mannose to four units of galactose. However, this substitution is not regular and regions of the locust bean gum are unsubstituted. The mannose-free regions of the locust bean gum are able to associate with the repeating helical structure of carrageenan dimers to form gels. The maximum interaction, and hence peak rupture gel strength, occurs at a ratios between 60:40 and 40:60 kappa carrageenan to locust bean gum as shown in Fig. 5.6. These polymer combinations are used in very large quantities in cooked meats and in gelled petfoods. Kappa carrageenan and clarified locust bean gum mixtures can be used for cake glaze and flan gels or formulated to give clear water dessert gels with an elastic cohesive gel texture like gelatin. Recent improvements in formulations of kappa and iota carrageenan blends are also able to give elastic cohesive gels similar to gelatin in texture. Konjac flour

Fig. 5.6 Gel strength of blends of kappa carrageenan with locust bean gum or konjac mannan.

Carrageenan

95

Fig. 5.7 Kappa carrageenan-kappa casein milk protein interaction.

(E425i) interacts even more strongly than locust bean gum to form strong elastic gels with kappa carrageenan which are at least four times the rupture strength of kappa carrageenan alone. Probably the best known synergistic carrageenan interaction is that involving milk proteins. Some of the first uses of carrageenan were in milk gels and flans, and in the stabilisation of evaporated milk and ice cream mixes. In these applications the kappa carrageenan forms a weak gel in the aqueous phase and it also interacts with positively charged amino acids in the proteins in the surface of the casein micelles. The specific kappa carrageenan-kappa casein interaction is shown diagramatically in Fig. 5.7. Very low levels of 150–250ppm of carrageenan are sufficient to prevent whey separation from a range of dairy products during manufacture and storage. These include ice cream and milk shake mixes, cream cheese and dairy desserts. In chocolate milks, this low level of carrageenan is able to prevent separation and also generate a stabilising network which maintains the cocoa particles in suspension. Iota carrageenan, in combination with starch, gives dessert products with a body that is equivalent to four times that of starch alone. A summary of the solution and gelation properties of carrageenan and its synergy with other materials is given in Table 5.2.

5.5

Food applications

Carrageenans which are able to hydrate at low temperatures present problems for their efficient use. Any lumps which are produced when the carrageenan is dispersed in water or milk greatly reduce the rate of hydration and may limit the development of full viscosity or gel strength. Various methods may be used to ensure that the carrageenan particles are fully dispersed before hydration commences. Mixing the carrageenan with

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Table 5.2

Summary of carrageenan properties Lambda

Solubility Hot (80ºC) water Cold (20ºC) water Hot (80ºC) milk Cold (20ºC) milk Cold milk (TSPP added) 50% sugar solutions 10% salt solutions Gelation Effect of cations Gel texture Shear reversible gel Syneresis Hysteresis Freeze-thaw stable Synergy with locust bean gum Synergy with konjac flour Synergy with starch

Iota

Soluble All water soluble

Soluble Na+ salt soluble Ca++ salt gives thixotropic sols Soluble Soluble Thickens Insoluble Increased thickening Thickens or gels or gelling Soluble Insoluble Soluble hot Soluble hot

Kappa Soluble Na+ salt soluble Limited swelling of K+, Ca++ salts Soluble Insoluble Thickens or gels Soluble hot Insoluble

Non-gelling – – – – Yes No

Strongest gels with Ca++ Elastic Yes No 5–10ºC Yes No

Strongest gels with K+ Brittle No Yes 10–20ºC No Yes

No

No

Yes

No

Yes

No

Salt tolerance Stability in acid

Good Hydrolysis

Protein reactivity

Strong interaction increasing at acid pH

Good Poor Hydrolysis of solution, accelerated by heat Gels are stable Specific reaction with kappa-casein

5–10 times its weight of an inert filler such as sugar, maltodextrin or salt physically separates the carrageenan particles. Slurrying the powder in oil gives a hydrophobic barrier around the particles so that the powder may be dispersed throughout the liquid before the particles start to hydrate. The powder may be dispersed into a salt solution, or in a sugar syrup or alcohol or similar medium which impedes the hydration of the particles or raises the temperature at which the carrageenan particles hydrate. Kappa-carrageenan may be processed with potassium chloride to assist gelation. The potassium ions prevent the hydrocolloid hydrating at ambient temperatures and these products may be readily dispersed in cold water or milk without lumping. High shear mixing may be used to break up the lumps produced during dispersion and vigorous agitation by high-speed or high-shear mixing will quickly hydrate the dispersed particles. The broad spectrum of thickening and gelling properties of carrageenan result in its use in a wide range of water- and milk-based food products described below.

5.5.1 Water gels Water dessert gels and cake glazes are one of the most traditional uses for carrageenan. These products are based on the firm, brittle gels properties of kappa carrageenan with

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97

Formulation 5.1 Fruit-flavoured water dessert jelly Ingredients Sugar Carrageenan (kappa-iota blend) Potassium citrate Citric acid Colour Flavour Water Total

% 15.00–20.00 0.60–0.90 0.20–0.35 0.30–0.45 as required as required to 100.00 100.00

Formulation 5.2 Cooked ham with 30% added brine Ingredients Meat, lean ham muscles Carrageenan (firm gelling kappa) Sodium tripolyphosphate Nitrate salt* Sodium chloride Dextrose Water Total

% 62.50 0.60 0.50 1.67 0.53 1.20 32.95 100.00

* Sodium chloride containing 0.6% sodium nitrite, giving a 100ppm sodium nitrite in the finished product. The total brine concentration of sodium chloride is 2.2%.

the texture modified as appropriate for elasticity, cohesiveness and syneresis control using iota carrageenan. Recent improvements in the combinations used for these applications has produced vegetarian products which have a similar appearance and texture to traditional gelatin products whilst giving additional benefits of fast setting and stability at ambient temperatures. An example recipe is given in Formulation 5.1. Similar gels are also used for aspics and gels in canned meats and petfoods and in cooked sliced meats. In these latter products the carrageenan is incorporated to improve moisture retention, cooking yields, slicing properties and mouthfeel and succulence. A typical formulation for a 30% added-water sandwich ham is given in Formulation 5.2. In this product the carrageenan disperses readily in the meat brine when it is added after phosphate and salt addition. The brine has a very low viscosity and is easily injected and distributed within the meat. During cooking the carrageenan hydrates above about 50–55ºC to bind moisture as the ham is cooked to 72–74ºC. When the product is cooled a cohesive gel forms which maintains product integrity during high-speed slicing operations and reduces moisture loss throughout the product shelf life. Other water gel applications for carrageenan include gelled petfoods and meat and fish aspics. Processed Euchema seaweed is principally used in cooked sliced meats. This material is very cost effective and it disperses readily without lumping in meat brines. During processing some differences are apparent between this and traditional carrageenan extracts. The small particles do not swell in the brine and may cause less damage when injected into the meat (Philp et al., 1998). The cellulose network in processed Euchema seaweed reduces the rate of hydration during heating so that solutions develop viscosity

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after longer heating periods or after heating to higher temperatures. The presence of cellulose in the finished gel gives a lower rupture strength with a less cohesive and more fragile structure. The dispersed cellulose particles make the product cloudy and any gel spots in the injected meat will be masked against the background of the meat. As a consequence, meat brines may include both carrageenan and processed Euchema seaweed to optimise properties and costs. In markets such as the EU, where carrageenan and processed Euchema seaweed have distinct E numbers and must be labelled separately, companies may prefer to use carrageenan alone. In the US and other markets where no distinction is made between carrageenan and processed Euchema seaweed, meat brines often contain both materials. The synergy of kappa carrageenan and locust bean gum is also used in water dessert gels and glazes, cooked, sliced hams and poultry products, canned meats and petfoods, air fragrance gels and similar very firmly gelled products. The gel produced by this gum combination exhibits benefits of high gel strength, a cohesive, elastic texture, excellent syneresis control and cost-effectiveness. The gums also have an increased hot viscosity to retain juices better in cooked meats and to reduce emulsion separation and splashing during filling and cooling in fragrance gels. Petfood is the largest single use for processed Euchema seaweed. It is used in combination with locust bean gum for gelled products or with guar gum for gravies. The interaction between kappa carrageenan and konjac gum is used to a lesser extent at present as a result of the relatively short time this latter material has been available in a purified form. However, the greater synergy of this gum combination will be utilised in new food developments and initial products have been commercialised in petfoods, surimi and air freshener gels. The reversible gel properties of dilute iota carrageenan are used to stabilise suspended herbs and vegetables in vinaigrette dressings. Firstly the carrageenan is dispersed into water at ambient temperature to give a viscous solution. Salt (sodium chloride) is then added to produce a reversible gel which is very effective for suspending particulates over the long shelf life of such products. A recipe is given in Formulation 5.3. Other applications which utilise the stabilising properties of this reversible gel network include soy milks and sterilised milk drinks. Higher concentrations of this carrageenan give soft elastic gels suited to gravies for canned meats and petfoods and for various toothpastes. Beer and wine fining are applications which rely on the protein reactivity of carrageenan. Coarse particles of kappa carrageenan or processed Euchema seaweed are used to interact Formulation 5.3

Vinaigrette-style salad dressing

Ingredients 7% spirit vinegar Sugar Salt Carrageenan (iota) Xanthan gum Chopped spice pieces Colour and preservative Water Total

% 12.50 9.50 3.20 0.30 0.15 1.00 as required to 100 100.00

Carrageenan Table 5.3

99

Typical applications for carrageenan in water

Use

Function

Dessert gels

Gelation

Carrageenan type

Use level (%)

kappa + iota 0.5–1.0 kappa + iota + locust bean gum Low calorie gels Gelation kappa + iota 0.5–1.0 Non-dairy puddings Emulsion stabilisation kappa 0.1–0.3 Syrups Suspension, bodying kappa, lambda 0.3–0.5 BBQ and pizza sauces Bodying kappa 0.2–0.5 Whipped toppings Emulsion stabilisation kappa, iota 0.1–0.3 Imitation coffee creams Emulsion stabilisation lambda 0.1–0.2 Petfoods Thickening, suspending, iota + guar gum 0.5–1.0 Gelation, fat stabilisation kappa + locust bean gum 0.5–1.0

with proteinaceous materials and small protein fragments produced during pasteurisation to form aggregates which can be readily filtered to clarify the beer and reduce chill haze. Applications for carrageenan in water-based products are shown in Table 5.3.

5.5.2 Dairy applications Milk-based puddings were one of the original uses for carrageenan from Chondrus crispus harvested off Ireland. The seaweed was boiled in milk and the extracted carrageenan formed a gel on cooling. These properties are now widely adopted worldwide for a large number of instant and ready-to-eat flans, cre`me dessert and mousse. These products utilise the complete range of carrageenan types for thickening and gelling products. In many cases lambda carrageenan is used with kappa in milk systems to obtain a suspension or creamy gel. Iota carrageenans are often used in combination with reduced levels of starch to lower process viscosity and improved body with a soft spoonable texture to desserts. Textures may range from firm gels in cre`me caramel to soft gels in ready-to-eat spoonable desserts to thickened custards, vla and cream desserts. Extremely low levels of carrageenan, around 100–200ppm, are used to stabilise and prevent whey separation in a number of dairy products. These include milk shake and ice cream mixes, chocolate milks, and pasteurised and sterilised creams. In these applications Formulation 5.4 Typical ice cream mix Ingredients Butterfat Milk solids non-fat Sugar Corn syrup solids Carrageenan (kappa) Other hydrocolloids (guar gum, locust bean gum, xanthan gum, sodium alginate) Emulsifier (glyceryl monostearate) Vanilla flavour Water Total (Total solids)

% 8.00–10.00 11.10–10.80 10.00 3.50 0.015–0.025 0.10–0.20 0.20–0.50 as required to 100.00 100.00 33.3–35.0

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Table 5.4

Typical applications for carrageenan in dairy products

Use

Function

Carrageenan type

Use level (%)

Gelation, mouthfeel Thickening, gelation Reduced starch, lower burn-on Syneresis control, mouthfeel

kappa, kappa + iota kappa, iota, lambda kappa

0.2–0.3 0.2–0.3 0.1–0.2

iota

0.1–0.2

Stabilise overrun Stabilise overrun, emulsion stabilisation

lambda kappa

0.05–0.15 0.02–0.05

Suspension, mouthfeel, stabilise overrun

lambda

0.1–0.2

Whey prevention, control meltdown

kappa

0.01–0.02

Soy milks

Suspension and mouthfeel Suspension and mouthfeel Suspension and mouthfeel

kappa kappa + lambda kappa + iota

0.015–0.03 0.03–0.10 0.02–0.04

Sterilised milks Chocolate milks Evaporated milks

Suspension and mouthfeel Emulsion stabilisation

kappa, lambda kappa

0.01–0.03 0.005–0.015

Milk gels Cooked flans Cold-prepared custards Pudding and pie fillings Ready-to-eat desserts Whipped products Whipped cream Aerosol cream Cold-prepared milks Shakes Frozen desserts Ice cream, ice milk Pasteurised milks Chocolate milks

Processed cheese Cheese slices and blocks

Improve slicing and grating kappa Control melting Cream cheese and spreads Gelation, moisture binding kappa + locust bean gum

0.5–3.0 0.3–0.6

the carrageenan interacts with the dairy proteins to form a stabilising network which is able to suspend particulates such as cocoa in chocolate milks. The network prevents protein-protein interaction and aggregation during storage. This avoids whey separation in fluid products and reduces shrinkage in ice cream. Processed cheese is another application where the protein reactivity and gelling properties of carrageenan are used. Processed cheese is made by incorporating ‘melting’ or ‘emulsifying’ salts into the cheese mix to control the melting temperature whilst maintaining firmness and mouthfeel, ensuring slice integrity and producing a strong block suitable for grating. It is possible to reduce the cheese content in such products and substitute a gel containing 0.5–3% carrageenan to give a product with excellent mouthfeel properties and good melting, grating and slicing properties. Acidic dairy products, such as soft cheese and yoghurt, are generally unsuitable for carrageenan; the low pH increases the electrostatic interactions between protein and carrageenan producing unstable aggregates which flocculate and separate. However, careful selection of appropriate carrageenan-galactomannan blends is able to control this aggregation. These blends are effective stabilisers which prevent moisture separation whilst conferring a smooth, creamy mouthfeel to the finished product. The many dairy products which utilise the properties of carrageenan are shown in Table 5.4.

Carrageenan

5.6

101

Regulatory status

Material extracted from Furcellaria species, furcellaran, also known as Danish agar, was given a separate classification of E408 under EC food legislation. However, a later reassessment of carrageenan and furcellaran recognised the structural and functional similarity of the two materials and reclassified them together as E407. In the EU, carrageenan and processed Euchema seaweed are listed in Annex 1 of the European Parliament and Council Directive 95/2/EC on Food Additives other than Colours and Sweeteners (Anon., 1995). Carrageenan and processed Euchema seaweed are approved as E407 and E407A respectively in the list of permitted emulsifiers, stabilisers, thickening and gelling agents for use ‘quantum satis’, the level required to achieve a given technological benefit. Purity standards for food-grade carrageenan and processed Euchema seaweed have been amended recently under Council Directive 98/86/ EC which revises the heavy metal limits and defines the limits for acid insoluble matter in food-grade carrageenan and processed Euchema seaweed (Anon., 1998). Toxicological reviews have considered the implications of the low molecular weight material found in all carageenans, including native carrageenan, and the possible degradation of carrageenan during food processing and digestion. On this latter point, the presence of associated cations prevents carrageenan hydrolysis in the gastric environment (Marrs, 1998) and recent EU purity standards do not specify any limit for material below 100kDa (Anon., 1998). A study on the fate of carrageenan subjected to a range of conditions revealed that normal food processes do not substantially increase the proportion of low molecular weight material (Marrs, 1998). The level of this material only increases significantly when processing combines the effects of high temperatures, low pH and and long process times. For instance, heating kappa carrageenan solutions at pH4 at 120ºC for 15 seconds does not significantly increase the low molecular weight fraction but a gel strength reduction of over 25% is seen when heating this solution at 135–140ºC for 10 seconds. In fact, as material with a molecular weight below 100kDa has little or no thickening or gelling properties it is not valued for food applications and processes are designed to minimise degradation. In the US, no distinction is made between carrageenan and processed Euchema seaweed; both are described as carrageenan and are generally recognised as safe (GRAS) in accordance with experts of the Food and Drug Administration.

5.7

References and further reading

and REES, D. A. (1965) ‘Evidence for a common structural pattern in the polysaccharide sulphates of the Rhodphyceae’. Nature 205, 1060–2. ANON. (1988) General Carrageenan Application Technology. FMC Corporation, USA, pp. 1–18. ANON. (1995) Commission Directive 95/2/EC Official Journal of the European Communities, L 61, 18.3.95, p. 1. ANON. (1998) Commission Directive 98/86/EC Official Journal of the European Communities, L 334, 11.11.98, p. 1. BIXLER, H. J. (1996) ‘Refined and semi-refined carrageenan: room for both’, Food Hydrocolloids 96, San Diego, March 1996. HOFFMANN, R. A., RUSSELL, A. R. and GIDLEY, M. J. (1996) ‘Molecular weight distribution of carrageenans’ in Gums & Stabilisers for the Food Industry 8, G. O. Phillips, P. J. Williams and D. J. Wedlock eds, IRL Press at the Oxford University Press, Oxford, pp. 137–48. MCCANDLESS, E. L., CRAIGIE, J. S. and WALTER, J. A. (1973) Carrageenans in the gametophytic and sporophytic stages of Chondrus crispus, Planta, Berlin. MARRS, W. M. (1998) ‘The stability of carrageenans to processing’ in Gums & Stabilisers for the Food Industry 9, P. A. Williams and G. O. Phillips eds, The Royal Society of Chemistry, Cambridge, pp. 345–57. ANDERSON, N. S., DOLAN, T. C. S.

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(1996) ‘The chemical identification of PNG-carrageenan’ in Gums & Stabilisers for the Food Industry 8, G. O. Phillips, P. A. Williams and D. J. Wedlock eds, IRL Press at the Oxford University Press, Oxford, pp. 403–21. PHILP, K., DEFREITUS, Z., NICHOLSON, D. and HOFFMANN, R. (1998) ‘Tiger striping in injected poultry’ in Gums & Stabilisers for the Food Industry 9, P. A. Williams and G. O. Phillips eds, The Royal Society of Chemistry, Cambridge, pp. 276–85. REES, D. A. (1963) ‘The carrageenan system of polysacccharides, Part 1. The relation between kappa and lambda components’. J. Chem. Soc. pp. 1821–32. THOMAS, W. R. (1997) ‘Carrageenan’ in Thickening and Gelling Agents for Food 2nd edn, A. P. Imeson ed, Blackie, London, pp. 45–59. TYE, R. (1994) ‘Philippine natural grade carrageenan’ in Gums & Stabilisers for the Food Industry 7, G. O. Phillips, P. A. Williams and D. J. Wedlock eds, IRL Press at the Oxford University Press, Oxford, pp. 125–37. PHILLIPS, G. O.

6 Xanthan gum G. Sworn, Monsanto (Kelco Biopolymers, Tadworth)

6.1

Introduction

Xanthan gum is an extracellular polysaccharide secreted by the micro-organism Xanthomonas campestris. Xanthan gum is soluble in cold water and solutions exhibit highly pseudoplastic flow. Its viscosity has excellent stability over a wide pH and temperature range and the polysaccharide is resistant to enzymatic degradation. Xanthan gum exhibits a synergistic interaction with the galactomannans guar gum and locust bean gum (LBG) and the glucomannan konjac mannan. This results in enhanced viscosity with guar gum and at low concentrations with LBG. At higher concentrations soft, elastic, thermally reversible gels are formed with locust bean gum and konjac mannan.

6.2

Manufacture

The bacterium Xanthomonas campestris produces the polysaccharide at the cell wall surface during its normal life cycle by a complex enzymatic process.1 In nature the bacteria are found on the leaves of the Brassica vegetables such as cabbage. Commercially, xanthan gum is produced from a pure culture of the bacterium by an aerobic, submerged fermentation process. The bacteria are cultured in a well-aerated medium containing glucose, a nitrogen source and various trace elements. To provide seed for the final fermentation stage, the process of inoculum build-up is carried out in several stages. When the final fermentation has finished the broth is pasteurised to kill the bacteria and the xanthan gum is recovered by precipitation with isopropyl alcohol. Finally, the product is dried, milled and packaged.

6.3

Structure

The primary structure of xanthan gum shown in Fig. 6.1, is a linear (1 ! 4) linked -Dglucose backbone (as in cellulose) with a trisaccharide side chain on every other glucose

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Fig. 6.1

Primary structure of xanthan gum.

at C-3, containing a glucuronic acid residue linked (1 ! 4) to a terminal mannose unit and (1 ! 2) to a second mannose that connects to the backbone.2,3 Approximately 50% of the terminal mannose residues are pyruvated and the non-terminal residue usually carries an acetyl group at C-6. X-ray diffraction studies on orientated xanthan gum fibres identified the molecular conformation as a right-handed, five-fold helix with a rise per backbone disaccharide residue of 0.94nm, i.e. a five-fold helix with a pitch of 4.7nm.4 In this conformation the trisaccharide side chain is aligned with the backbone and stabilises the overall conformation by non-covalent interactions, principally hydrogen bonding. In solution the side chains wrap around the backbone thereby protecting the labile -(1 ! 4) linkages from attack. It is thought that this protection is responsible for the stability of the gum under adverse conditions. Xanthan gum solutions at low ionic strength undergo a thermal transition. This was first observed as a sigmoidal change in viscosity of 1% salt-free solutions.5 Subsequent studies using optical rotation and circular dichroism show transitions coincident with the viscosity change.6,7 These results are consistent with a helix coil transition. It has been proposed that the xanthan helix in solution should be considered as a rigid rod. The transition is thermally reversible with the structure returning to its original state upon cooling. The transition temperature increases with increasing salt concentration as shown in Fig. 6.2. Xanthan gum in solution is also able to form intermolecular associations that result in the formation of a complex network of weakly bound molecules.

Xanthan gum

Fig. 6.2

6.4

105

Melting temperature of 1.0% xanthan gum solutions as a function of sodium chloride concentration.

Technical data

The functionality of xanthan gum is dependent upon the correct preparation of the gum solution. Poor solution preparation can lead to poor functionality in the final application. Therefore, before discussing the properties of xanthan gum in more detail, it is important to understand how to prepare a gum solution correctly.

6.4.1 Preparing xanthan gum solutions To obtain optimum functionality, xanthan gum must be properly hydrated before use. Hydration depends on four factors: 1. 2. 3. 4.

dispersion agitation rate of the solvent composition of the solvent particle size.

To hydrate properly the gum particles must be well dispersed. Poor dispersion leads to clumping of particles during mixing which results in formation of swollen lumps (sometimes called ‘fisheyes’). Severe lumping prevents complete hydration and reduced functionality. The ideal way to disperse xanthan gum is through the use of a dispersion funnel and mixing eductor (Fig. 6.3). Using this apparatus may eliminate the need for high shear mixing equipment. Before xanthan is added, the tank should be filled with sufficient water to cover the mixer blades when a vortex is developed. In small tanks, the water may be run through the eductor. Larger tanks may be filled faster directly. When enough water has been added, the agitator is turned on, and water flow is started in the eductor at a rate of 80–120 litres per minute. Dry xanthan gum powder is poured into the funnel attached

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Fig. 6.3 Diagram of dispersion funnel and mixing eductor.

to the top of the eductor. While xanthan gum is being added, and until it is completely dissolved, the liquid must be stirred continuously to prevent settling of dispersed granules. A minimum water pressure of 50psi is required. If water pressure exceeds 100psi, flow will be too fast, and particles will lump. An alternative method of dispersion is to blend the xanthan gum with other ingredients in the formulation such as sugar or starch. In this case the gum particles are kept separated by the dispersant and the blend can be added directly to the vortex created by an agitator. An ideal blend ratio is approximately 10:1 dispersant:xanthan. Dispersion can also be improved by separating the xanthan gum particles with non-solvents such as miscible, non-aqueous liquids (alcohol or glycol), or non-miscible liquids (vegetable or mineral oil). The xanthan gum can be slurried in the non-aqueous liquid and poured into water that is being agitated. Glycol dispersions should be used within a few minutes of being prepared because xanthan gum tends to solvate and swell in these liquids. In some applications the use of a stock paste may be desirable. Xanthan gum pastes of at least 6% solids can be prepared and, with the addition of a preservative, stored indefinitely at room temperature. When needed, the appropriate amount of paste can be weighed into a container equipped with an agitator and dilution water added slowly with agitation. Note that addition of stock paste to the water results in poor dispersion and hydration. Several grades of xanthan are specifically processed for ease of dispersion. These can be used where only relatively low shear mixing equipment is available. Examples are

Xanthan gum

107

Fig. 6.4 Effect of particle size (m) on the viscosity development profile of xanthan gum.

KELTROLÕ RHD and KELTROLÕ RD xanthan gum. Where dispersion and mixing conditions are good, fine particle size food grades of xanthan such as KELTROLÕ F and KELTROLÕ TF are available. When the dry powder particles are dispersed correctly a solution can be achieved in a few minutes with the hydration rate being dependent on the particle size as shown in Fig. 6.4.

6.4.2 Rheology of xanthan gum solutions Present knowledge of the structure and conformation of xanthan gum explains many of its unique solution properties. The relationship between the structure of xanthan gum and its properties is summarised in Table 6.1. Xanthan gum solutions are highly pseudoplastic. When shear stress is increased, viscosity is progressively reduced. Upon the removal of shear, the initial viscosity is recovered almost instantaneously. This behaviour results from the ability of xanthan molecules, in solution, to form aggregates Table 6.1

Structure/property relationship for xanthan gum

Structural features

Properties

Complex aggregates, with weak intermolecular forces

High viscosity at low shear rates (suspension stabilising properties) High viscosity at low concentrations High elastic modulus Pseudoplastic rheology Temperature insensitivity and salt compatibility Stability to acids, alkalis and enzymes

Rigid helical conformation, hydrogen bonded complexes, anionic charge on side chains Backbone protected by large overlapping side chains

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Fig. 6.5 Flow curve of 0.5% xanthan gum solution in standardised tap water. Standardised tap water is prepared by dissolving 1.00g NaCl and 0.15g CaCl2.2H2O in 1 litre of deionised water.

through hydrogen bonding and polymer entanglement. This highly ordered network of entangled, stiff molecules results in high viscosity at low shear rates, and in practical terms, accounts for the outstanding suspending properties of xanthan gum solutions. These aggregates are progressively disrupted under the influence of applied shear, hence the highly pseudoplastic flow characteristics of xanthan gum solutions.8 Figure 6.5 shows the effect of shear rate on a 0.5% xanthan gum solution. Eleven orders of magnitude are covered, and xanthan gum shows pseudoplastic properties over most of the range. This solution varies in viscosity from 1 million mPa.s at low rates of shear to about 1.7 mPa.s at the highest rates of shear. At both the highest and lowest shear rates there is evidence of a levelling off of the viscosity. These regions are known respectively as the upper and lower Newtonian regions. Solutions of xanthan gum at 1% or higher concentration appear almost gel-like at rest yet these same solutions pour readily and have low resistance to mixing and pumping. These same qualities are observed at typical use levels of about 0.1–0.3%. The high viscosity of xanthan gum solutions at low shear rates accounts for their ability to provide long-term stability to colloidal systems. The reduction in viscosity in response to increasing shear is important to the pouring properties of suspensions and emulsions and to the efficacy of xanthan gum as a processing aid. In Fig. 6.6, the viscosity of some common gums is compared over a range of shear rates relating to specific functions or processes. At low shear rates, solutions of xanthan gum have approximately 15 times the viscosity of guar gum and significantly more viscosity than carboxymethylcellulose (CMC) or sodium alginate which accounts for its superior performance in stabilising suspensions. At a shear rate of approximately 100sÿ1 all the hydrocolloids have similar viscosity. Above 100sÿ1, however, the viscosity of xanthan gum solutions drops sharply compared to the other gums making it easy to pour, pump or spray.

Xanthan gum

Fig. 6.6

109

Comparison of the flow behaviour of xanthan gum to other hydrocolloid solutions (0.5% concentration).

Effect of salts on viscosity How salts affect viscosity depends on the concentration of xanthan gum in solution. At or below about 0.25% gum concentration, monovalent salts such as sodium chloride cause a slight decrease in viscosity. At higher gum concentrations, viscosity increases with added salt. At a sodium chloride level of 0.1%, a viscosity plateau is reached, and further addition of salt has little effect on viscosity. Many divalent metal salts, including those of calcium and magnesium, have a similar impact on viscosity. To develop optimal rheological and uniform solution properties, some type of salt should be present; usually the salts found naturally in tap water are sufficient to generate these effects. Salt concentrations of greater than 1–2% in the water can slow down the hydration of xanthan gum and it is therefore recommended to hydrate the gum in the absence of excess salt. Once hydrated, additional salt can be added without adverse effects. Special grades of xanthan gum such as KELTROLÕ BT are available that are able to hydrate in up to 20% salt solutions. Effect of pH on viscosity Generally, pH has little effect on the viscosity of xanthan gum solutions over the range encountered in food systems. Uniform and high viscosity is maintained over the pH range 2–12, with some reduction at extreme pH values. However, differences in viscosity are more evident at low concentrations of xanthan gum. The solutions have excellent stability at low pH over long time periods. Xanthan gum hydrates in many acidic solutions. For example, it is directly soluble in 5% acetic acid, 5% sulfuric acid, 5% nitric acid and 25% phosphoric acid. Additionally, xanthan gum will hydrate in up to 5% sodium hydroxide. Hydration rate, however, is improved when it is dissolved in water before adding the acid or alkali. In the presence of most organic acids, stability is excellent. At elevated temperatures, however, acid hydrolysis of the polysaccharide is accelerated and a reduction in viscosity will occur.

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Table 6.2

Stability of xanthan gum solutions to acid at ambient temperatures

Acid

Acetic acid Citric acid Hydrochloric acid Phosphoric acid Sulfuric acid Tartaric acid

Acid concentration (%)

Xanthan gum concentration (%)

Viscosity retained after 90 days

20 20 5 40 10 20

2 1 2 2 2 1

100 75 80 100 80 75

Table 6.2 shows the stability of xanthan gum in several organic and mineral acids at ambient temperatures. Effect of temperature on viscosity Xanthan gum solutions are unique in their ability to retain their viscosity until a definite ‘melting temperature’ is reached. At this temperature, the viscosity drops sharply due to a reversible molecular conformation change. The melting temperature is dependent on the ionic strength of the solution as shown in Fig. 6.2. Above approximately 5% NaCl the melting temperature is greater than 100ºC. The viscosity loss is reversible and upon cooling the original high viscosity is recovered.

6.4.3 Compatibility of xanthan gum Alcohol While xanthan gum will not dissolve directly into alcohol, solutions of xanthan gum are compatible with alcohol. Products containing alcohol can be formulated, to contain up to 60% water-miscible solvents such as ethanol. This enables its use as a thickener in alcohol-based products such as cocktails and chocolate liqueurs. Enzymes Most hydrocolloids are degraded to some extent by enzymes normally present in some foodstuffs. Enzymes commonly encountered in food systems such as proteases, cellulases, pectinases and amylases, however, do not degrade the xanthan gum molecule. It is thought that this enzyme resistance is due to the arrangement of the side chains attached to the backbone. This arrangement prevents the enzymes from attacking the (1!4) linkages in the backbone, thereby preventing depolymerisation by enzymes, acid and alkali. In practice the enzyme resistance of xanthan gum is exploited in food systems such as pineapple products, starch-based systems, spice mixes and many other products containing active enzymes.

6.4.4 Interaction with galactomannans/glucomannans A synergistic interaction occurs between xanthan gum and galactomannans such as guar gum, locust bean gum and cassia gum and glucomannans such as konjac mannan. This interaction results in enhanced viscosity or gelation. Galactomannans are hydrocolloids in which the mannose backbone is partially substituted by single-unit galactose side chains.9,10,11 The degree and pattern of

Xanthan gum

111

substitution varies between the galactomannans and this strongly influences the extent of interaction with xanthan gum. Galactomannans with fewer galactose side chains and more unsubstituted regions react more strongly. Thus locust bean gum, which has a mannose to galactose ratio of around 3.5:1, reacts more strongly with xanthan than does guar gum, which has a mannose to galactose ratio of slightly less than 2:1. Although there is still much debate as to the exact nature of this interaction, it is generally accepted that the xanthan gum interacts with the unsubstituted ‘smooth’ regions of the galactomannan molecules. Xanthan/guar mixtures exhibit a synergistic increase in viscosity as do low concentration mixtures with locust bean gum (< 0.03%) At higher concentrations soft, elastic gels are formed with LBG. Xanthan gum/LBG gels are thermally reversible setting and melting at approximately 55–60ºC. Solutions of xanthan/guar mixtures can be prepared at room temperature using the guidelines outlined in Section 6.4.1. However, heating of the solutions above the transition temperature of the xanthan gum does result in a greater synergistic interaction. Mixtures of xanthan and LBG require heating to approximately 90ºC to 95ºC to fully hydrate the LBG and maximise the synergistic interaction. The interaction of xanthan gum with galactomannans is dependent on the ratio of the mixture, pH and ionic environment. Optimum gum ratios are approximately 80:20 guar gum:xanthan gum (Fig. 6.7) and 50:50 for LBG:xanthan gum (Fig. 6.8). Generally, the synergistic interaction with galactomannans is at its maximum in deionised water at neutral pH and is reduced at high salt concentrations and low pH.

Fig. 6.7 Theoretical (dashed line) and observed (solid line) viscosities for blends of guar and xanthan gum. Data were collected with a Brookfield LVT viscometer at 60rpm at 25ºC. The theoretical viscosity line is calculated on the assumption that non-interacting hydrocolloids will obey the log mean blending relationship.

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Fig. 6.8

6.5

Effect of gum ratio on the hardness (force required to break the gel) of xanthan/locust bean gum gels (1% total gum in 0.05M NaCl).

Uses and applications

The following is a summary of the many applications for xanthan gum or blends of xanthan gum and galactomannans and their related functions and benefits.

6.5.1 Batters In wet prepared batters, xanthan gum reduces flour sedimentation; improves gas retention; imparts enzyme, shear and freeze-thaw stability; and provides uniform coating and good cling. In pre-dusts, xanthan gum improves adhesion and controls moisture migration during frying. In pancake batters, xanthan gum improves spread control, volume and air retention. Xanthan gum is used in batter coatings for onion rings where inconsistent adhesion, caused by the waxy coating found in onion rings, can be eliminated. Variations in waxiness between varieties and seasons occur and can increase this problem. Cling properties associated with the high at-rest viscosity of xanthan gum solutions contribute to the increased adhesion of the batter. Use levels of approximately 0.15% of the batter weight are effective in this application. Batters for fish can be stabilised with 0.06% xanthan gum whereas 0.1–0.15% is recommended for thin batters such as those used with shrimps (tempura). Xanthan can also be used in batters for frozen products such as chicken, shrimp or fish.

6.5.2 Baked goods, bakery and pie fillings Xanthan gum contributes smoothness, air incorporation and retention, and recipe tolerance to batters for cakes, muffins, biscuits and bread mixes. Baked goods have

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113

increased volume and moisture, higher crumb strength, less crumbling and greater resistance to shipping damage. Xanthan gum improves volume, texture and moisture retention in refrigerated dough, reduced calorie baked goods and gluten-free breads. Moisture control is essential at all stages of cake production and also when formulating a dry cake mix. Improper moisture control can result in lumpy cake batters and uneven mixing, giving poor structure which results in collapsed cakes during or after baking. The overall quality of the finished cake, particularly after storage, can be affected by poor hydration characteristics of the dry ingredients. For example, volume can be reduced and texture may be non-uniform or fragile when moisture is not evenly distributed throughout the cake. Xanthan gum blended with the other dry cake ingredients, hydrates rapidly and evenly to aid in preventing lumping during the critical initial mixing stage. This even hydration aids in the uniform distribution of moisture in the batters, which in turn helps stabilise the fine air cells formed during the mixing process. The stabilisation of air cells improves volume and symmetry in the finished cake. Xanthan can be added to the cake batter at 0.05% (total batter weight) without the need for any other formulation change. Adding xanthan gum to either cold or hot processed bakery and fruit pie fillings improves texture and flavour release. The added benefits in cream and fruit fillings are extended shelf stability, freeze-thaw stability and syneresis control.

6.5.3 Dairy products Blends of xanthan gum, carrageenan and galactomannans are excellent stabilisers for a range of frozen and chilled dairy products such as ice cream, sherbet, sour cream, sterile whipping cream and recombined milk. These economical blends are available preprepared and provide optimal viscosity, long-term stability, improved heat transfer during processing, heat shock protection and ice crystal control.

6.5.4 Dressings This is arguably the largest single application of xanthan gum in the food industry. Xanthan gum’s stability to acid and salt, effectiveness at low concentrations and highly pseudoplastic rheology make it the ideal stabiliser for pourable, no-oil, low-oil and regular salad dressings. Dressings with xanthan gum have excellent long-term stability and a relatively constant viscosity over a wide temperature range. They pour easily but cling well to the salad. Use level is typically between 0.2–0.4% xanthan depending on the oil content. Generally, as the oil content of the dressing increases less xanthan gum is required for stabilisation and a guide to use levels is given in Table 6.3. Dressings can be formulated over a wide range of oil contents as shown in Formulation 6.1.

Table 6.3

Suggested stabiliser level for salad dressing formulation

% Oil used

10

20

30

40

% Starch % Xanthan gum

2.0 0.35

2.0 0.3

1.5 0.25

1.5 0.25

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Handbook of hydrocolloids Formulation 6.1 gum.

Recipe for salad dressings using KELTROLÕ T xanthan

Ingredients Water Distilled malt vinegar Vegetable oil Sugar Egg yolk powder Salt Mustard powder Lemon juice Potassium sorbate Waxy maize starch Xanthan gum

(%) to 100.0 15.0 see Table 6.3 10.0 1.35 1.0 0.8 0.8 0.1 see Table 6.3 see Table 6.3

Preparation 1. Slurry the xanthan and starch in a little of the oil. Use approximately 2 parts oil to 1 part gum. This will help to disperse the gum easily into the water without lumping. 2. Add the slurry to the water and continue mixing with a high shear mixer until a smooth, lump free solution is obtained. 3. Pre-blend the other dry ingredients and dissolve them into the gum solution. 4. Slowly add the oil using vigorous agitation to achieve an homogeneous emulsion. 5. Slowly add the distilled malt vinegar and lemon juice whilst stirring. 6. Homogenise at 105kg/cm2 (1500psi) and fill into bottles.

6.5.5 Dry mixes Fine particle size xanthan gum provides rapid, high viscosity development in cold or hot systems and yields excellent texture and flavour release. It also permits easy preparation of desserts, salad dressings, dips, soups, milkshakes, sauces, gravies and beverages. In dry mix beverages, xanthan gum provides enhanced body and quality to the reconstituted drink. In addition, it uniformly suspends fruit pulp in prepared drinks to improve product appearance and texture.

6.5.6 Frozen foods Stability, syneresis control and consistent viscosity during freeze-thaw cycles and heating are achieved by adding xanthan gum to a variety of frozen products such as whipped toppings, sauces, gravies, batters, entre´es and souffle´s.

6.5.7 Retorted products Although xanthan gum provides stable, high viscosity over a range of temperatures, this viscosity is temporarily reduced at retort temperatures, ensuring good thermal penetration in retorted foods. At the same time the ability of xanthan gum to recover its viscosity upon cooling, provides a uniform, high-quality product. In retort pouch products, xanthan gum also improves filling and reduces splashing and fouling of the critical heat-seal area of the pouch. Xanthan gum can be used to partially replace starch in this application. This results in improved heat stability and a cleaner, less pasty mouthfeel. Typically, xanthan is used at 0.1–0.2% concentration.

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115

6.5.8 Sauces and gravies Low levels of xanthan gum provide high viscosity in sauces and gravies at both acid and neutral pH. Viscosity is also stable to temperature changes and is maintained under a variety of long-term storage conditions. Sauces and gravies containing xanthan gum cling to hot foods.

6.5.9 Syrups and toppings Xanthan gum promotes ease of pouring and excellent cling to ice cream, fruits and pancakes. Under refrigerated storage, syrups and toppings retain uniform consistency. Cocoa powder in chocolate syrups remains suspended. Frozen non-dairy whipped toppings and frozen whipped topping concentrates have firm texture, high overrun and excellent freeze-thaw stability.

6.6

Regulatory status

Xanthan gum is recognised as a food additive under the provisions of the US Food and Drug Administration regulations (21 CFT 172.695) for use as a stabiliser, thickener or emulsifier. Xanthan gum is designated by the European Union as E415 with a nonspecified acceptable daily intake (ADI). KELTROLÕ and KELTROLÕ F xanthan gum are approved for Kosher use.

6.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

References and CLEARY, J. M. (1995) ‘Genetics and biochemistry xanthan gum production by Xanthomonas campestris’ in Food Biotechnology Microorganisms, eds Y. H. Hui and G. C. Khachatourians. VCH Publishers, New York, pp. 495–514. JANSSON, P. E., KENNE, L. and LINDBERG, B. (1975) ‘Structure of the exocellular polysaccharide from Xanthomonas campestris’ Carbohydr. Res. 45, 275–82. MELTON, L. D., MINDT, L., REES, D. A. and SANDERSON, G. R. (1976) ‘Covalent structure of the polysaccharide from Xanthomonas campestris: evidence from partial hydrolysis studies’ Carbohydr. Res. 46, 245–57. MOORHOUSE, R., WALKINSHAW, M. D. and ARNOTT, S. (1977) ‘Xanthan gum-molecular conformation and interactions’ in Extracellular Microbial Polysaccharides, ed. P. A Sandford. ACS symposium series no. 45, American Chemical Society, Washington, DC, pp. 90–102. JEANES, A., PITTSLEY, J. E. and SENTI, F. R. (1961) ‘Polysaccharide B-1459: a new hydrocolloid polyelectrolyte produced from glucose from bacterial fermentation’ J. App. Polym. Sci., 5, 519–26. MORRIS, E. R., REES, D. A., YOUNG, G., WALKINSHAW, M. D. and DARKE, A. (1977) ‘Order-disorder transition for a bacterial polysaccharide in solution: a role for polysaccharide conformation in recognition between Xanthomonas pathogen and its plant host’ J. Mol. Biol., 110, 1–16. KAWAKAMI, K., OKABE, Y. and NORISOYE, T. (1991) ‘Dissociation of dimerized xanthan in aqueous solution’ Carbohydr. Polym., 14, 189–203. OVIATT, H.W. and BRANT, D. A. (1993) ‘Thermal treatment of semi-dilute xanthan solutions yields weak gels with properties resembling hyaluronic acid’ Int. J. of Biol. Macromol., 15, 3–10. BAKER, C. W. and WHISTLER, R. L. (1975) ‘Distribution of D-galactosyl groups in guar and locust bean gum’ Carbohydr. Res., 45, 237–43. COURTOIS, J. E. and LEDIZET, P. (1966) ‘Action de a-galactosidase du cafe´ sur quelques galactomannaes’ Carbohydr. Res., 3, 141–51. COURTOIS, J. E. and LEDIZET, P. (1970) ‘Recherches sur les galactomannanes. VI Action de quelques mannase sur diveres galactomannanes’ Socie´te´ de Chemie Biologique, Paris, Bulletin 52, 15–22. HARDING, N. E., IELPI, L.

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7 Gellan gum G. Sworn, Monsanto (Kelco Biopolymers, Tadworth)

7.1

Introduction

Gellan gum is an extracellular polysaccharide secreted by the micro-organism Sphingomonas elodea (ATCC 31461) previously referred to as Pseudomonas elodea. Gellan gum forms gels at low concentrations when hot solutions are cooled in the presence of gel promoting cations. It is available in a substituted or unsubstituted form. Gel properties depend on the degree of substitution with the substituted form producing soft, elastic gels whilst the unsubstituted form produces hard, brittle gels.

7.2

Manufacture

Commercially, gellan gum is manufactured by inoculating a fermentation medium with the micro-organism. The medium contains a carbon source, such as glucose, phosphate and nitrogen sources, and appropriate trace elements. The fermentation is carried out under sterile conditions with strict control of aeration, agitation, temperature and pH. After fermentation, the viscous broth is pasteurised to kill viable cells. The polysaccharide can then be recovered in several ways. Direct recovery by alcohol precipitation from the broth yields the substituted, high acyl form. Alternatively, treatment of the broth with alkali prior to alcohol precipitation results in deacylation and yields the unsubstituted, low acyl form. Gellan gum is currently sold commercially in three forms namely, high acyl, unclarified (KELCOGELÕ LT100), low acyl, unclarified (KELCOGELÕ LT) and low acyl, clarified (KELCOGELÕ and KELCOGELÕ F).

7.3

Structure

The primary structure of gellan gum is composed of a linear tetrasaccharide repeat unit: !3)-b-D-Glcp-(1!4)-b-D-GlcpA-(1!4)-b-D-Glcp-(1!4)-a-L-Rhap-(1!.1, 2 The polymer is produced with two acyl substituents present on the 3-linked glucose, namely L-glyceryl, positioned at O(2) and acetyl at O(6). On average there is one glycerate per

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repeat unit and one acetate per every two repeats.3 X-ray diffraction of low acyl gellan gum has shown that the polymer exists in the solid state as a co-axial three-fold double helix.4, 5 Computer modelling of the high acyl structure, based on the knowledge of the low acyl form concluded that the acetate substituents would be positioned on the outside of the double helix.6 Subsequent X-ray diffraction data revealed that the glycerate substituent was accommodated by an approximately 14º rotation of the carboxyl group about the C(5)-C(6) bond on the adjacent glucuronate residue.7 This was accompanied by a 173º rotation of the glucuronate residue itself to maintain the required spacing between the carboxyl and glycerate groups. It is proposed that this structure results in helices stabilised by interchain associations involving the glycerate groups, with the acetyl substituents positioned on the periphery of the helix.8

7.4

Technical data

There are three steps to consider for the successful formulation of gellan gum systems, these being: 1. 2. 3.

dispersion hydration gelation

and these will now be considered in turn for both low acyl (LA) and high acyl (HA) forms of the gum.

7.4.1 Dispersion The first step in preparing any gum solution is to ensure that the gum particles are properly dispersed in the solvent and do not clump together. Poor dispersion will result in incomplete hydration and loss of gum functionality. Both forms of gellan gum are insoluble in cold water although it will tend to swell in water of low calcium content. The gum can therefore be readily dispersed in deionised water by stirring and adding the powder slowly to the vortex. As the ion concentration in the water increases, dispersion becomes even easier. For example, in moderately hard water (~180ppm hardness expressed as CaCO3), the gum can be added to the surface of the water and then dispersed with gentle agitation. By blending the gum with dispersants such as sugar (5–10 times weight of gum) or glycerol, alcohol, or oils (3–5 times weight of gum), it is possible to add the gum directly to hot water. Both forms of gellan gum are also readily dispersible in milk and reconstituted milk systems.

7.4.2 Hydration Low acyl gellan gum The temperature at which LA gellan gum hydrates is dependent on the type and concentration of ions in solution. The presence of ions such as sodium, and in particular calcium, in solution will inhibit the hydration of LA gellan gum as shown in Table 7.1. It is therefore necessary, in most circumstances, to use a sequestrant to bind the soluble calcium and so aid hydration. Typically, between 0.1–0.3% of a sequestrant such as sodium citrate is sufficient to allow complete hydration at 90 to 95ºC in water of up to

Gellan gum Table 7.1

Effect of dissolved salts on the hydration temperature of 0.25% low acyl gellan gum

Water hardness (ppm CaCO3) 0.000 100 200 300 – – – – – – – Table 7.2

Dissolved NaCl (%)

Hydration temperature (ºC)

– – – – 0.10 0.25 0.45 0.70 0.90 1.00 1.30

75 88 > 100 > 100 50 52 60 70 82 89 > 100

Effect of sodium citrate on the hydration of 0.25% low acyl gellan gum

Water hardness (ppm CaCO3) 0 100 300 600 900 Table 7.3

119

Sodium citrate (%)

Hydration temperature (ºC)

0.00 0.05 0.10 0.20 0.40

75 25 65 65 68

Relative sequestering power of sequestrants commonly used in foods

Sequestrant

Sodium hexametaphosphate Tetra sodium diphosphate Di sodium orthophosphate Tri sodium citrate dihydrate

Parts of sequestrant required to sequester 1 part of available calcium pH6

pH4

7 10 130 20

7 20 180 40

600ppm as CaCO3 water hardness. Incomplete hydration will result if the sodium ion concentration exceeds 0.5% (approximately 1.3% sodium chloride). Once the gum is hydrated additional ions can be added to the hot solution and, provided the temperature is maintained above the gelation temperature, no gel will form. Table 7.2 provides details of the effects of the sequestrant sodium citrate on the hydration temperature of LA gellan gum and shows that hydration can be achieved at temperatures ranging from room temperature to boiling point. Table 7.3 lists the relative sequestering power and effective pH ranges of a number of commonly used sequestrants. In foods containing sugar, LA gellan gum should be hydrated in water and any sugars can be added to the hot gum solution. However, LA gellan gum can be hydrated directly in sugar solutions up to 80% total soluble solids (tss) by heating to boiling. In some cases a low level of sequestrant such as sodium citrate (less than 0.3%) is required to bind the free calcium often present in sugar syrups.

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Low acyl gellan gum will not fully hydrate below pH3.9. In this case, the acid should be added, preferably as a concentrated solution, to the hot gum solution. Prolonged heating in acidic conditions should be avoided as this leads to some hydrolytic degradation of the gum resulting in a reduction in the quality of the final gel. However, at pH3.5, LA gellan gum can be held for up to 1h at 80ºC with only minimal loss in quality of the final gel. In neutral conditions solutions can be held at 80ºC for several hours without a significant loss in the quality of the final gel. LA gellan gum is readily dispersible in milk and reconstituted milk systems and will hydrate upon heating to approximately 80ºC without the need for a sequestrant. High acyl gellan gum The hydration of HA gellan gum is much less dependent on the concentration of ions in solution than LA gellan gum and generally heating to 85–95ºC is sufficient to fully hydrate the gum in both water or milk systems. As a dispersion of high acyl gellan gum is heated it swells rapidly at approximately 40–50ºC to form a thick, pasty suspension. With continued heating the suspension loses viscosity suddenly at approximately 80–90ºC signifying complete hydration. The swelling stage can be avoided by adding the gum directly to hot water (> 80ºC) with the aid of a dispersant such as sugar, oil or glycerol, as described in the previous section. The hydration of HA gellan gum is inhibited by the presence of sugars; therefore it is recommended to hydrate the gum in less than 40% tss. Additional sugar can then be added to the hot gum solution. Unlike low acyl gellan gum, high acyl gellan gum will hydrate below pH4.0. Similar care must be taken under hot acidic conditions to avoid hydrolytic breakdown and loss of gel quality.

7.4.3 Gelation The proposed gelation mechanism of gellan gum is based on the domain model.9 As a hot solution cools gellan gum undergoes a disorder–order transition. This transition has been attributed to a coil-helix transition.10 In the case of low acyl gellan gum, gel promoting cations such as sodium, potassium, calcium and magnesium promote aggregation of the gellan double helices to form a three-dimensional network and the subsequent gels are hard and brittle. The acyl substituents have a profound effect on the structure and rheological characteristics of high acyl gellan gum gels. The high acyl gellan gum undergoes a similar disorder to order transition as the solution is cooled, but further aggregation of the helices is limited by the presence of the acetyl group.8 The subsequent gels are, therefore, soft and elastic. Low acyl gellan gum The easiest and most common method of making LA gellan gum gels is to cool hot solutions. LA gellan gum forms gels with a wide variety of cations, notably calcium (Ca2+), magnesium (Mg2+), sodium (Na+), and potassium (K+) as well as acid (H+).10,11 Divalent cations are more efficient at promoting gelation of LA gellan gum than monovalent ions. Gel strength increases with increasing ion concentration until a maximum is reached. Further addition of ions results in a reduction of gel strength due to the ‘over conversion’ of the LA gellan gum with excess ions. Ion concentrations for optimum gelation are generally independent of gum concentration but are reduced as the level of sugar is increased. Figure 7.1 compares the effect of both mono and divalent ions

Gellan gum

121

Fig. 7.1 Effect of calcium (squares), sodium (triangles) and potassium (circles) ion concentration on the modulus of 0.5% low acyl gellan gum gels in water (open symbols) and 60% sucrose (closed symbols).

on the modulus of 0.5% LA gellan gum gels in water and 60% sucrose. Below pH3.0 LA gellan gum is able to form a gel without the need for mono or divalent metal ions. Optimum gel modulus occurs for these acid gels at approximately pH2.8–3.0 regardless of the acid used. This optimum is not affected by the presence of sugars to the same extent as ion requirements. For example, in the presence of 60% sucrose the optimum shifts to approximately pH2.5–2.7. Acid gels are generally stronger than ion-mediated gels in both water and sugar. Addition of other gelling ions such as sodium or calcium generally result in a reduction in gel strength of the acid gels. Gel properties at optimum conditions for LA gellan gum gels in water and 60% sucrose are summarised in Tables 7.4 and 7.5 respectively. In many instances addition of gelling ions is not necessary since there are sufficient ions present in the water or other ingredients to promote gelation of the LA gellan gum. When required, the gel promoting ions can be added to the hot gum solution and, provided the solution is kept above its setting temperature no gel will form. This allows for the easy preparation of stock solutions (1–2% gum) which can be held at high temperature until required. LA gellan gum is often described as having a ‘snap set’ since gelation is very rapid once the setting temperature is reached. As with hydration temperature, setting and melting temperatures of the gels depend on the ion concentration in solution. The higher the ion concentration the higher the setting and melting temperature. Significant thermal hysteresis between the setting and melting temperature is observed in LA gellan gum gels, i.e., the gels melt at a higher temperature than that at which they set.12 In most conditions LA gellan gum gels are not thermally reversible

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Table 7.4 water

Properties of 0.5% LA gellan gum gels in optimum conditions for gel formation in

Gelling ion

Ion concentration (mM)

Modulus (Ncmÿ2)

Brittleness (%)

Setting temperature (ºC)

Calcium Sodium Potassium Acid

8–10 260–300 240–260 pH2.8–3.0

19.3 12.3 12.1 20.3

28.5 28.2 30.0 27.7

42 54 59 10

Table 7.5 sucrose Gelling ion Calcium Sodium Potassium Acid

Properties of 0.5% LA gellan gum gels in optimum conditions for gel formation in 60% Ion concentration (mM) 0.5–1.0 25–35 8–10 pH2.5–2.7

Modulus (Ncmÿ2)

Brittleness (%)

Setting temperature (ºC)

1.66 3.13 1.71 7.74

61.5 54.1 65.6 43.9

38 47 43 64

below 100ºC. The exceptions are gels formulated with a low level of monovalent ions, particularly potassium, and milk gels. Setting time is governed by the rate at which heat is removed which, in turn, depends on the dimensions of the system being cooled. Thin films on a cold surface, for example, set almost instantaneously. Once set, gel strength does not change markedly over time. LA gellan gum is capable of forming selfsupporting gels at concentrations as low as 0.05% gum. Low acyl gellan gum gels do not synerise unless cut or broken. High acyl gellan gum As with LA gellan gum the easiest way to form HA gellan gum gels is to cool hot solutions. Addition of cations is not necessary for the formation of HA gellan gum gels and their properties are much less dependent on the concentration of ions in solution. Gels typically set and melt between 70–80ºC and show no thermal hysteresis, i.e., they melt at the same temperature at which they set. HA gellan gum is capable of forming selfsupporting gels at concentrations above approximately 0.2% gum. High acyl gellan gum gels do not synerise.

7.4.4 Texture of gellan gum Texture is generally regarded as a multifarious property.13 Texture profile analysis (TPA) is a technique based on compression of free-standing gels twice in succession and is capable of providing both fundamental and empirical data on the mechanical properties of gels. It has the advantage of providing data at both low and high strains allowing gels to be characterised by multiple parameters. These include • modulus: a measure of gel firmness when lightly squeezed • hardness: a measure of the force required to rupture the gel

Gellan gum

Fig. 7.2

123

Schematic comparison of the gel texture of high and low acyl gellan gum with other common gelling agents.

• brittleness: a measure of how far the gel can be squeezed before it ruptures. It is important to note that the higher the brittleness value the less brittle the gel is, i.e., it has to be compressed further to break • elasticity: a measure of how far the gel springs back after the first compression cycle • cohesiveness: indicates the degree of difficulty in breaking down the gel in the mouth. Texture profile analysis has been used to characterise a diversity of foods and hydrocolloid gels. A recent review by Pons and Fiszman provides a more detailed account of the technique.14 High and low acyl gellan gum gels have very different textures that can be considered to be at opposite ends of the textural spectrum of hydrocolloid gels and Fig. 7.2 shows schematically how gellan gum gels compare to other common gelling systems. LA gellan gum forms hard, non-elastic, brittle gels whereas HA gellan gum gels are soft, elastic and non-brittle. A comparison of the texture of HA and LA gellan gum gels, made using texture profile analysis, is shown in Fig. 7.3. It is immediately apparent that through blending of the two forms, a diverse range of textures can be achieved that encompass many of the textures produced by other hydrocolloids (Fig. 7.4). It has been demonstrated by differential scanning calorimetry (DSC), and rheological measurements that mixtures of the high and low acyl forms exhibit two separate conformational transitions at temperatures coincident with the individual components.8,12 This is important to note since in mixtures consideration for the high setting temperature of the HA gellan gum will need to be made. No evidence for the formation of double helices involving both high and low acyl molecules has been found. Properties of the blended system can be varied through control of the blend ratio and level of ions in the mixture. At low ionic concentrations the high acyl form predominates but as the ionic concentration increases the contribution of the low acyl form to the texture increases.

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Fig. 7.3

Texture profile of high acyl (solid line) and low acyl (dotted line) gellan gum gels measured on 1% gels at 70% strain.

Fig. 7.4 Effect of high and low acyl gellan gum blend ratio on the modulus and brittleness of gels prepared at 0.5% total gum concentration.

7.5

Uses and applications

Before discussing the main applications of gellan gum, an overview of the key properties of both the low and high acyl forms is given in Table 7.6. This provides a useful frame of reference from which existing applications can be understood and new opportunities visualised.

Gellan gum Table 7.6

125

Comparison of the key properties of low acyl and high acyl gellan gum

Hydration Sequestrants Viscosity Gelling ions Setting temperature Melting Clarity Texture

Low acyl gellan gum

High acyl gellan gum

> 80ºC Yes Low Yes (mono or divalent or acid) 10–60ºC No (except low ionic strength and in milk) Clear Firm, brittle

> 70ºC No High Not required 70–80ºC Yes Opaque Soft, elastic

7.5.1 Dessert jellies Water-based dessert jellies are popular throughout the world and have a range of textures. The firm, brittle texture of LA gellan gum for example, complements the flavour of fruit juice jellies. Alternatively, combinations of HA and LA gellan gum can be used to produce jellies with a variety of textures. Products can be ‘ready-to-eat’ (RTE) or in dry mix form. Example formulations are provided below. Formulation 7.1 is an example of a fruit juice jelly prepared with LA gellan gum. It can be made with apple, orange, grape, pineapple or grapefruit juice and will hydrate as a dry mix in a water hardness of up to 600ppm (as CaCO3). The pH and solids vary according to the juice used, but are typically pH3.5–3.7, and 17% total soluble solids. Alternatively, blends of LA and HA gellan gum can be used to give a range of textures. Blend ratio and gum concentration will depend on the final texture required but a 3:1 HA:LA gellan gum blend at approximately 0.3% is recommended as a starting point for textural evaluation. LA gellan gum can also be used to modify the properties of traditional gelatin dessert jellies and an example is given in Formulation 7.2. The LA gellan gum in this formulation raises the initial set temperature of the dessert to around 35ºC, allowing more rapid processing of an RTE product. The formulation can also be used in dry-mix composite desserts allowing further layers to be added more quickly than with gelatin alone. The time to consumption is, however, not reduced as the maturation time of the Formulation 7.1

Recipe for fruit juice jelly using LA gellan gum.

Ingredients Water Fruit juice Sugar Citric acid anhydrous Tri sodium citrate dihydrate LA gellan gum

Weight (g)

(%)

250.0 250.0 90.0 2.4 1.8 0.9

42.00 42.00 15.15 0.40 0.30 0.15

Preparation 1. Pre-blend all the dry ingredients. 2. Heat the water to boiling and dissolve the dry ingredients in the hot water. 3. Add the fruit juice, mix and chill. The gel sets at approximately 40–45ºC and the use of chilled fruit juice with dry-mix desserts ensures a rapid set.

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Formulation 7.2

Recipe for a dessert jelly using LA gellan gum and gelatin.

Ingredients

Weight (g)

(%)

Water Sugar Gelatin (type B, 240 Bloom) Citric acid anhydrous Tri sodium citrate dihydrate LA gellan gum Colour and flavour

500.0 90.0 10.2 2.3 1.6 0.35 as required

82.6 15.0 1.7 0.38 0.26 0.06

Preparation 1. Blend all the dry ingredients. 2. Heat the water to boiling and dissolve blend into the hot water by stirring for 1–2 minutes. 3. Deposit and chill.

gelatin gel remains unchanged. The gellan gum also raises the melting point of the gel so that desserts maintain their shape for longer, when removed from the fridge. This approach can also be used in savory gelled products such as aspics. Gellan gum is an anionic polysaccharide (ÿve charge) whereas gelatin is a protein and as such its overall charge will be dependent on the pH of the system. Below its isoelectric point the gelatin will carry an overall positive charge and will therefore interact with the negatively charged polysaccharide. This can lead to cloudiness in the gel or even precipitation. For this reason it is recommended to use type B gelatins since these have the lowest isoelectic point (pH4.5–5.5). The extent of the interaction will depend on the pH and the ratio of gelatin to gellan gum.

7.5.2 Suspending agent Gellan gum is commonly used as a gelling agent; however, it can be used to prepare structured liquids which are extremely efficient suspending agents. These structured liquids are gelling systems which have been subjected to shear either during or after the gelation process. The application of shear disrupts normal gelation and results, under certain conditions, in smooth, homogeneous, pourable systems often referred to as ‘fluid gels’.15 To produce smooth homogeneous fluid gels with gellan gum, systems must be formulated to give weak gelation, either by manipulating the ion type and concentration or gellan gum concentration. The viscosity and structure of the system correlates with the gel strength of the unsheared gel. Therefore, the greater the gel strength of the unsheared gel, the greater the viscosity and structure will be when the system is sheared. Systems which gel too strongly, however, can give rise to a grainy appearance in the final fluid. Gellan gum fluid gels can be prepared using a variety of processes. Three potential processes are outlined schematically in Fig. 7.5. The first step in each case is to hydrate the gellan gum through a combination of heat and sequestrants. Method 1 simply involves continually stirring the solution as it cools to form the fluid gel or allowing the weak gel to form undisturbed, then shearing to form the fluid gel. Alternatively, in method 2 the hot gellan gum solution can be added to cold water whilst mixing. This results in cooling of the solution and formation of a fluid gel. In method 3 it is possible to prepare gellan gum solutions that will not gel on cooling. Addition of ions to these cold solutions results in gelation and formation of a fluid gel. The application of shear can be achieved by using

Gellan gum

Fig. 7.5

127

Outline of potential processes for the preparation of gellan gum fluid gels.

stirring, homogenisation, filling or even ‘shake before use’. Shear can also be applied during or after gelation. UHT, HTST and processes involving scraped surface heat exchangers (i.e. for the production of custards, gravies and ketchup) are an ideal way to shear during gelation as the solution cools. Generally, HA gellan gum should be used at approximately 0.02–0.05% to give a smooth fluid gel. For LA gellan gum the use level is dependent on the ionic concentration in the system and a guide to the formulation of fluid gels using LA gellan gum is given in Table 7.7. Example formulations for beverages using LA or HA gellan gum which can be used to suspend gelled beads or fruit pulp are given below (ingredients at end of chapter). Formulation 7.3 provides a starting point for a beverage type fluid gel. It has a pH of 2.9 and a setting temperature of approximately 15ºC. It can be used to suspend jelly beads and is prepared as outlined in method 1 of Fig. 7.5 by shearing after the weak gel has been allowed to form. Formulation 7.4 is an example of a fluid gel formed with HA gellan gum. HA gellan gum fluid gels are less sensitive to the ionic conditions and have longer, more elastic flow properties when compared to LA gellan gum fluid gels. Table 7.7

Guidelines for formulation of low acyl gellan gum fluid gels

Ion

Concentration

Calcuim

Low (< 50ppm) Optimum (100–600ppm) High (> 600ppm) Low (< 0.25%) Optimum (0.5–2.0%) High (4.0–10%)

Sodium Milk Sugars

(40–60%)

LA gellan gum concentration (%) 0.05–0.2 0.03–0.05 0.05–0.2 0.05–0.2 0.03–0.05 0.05–0.2 0.05–0.2 0.1–0.3

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Formulation 7.3

Recipe for a fluid gel for beverages using LA gellan gum.

Ingredients Part 1 Sucrose Tri sodium citrate dihydrate LA gellan gum Sodium benzoate Deionised water Part 2 Citric acid Calcium lactate Deionised water

Weight (g)

(%)

112.0 0.60 0.28 0.20 862.0

11.25 0.06 0.028 0.02 86.60

5.00 0.25 15.00

0.50 0.025 1.517

Preparation 1. Blend the sucrose, tri sodium citrate dihydrate, LA gellan gum and sodium benzoate and disperse in the deionised water of Part 1. 2. Heat the dispersion to 70–80ºC to hydrate. 3. Dissolve the citric acid and calcium lactate in the deionised water of Part 2 and add to the hot gum solution. 4. Cool the sample to below 15ºC undisturbed. 5. Gently agitate the sample to form a fluid gel. Formulation 7.4

Recipe for a pulp suspension beverage using HA gellan gum.

Ingredients Water Fruit juice Sugar HA gellan gum Tri sodium citrate dihydrate Citric acid anhydrous Potassium citrate

Weight (g)

(%)

338.10 100.0 60.0 0.25 0.25 0.9 0.5

67.62 20.0 12.0 0.05 0.05 0.18 0.1

Preparation 1. Blend the HA gellan gum with the tri sodium citrate dihydrate and disperse in the water. 2. Heat the dispersion to 90ºC to hydrate the gum. 3. At 90ºC add the remaining dry ingredients and the fruit juice. 4. Cool to room temperature whilst mixing to form the fluid gel.

7.5.3 Dairy Unlike water systems, much of the calcium in milk is associated with the milk proteins. During heating any remaining free calcium is also bound by the proteins and therefore does not interfere with the hydration of the gellan gum. Because of this both HA and LA gellan gum will hydrate in milk above approximately 80ºC without the need for a sequestrant. Milk also contains sodium and potassium ions and it is therefore not usually necessary to add additional gelling ions to milk systems. Because the LA gellan gum is gelled with a low level of monovalent ions (predominantly K+), milk gels are thermally reversible melting at approximately 95ºC. Thermal stability of LA gellan gum milk gels can be improved by the addition of calcium. Care must be taken when adding calcium to hot milk since it can result in precipitation of the milk proteins if added above 70ºC. It is

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129

recommended to cool the gellan/milk mixture to between 55–65ºC before adding the calcium. This temperature range is above the gelation temperature of the LA gellan gum but below the temperature at which milk protein precipitation occurs. In many dairy systems milk powders are used. These powders are natural sequestrants and will bind calcium from the water used for reconstitution. Therefore, it is not usually necessary to add a sequestrant when water of hardness up to 400ppm (as CaCO3) is used for reconstitution. Some sequestrant may be required if less than 2% milk powder is being used or harder water is used to reconstitute the milk powder. Yoghurt The standard yoghurt process can be followed when using LA gellan gum for both set and stirred yoghurt. There are various ways in which yoghurt containing LA gellan gum can be made depending on the usual manufacturing process and on the desired properties of the final yoghurt. In all cases the initial steps are the same: The LA gellan gum should be blended with skimmed milk powder and other stabilisers (if required) and dispersed in cold milk before heating, homogenising and pasteurising. Generally, the fermentation time is not affected by the presence of LA gellan gum. The important factor to remember is that the setting temperature of LA gellan gum in skimmed milk is about 41ºC. If shear is applied through the setting temperature a fluid gel will be formed. This acts like a gel under static conditions, but flows like a liquid when shear is applied. If however, shear is applied below the setting temperature in yoghurt systems, a broken gel may result which can lead to unsatisfactory lumps in the final product. Typical use levels for LA gellan gum in yoghurt are 0.04%. It can be used in combination with other stabilisers such as starch depending on the final texture required.

7.5.4 Sugar confectionery One of the fundamental techniques for the manipulation of the texture of sugar confectionery is to use combinations of a variety of sugars such as sucrose, glucose, fructose and various corn syrups. Combinations of sugars produce desirable textures, as well as prevent crystallisation of individual sugars. Another critical ingredient is the hydrocolloid. These impart structure to the product and provide the characteristic jelly texture. Before describing how to make confectionery jellies with gellan gum, it is worth discussing the influence of sugars on the properties of gellan gum. Effect of sugars on low acyl gellan gum The presence of sugars has two major effects on the properties of LA gellan gum gels. Firstly, the ion requirements for optimum gel properties are reduced, and secondly, above approximately 40% sugar, gels become less firm and less brittle, i.e. softer and more elastic (Fig. 7.6). These effects are believed to be the result of the sugars inhibiting the aggregation step of the gelation process.16,17 These effects are also influenced by the type of sugar. Sucrose has a greater inhibitory effect than glucose, fructose or corn syrups.18 The effect of a number of sugars commonly used in confectionery manufacture on the texture and calcium required is given in Table 7.8. This shows that the presence of 40% w/w sugar approximately halves the calcium required for maximum gel modulus, from 8– 10mM in water gels to 4–5mM in the sugar gels. Addition of 60% w/w sugar results in an approximately tenfold reduction in the requirement for calcium, with only 0.5–1.0mM added calcium required for maximum gel modulus. Similar reduction in the requirements for sodium and potassium are also seen (Fig. 7.1). Texturally, increasing the sugar

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Fig. 7.6

Effect of sucrose concentration on the modulus (❍), hardness (❑) and brittleness (■) of 0.5% low acyl gellan gum gels.

Table 7.8 Effect of sugars at 40% w/w and 60% w/w on the textural properties of 0.5% w/w low acyl gellan gum gels prepared at ion concentrations giving maximum gel modulus Sugar

Fructose Glucose Sucrose Maltose 42DE corn syrup 14DE maltodextrin

Modulus (Ncmÿ2)

Brittleness (%)

40% w/w

60% w/w

40% w/w

60% w/w

14.1 12.9 13.5 15.5 19.0 16.1

3.70 2.17 1.60 3.83 5.06 5.88

31.3 36.7 30.2 30.7 27.9 24.1

53.6 62.9 63.3 51.4 53.0 43.1

content from 40–60% results in softer more elastic gels as indicated by the reduction in modulus and increase in brittleness values (Table 7.8). The differences observed between sugars mean that texture can, to some degree, be varied by manipulating the sugar composition of the system. For example, partial replacement of sucrose with fructose or corn syrup, a common practice to control crystallisation in confectionery manufacture results in firmer more brittle gels.19 Effect of sugars on high acyl gellan gum Less is known about the specific effects of sugars on HA gellan gum. However, addition of sugars to high acyl gellan gum gels generally results in an increase in the force required to break the gel. Setting and melting temperatures also increase with increasing sugar concentration. In the presence of high levels of sugar (70–80%), HA gellan gum

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131

has very high viscosity even when hot. This can make processes such as mixing and depositing difficult. This is often compounded by the high setting temperature which can result in pre-gelation, i.e., gel formation prior to deposition of the confectionery mix. However, incorporation of low levels of HA gellan gum into confections made with LA gellan gum will increase the chewiness of the jellies. Preparation of confectionery jellies LA gellan gum may be used alone or in combination with other gelling agents to produce jelly confectionery by traditional processes. Examples are provided in Formulations 7.5 and 7.6. When prepared with LA gellan gum as the sole gelling agent (Formulation 7.5), the jellies are firm with a short, clean bite and flavour. The jellies can be removed from the starch moulds after about two hours but are usually stoved for up to 72h before demoulding. Addition of a thin boiling starch as outlined in Formulation 7.6 results in a chewier texture. Pre-gelation is the premature gelation of the confectionery mix prior to, or during depositing. This makes depositing difficult and results in a weaker gel structure and grainy texture. Table 7.9 provides a guide to preventing pre-gelation in gellan gum confections.

7.5.5 Fruit preparations This application covers a wide variety of systems from 30–75% total soluble solids. Much of the understanding of the effects of sugars described in confectionery applications can be applied to these systems. In addition, the type of fruit used in the formulation is a key consideration when using LA gellan gum since the ion content and pH will vary. Fruit composition may also vary during the season. Table 7.10 shows that the ionic composition varies considerably between different fruits with most fruits containing significant levels of potassium ions. These ionic concentrations become Formulation 7.5

Recipe for jelly sweets using LA gellan gum.

Ingredients

Weight (g)

(%)

Sucrose Glucose syrup (42DE) Water Citric acid anhydrous Tri sodium citrate dihydrate LA gellan gum Calcium hydrogen orthophosphate Flavour and colour

159.0 159.0 120.0 5.00 5.00 3.75 0.20 as required

35.20 35.20 26.51 1.11 1.11 0.83 0.04

Preparation 1. Blend the LA gellan gum and calcium hydrogen orthophosphate with 1.0g of tri sodium citrate dihydrate and 40g of sucrose and disperse in the water. 2. Heat to boiling to hydrate the gellan gum then add the remainder of the sugar while continuing to boil. 3. Add pre-warmed glucose syrup while maintaining the temperature above 90ºC. 4. Cook the liquor to 80–82% total solids then cool to 90ºC. 5. Dissolve the citric acid and remainder of the tri sodium citrate dihydrate, colour and flavour in 20cm3 of water and stir into the liquor. 6. Deposit at 76–78% total solids into starch moulds. 7. Stove to final solids as required.

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Formulation 7.6

Recipe for jelly sweets using LA gellan gum and thin boiling starch.

Ingredients

Weight (g)

(%)

Water Glucose syrup (42DE) Sugar Thin boiling starch (FLOGEL 60) LA gellan gum Tri sodium citrate dihydrate Citric acid anhydrous Calcium hydrogen orthophosphate Flavour and colour

220.0 159.0 148.5 18.8 3.5 1.8 1.8 0.2 as required

39.0 28.2 28.1 3.4 0.62 0.32 0.32 0.04

Preparation 1. Slurry the starch in 50g of water. 2. Blend the LA gellan gum, calcium hydrogen orthophosphate and tri sodium citrate dihydrate with 40g of sugar and disperse in the remainder of the water. 3. Heat the dispersion to boiling to hydrate the gellan gum then add the remaining sugar and continue to boil. 4. Add pre-warmed glucose syrup and cook to boiling. 5. Add the starch slurry, breaking the boiling point and continue to cook to 78% total solids. 6. Add colour, flavour and citric acid pre-dissolved in a small amount of water. 7. Deposit into starch moulds at 74% total solids and stove to final solids as required.

Table 7.9

A guide to the prevention of pre-gelation in confectionery mixes

Problem

Possible causes

Solution

Pre-gelation when acid added

Hard water

Add sodium hexameta phosphate

Depositing soluble solids too high

Lower depositing solids

pH too low

Add sodium citrate with citric acid

Hard water

Increase sequestrant level

Soluble solids too high

Boil to lower soluble solids

Pre-gelation before acid added

Table 7.10 Fruit Apple Blackcurrant Raspberry Strawberry Apricot Peach

Ionic composition of raw fruits20 Ca++ (mg/100g)

Mg++ (mg/100g)

Na+ (mg/100g)

K+ (mg/100g)

4 60 25 16 15 7

3 17 19 10 11 9

2 3 3 6 2 1

88 370 170 160 270 160

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133

increasingly significant in medium to high solids systems where gellan gum ion requirements are greatly reduced. Therefore, formulations optimised for one fruit will often need modification to accommodate different fruits. Formulation 7.7 is an example of a low solids (36%) jam which can be formulated to achieve a range of textures. HA gellan gum provides a soft, spreadable jam with excellent sheen. The addition of a proportion of LA gellan gum may be used where a firmer textured jam is required. LA gellan gum alone can be used to give a more bake stable jam. Various fruits can be used including strawberries, raspberries and blackcurrants. Formulation 7.8 is slightly higher in solids than Formulation 7.7 and produces a lightly gelled yogfruit with evenly suspended fruit pieces. The gelled structure can be broken down by pumping to give a smooth, viscous yogfruit (pH3.9, soluble solids 40%). Finally, Formulation 7.9 with 55% fruit and no added water demonstrates the properties of a gellan gum and starch-based preparation. The LA gellan gum filling has a glossy appearance, good flavour release and excellent bake stability (pH 3.4, tss 56%). Formulation 7.7

Recipe for a reduced sugar jam using HA or LA gellan gum blend.

Ingredients Frozen strawberries Sugar Water Gellan gum* Tri sodium citrate dihydrate Potassium sorbate Citric acid solution (50% w/w)

Weight (g)

(%)

450.0 283.5 260.0 2.5 0.5 1.0 2.5

45.0 28.35 26.0 0.25 0.05 0.10 0.25

* High acyl and/or low acyl gellan gum can be used depending on desired final texture.

Preparation 1. Dry blend the gellan gum, tri sodium citrate dihydrate and potassium sorbate with the sugar and disperse into the water. 2. Add the fruit and heat to boiling. Cook for 1–2 minutes to ensure hydration of the gellan gum. Check the soluble solids. 3. Remove from the heat and add the citric acid solution. Fill into jars and cap immediately. Formulation 7.8

Recipe for a peach yogfruit using LA gellan gum.

Ingredients Peach pure´e Diced peach Glucose syrup (42DE) LA gellan gum Tri sodium citrate dihydrate Sodium benzoate

Weight (g)

(%)

200.0 200.0 300.0 0.35 1.80 0.25

28.50 28.50 42.65 0.05 0.26 0.04

Preparation 1. Combine the fruit and glucose syrup. 2. Add the tri sodium citrate dihydrate, LA gellan gum and sodium benzoate and heat to 90ºC with constant stirring. 3. Hold for 1 minute then cool, with stirring, to 60ºC. 4. Deposit and allow to cool undisturbed. Note: The tri sodium citrate dihydrate in the formulation is added to give a final pH of 3.9. The addition level may be varied depending on the fruit used, and the final pH required.

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Formulation 7.9

Recipe for a bake-stable fruit preparation using LA gellan gum.

Ingredients Apple (thawed) Sucrose Modified starch (THERMFLO) LA gellan gum Citric acid solution (50% w/w) Tri sodium citrate dihydrate

Weight (g)

(%)

210.0 160.8 8.00 0.32 0.80 0.88

55.2 42.2 2.10 0.12 0.20 0.18

Preparation 1. Pre-blend the dry ingredients, add to the apple and heat with stirring to boiling. 2. Remove from heat, add the citric acid solution, mix well and deposit. 3. Leave to gel before use. Shear, and use as required.

7.5.6 Other applications Gellan gum forms films and coatings that can be used in breadings and batters. Films offer several advantages, particularly their ability to reduce oil absorption by providing an effective barrier. Films can be prepared by applying a hot solution of gellan gum on to the surface of the food product, by spraying or dipping, and allowing to cool. Alternatively, in the case of LA gellan gum, the food can be dipped into a cold solution of the gum, allowing ions to diffuse into the solution, resulting in gelation or film formation. LA gellan gum can also be used to produce fat-free adhesion systems. Spraying of a cold solution of LA gellan gum onto the surface of products such as nuts, crisps and pretzels forms an instant thin layer of gel when it reacts with the salt thus facilitating adhesion of spice, flavour or sweetener blends.

7.6

Regulatory status

In Japan, gellan gum has been considered a ‘natural’ food additive since 1988. It is now approved for food use in the USA and the European Union as well as Canada, South Africa, Australia, most of South East Asia and Latin America. Gellan gum appears as E418 in the European Community Directive EC/95/2 in Annex 1. Both the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) and the European Community Scientific Committee for Food have given gellan gum a non-specified Acceptable Daily Intake (ADI). Combinations of high acyl and low acyl gellan gum have one name. A manufacturer may label a product made with a combination of both types of gellan gum simply E418 or gellan gum.

7.7 1. 2. 3. 4.

References and MORRIS, V. J. (1983) ‘Structure of the acidic extracellular gelling polysaccharide produced by Pseudomonas elodea’ Carbohydr. Res., 124, 123–33. JANSON, P.-E., LINDBURG, B. and SANDFORD, P. A. (1983) ‘Structural studies of gellan gum, an extracellular polysaccharide elaborated by Pseudomonas elodea’ Carbohydr. Res., 124, 135–9. KUO, M.-S., MORT, A. J. and DELL, A. (1986) ‘Identification and location of L-glycerate, an unusual acyl substituent in gellan gum’ Carbohydr. Res., 156, 173–87. CHANDRASEKARAN, R., MILLANE, R. P., ARNOTT, S. and ATKINS, E. D. T. (1988) ‘The crystal structure of gellan’ Carbohydr. Res., 175, 1–15. O’NEILL, M. A., SELVENDRAN, R. R.

Gellan gum 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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and ARNOTT, S. (1988) ‘Cation interactions in gellan: an X-ray study of the potassium salt’ Carbohydr. Res., 181, 23–40. CHANDRASEKARAN, R. and THIALAMBAL, V. G. (1990) ‘The influence of calcium ions, acetate and Lglycerate groups on the gellan double helix’ Carbohydr. Polym., 12, 431–42. CHANDRASEKARAN, R., LEE, E. J., RADHA, A. and THAILAMBALl, V. G. (1992) ‘Correlation of molecular architectures with physical properties of gellan related polymers’ in Frontiers in Carbohydrate Research2, ed. Chandrasekaran, R., Elsevier Applied Science, New York, pp. 65–84. MORRIS, E. R., GOTHARD, M. G. E., HEMBER, M. W. N., MANNING, C. D. and ROBINSON, G. (1996) ‘Conformational and rheological transitions of welan, rhamsan and acylated gellan’ Carbohydr. Polym., 30, 165–75. MORRIS, E. R., REES, D. A. and ROBINSON, G. (1980) J. Mol. Biol., 138, 349. GRAZDALEN, H. and SMIDSRØD, O. (1987) ‘Gelation of gellan gum’ Carbohydr. Polym., 7, 371–93. SANDERSON, G. R. and CLARK, R. C. (1984) ‘Gellan gum, a new gelling polysaccharide’ in Gums and Stabilisers for the Food Industry 2, eds G. O. Phillips, D. J. Wedlock and P. A. Williams. Pergamon Press, Oxford, pp. 201–10. KASAPIS, K., GIANNOULI, P., HEMBER, M. W. N., EVAGELIOU, V., POULARD, C., TORT-BOURGEOIS, B. and SWORN, G. (1999) ‘Structural aspects and phase behaviour in deacylated and high acyl gellan systems’ Carbohydr. Polym., 38, 145–54. BOURNE, M. C. (1978) ‘Texture profile analysis’ Food Technology 32, 67–72. PONS, M. and FISZMAN, S. M. (1996) ‘Instrumental texture profile analysis with particular reference to gelled systems’ Journal of Texture Studies, 27, 597–624. SWORN, G., SANDERSON, G. R. and GIBSON, W. (1995) ‘Gellan gum fluid gels’ Food Hydrocolloids, 9, 265– 71. SWORN, G. (1996) ‘Gelation of gellan gum in confectionery systems’ in Gums and Stabilisers for the Food Industry 8, eds G. O. Phillips, P. A. Williams and D. J. Wedlock, IRL Press, Oxford, pp. 341–9. SWORN, G. and KASAPIS, S. (1998) ‘The use of Arrhenius and WLF kinetics to rationalise the mechanical spectrum in high sugar gellan systems’ Carbohydrate Research 309, 353–61. SWORN, G. and KASAPIS, S. (1998) ‘Effect of conformation and molecular weight of co-solute on the mechanical properties of gellan gum gels’ Food Hydrocolloids, 12, 283–90. GIBSON, W. (1992) ‘Gellan gum’ in Thickening and gelling agents for food ed. A. Imeson, Blackie Academic & Professional, Glasgow, pp. 227–49. MCCANCE and WIDDOWSON’S The Composition of Foods Fifth Edition, RSC and MAFF, 1991. CHANDRASEKARAN, R., PUIGJANER, L. C., JOYCE, K. L.

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8 Galactomannans W. C. Wielinga, Maehall AG (Rhodia Food, Kreuzlingen)

8.1

Introduction

Galactomannans as reserve carbohydrates are found as cell wall storage polysaccharides of various albuminous or endospermic seeds. The endosperm of these seeds develops alongside the embryo or germ and completely envelopes it. The endosperm itself is protected by a seed coat. The seed endosperm contains very little cellulose and no lignin. Widely used are the seed galactomannans from the carob tree (Ceratonia siliqua), from the guar plant (Cyamopsis tetragonoloba) and to a lesser extent from the tara shrub (Cesalpinia spinosa). The galactomannans from these three sources are composed entirely of linear (1!4) -D-mannan chains with varying amounts of single D-galactose substituents linked to the main backbone by (1!6)--glycosidic bonds. These polydisperse galactomannans can be easily distinguished from each other by their overall mannose-galactose ratios between 1.6:1 and ca. 3.5:1. This large amount of galactose side stubs of about 20–40% wt. prevents strong cohesion of the main backbone, so that no extensive crystalline regions can be formed. Water of room or elevated temperatures can thus easily penetrate between the single molecules to hydrate or dissolve the accessible gum. Since certain galactomannans of carob bean gum self-associate under defined conditions, it is, however, possible that nano crystalline regions of 3–5nm are formed, which alternate with much bigger amorphous regions. These minute crystalline parts can be dissociated by hot water. During germination of the seeds the endosperm absorbs up to 75% wt. of water, based on its own dry weight to allow the diffusion of enzymes required for the germination process and to enable the transport of metabolic low molecular weight endproducts needed for the growth of the plant. The endosperm of Ceratonia siliqua seeds or kernels consists of living cells, which can synthesise the enzymes to hydrolyse the galactomannans (-galactosidase [EC.3.2.1.22], -mannanase [EC 3.2.1.25] and mannosidase). The endosperm of the Cyamopsis tetragonoloba seeds has – on the convex periphery – aleurone cells, the only living cell layers of the endosperm, which can also synthesise enzymes similar to those mentioned above for the carob endosperm (see Fig.

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Fig. 8.1

Scanned electron micrograph of a fractured guar split showing different cell structures between peripheral and inner endosperm cells.

8.1).1 The endosperm of the Cesalpinia spinosa seeds is morphologically and physiologically quite similar to that of the seeds of the carob tree. The hard and compact endosperm halves of the three mentioned seeds contain more than 88% wt. of galactomannans on a dry basis. Endosperm halves are obtained by either removing mechanically, physically or chemically to a very large extent the hull and also the more friable embryos of the seeds by submitting the peeled seeds to splitting, grinding and sifting operations. The recovered endosperm halves of the carob, guar and tara seeds are then processed into a fine off-white powder. Commercial products do not always consist of pure endosperm, but may contain residual hull and germ parts. The products are specified mainly according to viscosity,

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139

Table 8.1 Annual consumption of hydrocolloids per capita in grams per year for 1990 (for US citizens and for those in Germany, France, UK and Italy (D, F , GB , I)) Hydrocolloids

US

D, F, GB, I

Starch products Gelatin Agar Agar Alginates Carrageenans Guar gum Arabic gum Carob bean gum CMC Other cellulose derivatives Xanthan gum Pectin

1300 65 2 10 11 34 22 8 23 6 13 11

1281 122 3 10 18 39 59 18 6 1 3 19

Total

1505

1576

protein content, acid insoluble residue (an indication of the residual hull content) and particle size distributions. Seed varieties, weather conditions during their growth and harvesting, geographical influences, different morphological structures of the endosperm and processing conditions make an exact definition of these products difficult. Table 8.1 shows the annual (1990) consumption in grams/capita of different food additives including carob bean gum and guar gum in various countries. The functionality of the galactomannans is mainly due to their ability to change the rheology of aqueous systems. All three types are very efficient thickening agents, if dissolved in water, and are able to interact with agar-agar, Danish agar, carrageenans and xanthan gum to form or to fortify three-dimensional stabilising structures. The thickening power of the galactomannans depends, of course, upon the size or the length and association of the macromolecules and thus on their apparent molecular weights. These thickening and gelling agents are widely used in food products, mainly to make them appealing and attractive to the consumer and to improve their shelf life by binding water, to control the texture, to influence crystallisation, to prevent creaming or settling, to improve the freeze-thaw behaviour, to prevent syneresis, to prevent the retrogradation of starch products, to maintain turbidity in soft drinks and juices, and as dietary fibres. This means that these food additives find their applications in convenience food, dairy products including frozen products (ice cream), soft drinks and fruit juices, bread and pastry, fruit preserves, baby food and as household gelling agents in puddings, flans and pudding powder, as dietary fibres, and in pet foods. They also are used in pharmaceutical and cosmetic products. Other non-food applications of galactomannans are in the textile industry (carpet dyeing and textile printing), in the paper, mining, explosive, drilling, construction, oil field and chemical industries. Synthetic and natural high molecular weight substances consist of blends of macromolecules with different chain lengths. The distribution of molecular weights of these substances therefore is very important. Figure 8.2 shows these values for carob bean gum, guar gum, xanthan gum and carrageenan. It is not uncommon that polymeric materials contain macromolecules ranging in molecular weights from a few hundred to several million.2

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Fig. 8.2

SEC comparison of molecular weight distributions of hydrocolloids.

Kubal J. V. et al. (1948) published the average molecular weight of carob bean gum to be 310,000 daltons, which was determined at 20ºC with ultracentrifugation.3 However, the investigated non-industrial sample of their carob bean gum in 1948, thus prepared on bench scale showed an intrinsic viscosity [] of 5.0dl/g. Present industrial carob bean gum products (E410) on the market show intrinsic viscosities of about 12.0dl/g, thus significantly higher than the one used by Kubal. Hui. P. determined for a certain quality of guar gum according to the same ultracentrifugation method at 25ºC its average molecular weight of 1,900,000 daltons.4 If Hui had used a more realistic value for the partial specific volume this average molecular weight would have increased to 2,200,000 daltons. Present industrial guar gum products (E412) on the market show intrinsic viscosities of 0.7–15.0dl/g, which correspond to apparent viscosities of aqueous solutions at 2% wt. concentration of about 5–100,000mPa/s, if measured with a Brookfield RVT viscometer, 20 rpm at 25ºC. According to recent EC specifications (1998) as well as to FAO/WHO specifications, the molecular weight of carob bean gum (E410) used as a food additive has to be within a range of approx. 50,000–3,000,000 daltons. For guar gum (E412) these specifications call for molecular weights between 50,000–8,000,000 daltons.

8.2

Manufacture

8.2.1 Carob bean gum The evergreen carob tree can be planted in semi-arid or subtropical zones and grows in calcareous soils. It is important for the vegetation of the entire Mediterranean area, and especially for Morocco and Portugal. It can grow as high as 10–15m and the roots of the tree can reach 25m of depth. The carob tree can live for more than 100 years. Grafted carob trees can be interplanted with olives, grapes, almonds and barley in low-intensity farming systems. They are also grown as ornamentals and for landscaping, windbreaks and afforestations.5 Normally it yields fruits after 8–10 years. The fruits, i.e. carob pods, can be harvested once a year. The pods are 10–30cm long, 1.5–3.5cm wide and 1cm thick. Their colour is dark chocolate brown. Their shape is straight or curved. The pods normally contain 8–12 seeds or kernels, exceptionally up to 15 kernels. The fruits are

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141

collected when they show a moisture content of 12–18% wt. and are shaken from the trees by long stakes. Then the fruits are dried. Dry pods are leathery. The average yield amounts to 50–70 kilos of carob pods per tree, which can be collected from orchards with minimum management, producing 2,000–3,000kg/ha. In irrigated orchards an average of 250–300 kilos per tree/12,300 kilos per ha is possible. World production of carob pods is estimated at about 300,000–350,000 tons per year, produced on some 200,000ha, yields depending on cultivar, region and farming practice. The two main constituents of the carob pod, called St. John’s bread, are: • 90% wt. of pulp, containing a high amount of sugars (48–56% wt.) • 10% wt. of seeds. The kernels are set free in a process called kibbling. The pods are broken between two rollers with specific geometry, and the freed seeds are then removed from the pods using special screens. Due to the high content of sugar the carob pods are a staple in the diet of farm animals. They are also used to produce alcohol and as cacao substitute. People ate them in times of famine and children still like them as snacks. The main interest, however, lies in the kernels. The size of the kernel is oval–oblong, 8–10mm long, 7–8mm wide and 3–5mm thick. The glossy brown testa is hard and smooth. A kernel weighs ca. 0.2g with small deviations and was used as a weight unit for gold and precious stones (carat is derived from the Latin word ceratonia). The kernels are composed of 30– 35% wt. of hull, 20–25% wt. of germ, 40–45% wt. of endosperm. The hull is without value, the germ contains about 50% wt. of protein and is used as cattle feed. It also is used as a colouring agent for certain Japanese noodles and in cookies, etc. To obtain the endosperm halves it is necessary to remove the very hard hull of the seeds in a process called ‘peeling’ and then to separate the fragile germ. There are two different peeling processes: 1.

2.

Chemical peeling process The hull is carbonised by concentrated sulfuric acid solutions at high temperature. The advantage of this technology is an even peeling effect, allowing the production of white powder. The process also facilitates the separation of the germ from the endosperm halves. Thus, high grade qualities of carob bean gum with high viscosities can be made. A disadvantage of this technique is the effluent problem. Thermal mechanical peeling process The kernels are roasted at temperatures of up to 450ºC, so that the hull pops off to a large extent. The residual hull fragments are rubbed off mechanically. As this leads to a simultaneous cracking of the endosperm halves and germ parts, a clean separation of endosperm halves and germ/hull parts becomes difficult. The advantages of this technology are that it requires only relatively simple production equipment, no special effluent treatment and that the yield is high. The disadvantages are that this material leads to gums of lower quality and lower viscosities, all showing a light-yellowish colour due to the insufficient separation of the germ particles.

Endosperm halves recovered by either technique are ground to the desired fineness. Meyhall’s range of carob bean gums (CBG), also called locust bean gums (LBG) are sold under the following trademarks: • Meypro-FleurTM (with various particle size distributions) • MeyprodynTM (cold swelling carob bean gum) • PavitinTM (the tradename for Meyhall carob germs in powder form).

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8.2.2 Guar gum Guar gum is the name of the ground endosperm halves, called guar splits, from the seeds of the guar plant Cyamopsis tetragonoloba L. This plant is an annual summer legume that grows mainly in arid and semi-arid zones, since it has deep, fibrous tap roots. It enriches the soil with nitrogen and is an ideal rotation crop with cotton and grains. Guar has been grown for centuries in the Indian subcontinent and is used as human and animal food. The word guar comes from the Sanskrit word ‘Gau-ahar’, ‘Gau’ meaning cow and ‘ahar’ meaning food. The guar plant is bush-like with a height of 90cm. It is very drought resistant. Once it has germinated, it requires very little surface water during the main growth period, i.e. for 20–25 weeks. To induce maturation of the seeds water is needed again. The monsoon season of the Indian subcontinent, especially in the northwestern part of India and northeastern part of Pakistan usually provides the right amount of rainfall at the right time for optimal growth. The growing season starts in July or August in India and Pakistan and the harvest takes place in November or December. The guar pod is 5–8cm long, almost round and ca. 1cm wide. It contains 6–9 seeds, amounting to about 60% wt. of the pod. Green pods are used as cattle fodder and as a vegetable by poor people. Ripe and dry pods cannot be eaten any more. Empty pods have no commercial value. The yield per ha can be as high as 1,800kg of pods. On the subcontinent harvesting is done manually. The seeds are recovered by mobile threshing machines. Guar seeds as such are not exported, neither from India nor from Pakistan. Contrary to the seeds of the carob tree, which are called kernels, the guar seeds are in fact called ‘seeds’. The shape of the seeds can be seen in Fig. 8.3. The colour can be light amber to yellowish green to grey olive. Black seeds are the result of the onset of decomposition due to microbiological attack, induced by rain at the wrong time. Black seeds cause problems at manufacturing with regard to specks and colour of the finished powder. The average total amount of guar seeds worldwide is estimated at about 500,000 tons p.a. However, large fluctuations of annual availability occur, mainly due to weather conditions. To become less dependent on these fluctuations and to meet the constantly increasing demand for guar products, agronomy programmes have been carried out in different parts of the world, especially in the southern hemisphere. Plantings were made in Malawi, Australia, Colombia, Brazil and Argentina. Guar grows particularly well in parts of Texas, Oklahoma and Arizona. In Texas the harvest is carried out mechanically. The key factor for the success of all agronomy programmes outside the Indian subcontinent are economic conditions. The seeds are composed of: 20–22% wt. of hull, 43–44% wt. of germ and 34–36% wt. of endosperm. Table 8.2 shows an analysis of the above-mentioned seed components regarding protein content, ash and moisture contents, acid insoluble residue (AIR), other extractable matter and the calculated gum content. The AIR is determined after having hydrolysed the products for 6–8 hours in 0.4N H2SO4 at boiling temperature. The germ contains about 50–55% wt. of protein and is used as protein-rich cattle fodder, after the trypsin inhibitor has been reduced to an acceptable level by toasting.

8.2.3 Production of guar splits and guar gum powder The whole seeds can be fed into an attrition mill or to any other type of mill having two grinding surfaces travelling at different speeds. The seed is split into the endosperm halves covered with hull and fine germ material, which later can be sifted off. The crude

Galactomannans

Fig. 8.3 Table 8.2

143

Photograph of the pods and seeds of the guar plant.

Composition of guar seed Composition of each portion

Seed portion

Hull Endosperm Germ

Weight fraction %

Protein

Ash

Moisture

AIR

%

Ether soluble %

%

%

%

Gum content %

20–22 32–36 44–46

5.0 5.0 55.3

0.3 0.6 5.2

4.0 0.6 4.6

10.0 10.0 10.0

36.0 1.5 18.0

49.0 83.5 16.7

AIR = acid insoluble residue Gum content = 100% (% protein + % moisture + % AIR)

crack (endosperm + hull) is heated to soften the hull and fed into a mill, which can either abrade the hull from the endosperm, or into a hammermill, where the hull is shattered away. Any remaining germ particles are pulverised during this step and after a further sifting the resulting splits are essentially pure endosperm. The fine material sifted off is marketed as cattle feed and called guar meal. It has a minimum protein content of 35% wt. (N% 6.25). The guar endosperm or guar splits are processed to commercial powdered products by milling and screening techniques. Figure 8.4 illustrates the production of carob bean gum and guar gum, starting with endosperm halves as raw material. It also depicts the different

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Fig. 8.4

Flow sheet for producing guar and carob bean gum from their endosperm halves.

morphological structures of a fragment of the different endosperm halves. Meyhall’s range of guar gums are sold under the following trademarks: • Meypro-GuarTM CSAA (with high viscosity and various particle size distributions) • MeyprofinÕ (with high viscosity and various particle size distributions) (lower plate count than Meypro-GuarTM CSAA) • MeyprodorÕ (products of different viscosity specifications) • MeyprogatÕ (products of high or very low viscosity).

8.3

Structure

The basic structure of galactomannans is already mentioned in the introduction. These polycondensates consist of linear chains of mannose units linked by 1!4- -D-glycosidic bonds at which the hydrogen atom of several primary hydroxyl groups on C6 are substituted by single -D-galactose units by 1!6 linkages. The galactose content of

Galactomannans

Fig. 8.5

145

Basic structural fragment of carob bean-, guar-, tara gum and cellulose.

carob bean gum is 17–26% wt.; tara gum ca. 25% wt.; guar gum 33–40% wt. The common chemical structure of an average theoretical building block of the different galactomannans is shown in Fig. 8.5. The fine structure of these galactomannans can be quite irregular with respect to the distribution of the galactose units. As many as five unsubstituted mannose units in a row can occur in certain galactomannans of guar gum and as many as 10–11 in specific galactomannans of carob bean gum. There is thus some degree of block condensation. The fine structure of the galactomannans of tara gum lies most probably in between that of carob bean gum and guar gum. A possible model for a galactomannan with a molecular weight of about 68,000 of carob bean gum with about 20% wt. of galactose is shown in Fig. 8.6. The main chain of carob, tara and guar galactomannans is structurally similar to that of cellulose. Cellulose is composed of -D-glucopyranosyl groups polymerised by 1–4 linkages. Mannose is the epimer of glucose at the 2-position. Cellulose is completely insoluble in water as a result of chain association. Substitution of the mannan chain by more than 12% wt. of galactose makes the galactomannans water soluble.6,7

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Fig. 8.6

8.4

Schematic galactomannan molecule with a molecular weight of about 68,000 with about 20% wt. of galactose with a random distribution.

Technical data

Guar gum containing galactomannans with galactose contents of 33–40% wt. is soluble in water of 25ºC, provided the gum is accessible for the solvent water. The larger part of galactomannans of carob bean gum with galactose contents of about 17–21% wt. needs a heat treatment during 10 minutes at 86–89ºC under stirring to dissolve in water. If the galactose of the galactomannans of guar gum is removed enzymatically to less than 12% wt., the final products become insoluble even in hot water. The galactomannans of carob bean gum, tara and guar gum are non-ionic. The hydroxyl groups can be derivatised and thus provide nonionic, anionic, cationic and amphoteric derivatives. The primary and secondary hydroxyl groups show practically the same reactivity. Random distribution of substituents is usually obtained. The galactose side stubs each have four OH-groups, the substituted mannose unit two secondary OH-groups and the unsubstituted mannose three OH-groups. The maximum average degree of substitution (DS), therefore, is three. Introducing more substituents to one hydroxyl group leads to a molar substitution. Galactomannans are susceptible to strong acids, organic acids like citric, acetic and ascorbic acid, alkali in presence of air and strong oxidising agents, especially at elevated temperatures, and also towards irradiation with -rays, so that a depolymerisation to different extents may occur. Both mannose and galactose contain vicinal secondary cis-hydroxyl groups, 2, 3 and 3, 4 respectively. These vicinal cis-hydroxyl groups form cyclic complexes with appropriate reagents (such as borate). Semi-dry processes allow the production of derivatives and modified products, which can be water washed, if required. A special anaerobic waste-water treatment produces methane, which is a welcome additional energy source for the factory. Figure 8.7 illustrates some of the available guar derivatives. New technologies enable the production of ionic, non-ionic and amphoteric guar products, which upon dissolution in water yield water-clear solutions, even at an actual DS of about 0.1. Purified guar products are available for food applicatons. Dissolved in water, they yield solutions of excellent clarity. More than 90% wt. of the available carob bean gum and tara gum are not chemically derivatised any more. These products are used as straight gums in food additives and in the pet-food industry. Table 8.3 summarises some characteristics of two high-, one industrial- and one technical-grade of carob bean gum. Table 8.4 shows technical data for five high-grade guar products. Table 8.5 shows

Galactomannans

Fig. 8.7 Table 8.3

147

Guar derivatives.

Typical analysis of locust bean gum (carob bean gum)

Specification

% H2O % protein % AIR 1% viscosity 10’ at 86–89ºC RVT Brookfield 20 rpm, 25ºC mPas ÿM 80 (max.) ÿM 200 (max.) Metal As, ppm Pb, ppm Cu, ppm Zn, ppm Cd, ppm

High grade M 175/M 100

Industrial

Technical

10.0–12.0 6.5 2.0 min. 3,000

10.0–14.0 5.7–7.0 2.0–4.5 1,500–1,800

10.0–14.0 6.0–13.0 5.0–8.0 500–1,000

99% 25%

99% not specified

99% 25%

99% 10%

0.2 ca. 0.03 2.5 5.6 ca. 0.05

AIR = acid insoluble residue

the viscosity data of two types of tara gum for 1% solutions prepared at 25ºC, and at 86– 89ºC for 10 minutes under stirring. Table 8.6 shows the insolubles of straight (mainly hot water soluble) and cold-swelling carob bean gum, as well as their solubility in water of 25ºC and of 86–89ºC.

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Table 8.4

Typical analysis of high grade guar gum

Specification

M 100

M 175

M 200

M 225

M 200/50

% H2O % protein % AIR % ash pH 1% solution

12.0 5.0 3.0 1.0 6.5

12.0 5.0 3.0 1.0 6.5

12.0 5.0 3.0 1.0 6.5

12.0 5.0 3.0 1.0 6.5

12.0 4.5 2.5 1.0 6.5

1% viscosity fully hydrated RVT Brookfield 20 rpm, 25ºC mPas (min.)

3,000

3,000

3,000

3,600

5,000

+ M 100 (max.) + M 150 (max.) + M 200 (max.) ÿM 150 (max.) ÿM 200 ÿM 250 (min.)

1% – – 15% – –

1% – – – max. 25% –

– 1% – – min. 80% –

– 1% 15% – – 75%

– 1% – – min. 90% –

Table 8.5

Viscosity data of 1% solutions of tara gums

Cold viscosity Hot viscosity

M 175

M 200

3,000 mPas 4,400 mPas

3,000 mPas 4,000 mPas

Table 8.6 Product

CBG M-200 Cold swelling CBG

Insolubles wt%

17.0 14.0

Solubles wt% 86–89ºC

25ºC

44.0 16.0

39.0 70.0

Figure 8.8 shows the freeze/thaw behaviour of 0.5 and 1% aqueous solutions in demineralised water, as well as in the presence of some electrolytes, acids, sucrose and in some dairy model systems of guar gum, carob bean gum and their 1:1 blends. The solutions were quickly frozen at about ÿ78ºC and brought to 25ºC again, at which point a flow curve was made, if possible. The test solutions were frozen again in the same way for a second cycle and slowly brought to 25ºC to determine their rheological properties. This figure clearly demonstrates the different behaviour of both gums. Guar is freeze/ thaw stable, whereas carob bean gum does self-associate, forming weak gels. A hypothetical 3-D structure of such gels is drawn in Fig. 8.9. Table 8.7 summarises data of Meyprogat 150 (A), 120 (B), 90 (C), 60 (D), 30 (E), and 7 (F) with respect to viscosity, insoluble matters, intrinsic viscosities and average molecular weight. Meyprogat 90 to 7 are depolymerised guar products, which have a very low plate count. The same data are shown for some high-grade guar and carob bean gums at two pH levels. The mannose content of Meyprogat 90, 60 and 30 is 65.8, 65.5 and 65.8

Fig. 8.8

Tendency of rheological behaviour of galactomannans dissolved in different aqueous media upon two freeze-thaw cycles (x-axis = 0, 1 and 2 cycles; epsilon axis = viscosity at 25ºC).

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Fig. 8.9

Schematic model of self-association between gelation of galactomannan molecules of carob bean gum.

Table 8.7 Product

% wt.

Viscosity Brookfield RVT, 20 rpm 25ºC after 1h mPa.s

Insolubles % wt.

Conc. super natant % wt.

Intrinsic viscosity [] dl/g

Mol. wt. 106

Guar gum A B C D E F

0.96 1.10 1.60 3.00 3.63 9.22

4,550 5,050 3,850 4,600 2,250 600

26 29 21 19 16 19

0.70 0.79 1.27 2.40 3.09 7.96

14.58 13.31 8.23 4.69 3.41 0.74

2.19 1.93 0.99 0.46 0.29 0.035

LBG M-175 LBG M-200

1.06 1.16

3,850 4,760

14 15

0.85 0.93

11.81 11.43

1.70 1.64

Guar gum A pH4 (10 minutes)* pH4 (30 minutes)* pH9 (30 minutes)*

0.96 0.96 0.96 0.96

4,550 4,450 4,200 4,750

26 26 23 25

0.70 0.71 0.72 0.72

14.58 13.45 13.41 13.80

2.19 1.96 1.95 2.03

LBG M-200 pH4 (10 minutes)* pH4 (30 minutes)* pH9 (30 minutes)*

1.16 1.16 1.16 1.16

4,760 4,450 4,400 4,800

15 16 15 15

0.93 0.92 0.93 0.93

11.43 11.07 11.00 11.72

1.64 1.59 1.58 1.68

Guar gum 200/50 0.96 pH4 (30 minutes)* 0.96 pH9 (30 minutes)* 0.96

5,350 5,100 5,150

24 20 21

0.72 0.75 0.72

15.16 13.75 14.61

2.31 2.02 2.19

* Heating at 86–89ºC while stirring, then cooled down to 25ºC.

Galactomannans

Fig. 8.10

151

Effect of concentration on viscosity of solutions of CBG types prepared at 86–89ºC for 10 minutes in dimineralised water.

respectively, so that no galactose was split off during depolymerisation. Meyprogat 90 is slightly and Meyprogat 30 strongly depolymerised. Figure 8.10 shows the relationship of viscosity and concentration for six types of carob bean gums. Figure 8.11 shows the relationship of viscosity and concentration for the

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Fig. 8.11 Effect of concentration on viscosity of Meyprogat types (own pH) prepared at 86–89ºC for 10 minutes in dimineralised water. Viscosity measured at 25ºC after 1 hour.

Galactomannans Table 8.8

153

Functional properties of carob bean gum

Function

Example

Use level (%)

Adhesion Binding agent Body agent Crystallisation inhibitor Clouding agent Dietary fibre Foam stabiliser Gelling agent Moulding Protective colloid Suspending agent Swelling agent Synergistic agent Thickening agent

Glaces, juices Pet foods Dietetic beverages Ice cream, frozen foods, bread Fruit drinks, beverages Cereals, bread Whipped toppings, ice cream Pudding, desserts, confectionery Gum drops, jelly candies Flavour emulsions Chocolate milk Processed meat products Soft cheeses, frozen foods Jams, pie fillings, sauces, baby food

0.2–0.5 0.2–0.5 0.2–1.0 0.1–0.5 < 0.1 0.2–0.5 0.1–0.5 0.2–1.0 0.5–2.0 0.2–0.5 < 0.1 0.2–0.5 0.2–0.5 0.2–0.5

mentioned Meyprogat types (except for Meyprogat 7). Below a pH of 3.5, a certain depolymerisation of guar gum products must be expected at elevated temperatures. It goes without saying that the viscosity of 1% aqueous solutions of a high-grade guar gum decreases upon heating. An increase of temperature from 20–80ºC will be accompanied by a drop in viscosity of about 50%, from 4100 to 2050mPas. As a rule of thumb one may say that the viscosity of aqueous solutions increases by 10 when doubling the concentration.

8.5

Uses and applications

Functional properties of CBG are given in Table 8.8. Similar tables can be set up for the other two gums, i.e., guar gum and tara gum. More application examples are given in references nos. 8 and 9. P. H. Richardson et al. are particularly informative about the behaviour of CBG and guar gum in aqueous sucrose solutions.10

8.6

Regulatory status

All three gums are approved food additives with the following E-numbers: 410 for carob bean gum, 412 for guar gum and 417 for tara gum.

8.7 1. 2. 3. 4. 5. 6.

References (1977) ‘Galaktomannanabbau in keimenden Johannisbrotsamen (Ceratonia siliqua L.)’ Planta, 134, 209–21. SLADE, P. E. JR (1975) Polymer Molecular Weights, Part 1, New York, Marcel Dekker Inc. p. 2. KUBAL, J. V. and GRALEN, N. J. (1948) J. of Colloid Science, 3, 457. HUI, P. (1962) Prom. Nr 3297, Untersuchungen an Galaktomannanen, ETH, Juris Verlag, Zu¨rich. BATTLE, I. and TOUS, J. (1997) Carob tree, Ceratonia siliqua L., International Plant Genetic Resources Institute, Via della Sette Chiese 142, 00145 Rome, Italy. ALISTAIR, STEPHEN M. (1995) Food Polysaccharides and Their Applications, Ch. 6, New York, Marcel Dekker Inc. SEILER, A.

154 7. 8. 9. 10.

Handbook of hydrocolloids and COTTRELL, I. W. (1993) Emerging applications for guar and biopolymers in ‘green’ specialty chemical products, Rhoˆne-Poulenc Inc., Chemical Specialties USA 93-Symposium. PHILIPS G. O., WEDLOCK, D. J. and WILLIAMS P. A. (1984) Gums and Stabilisers for the Food Industry 2, 251–76, Oxford, Pergamon Press. PHILIPS G. O., WEDLOCK, D. J. and WILLIAMS P. A. (1990) Gums and Stabilisers for the Food Industry 5, 383–403, Oxford, Pergamon Press. RICHARDSON, P. H. et al. (1998) ‘Dilute solution properties of guar and locust bean gum in sucrose solutions’, Food Hydrocolloids, 12, 339–48. GOSWAMI, A.

9 Gum arabic P. A. Williams and G. O. Phillips, North East Wales Institute, Wrexham

9.1

Introduction

Gum arabic or gum Acacia is a tree gum exudate and has been an important article of commerce since ancient times. It was used by the Egyptians for embalming mummies and also for paints for hieroglyphic inscriptions. Traditionally the gum has been obtained mainly from the Acacia senegal species. The trees grow widely across the Sahelian belt of Africa situated north of the equator up to the Sahara desert and from Senegal in the west to Somalia in the east. The gum oozes from the stems and branches of trees (usually five years of age or more) when subjected to stress conditions such as drought, poor soil or wounding. Production is stimulated by ‘tapping’, which involves removing sections of the bark with an axe taking care not to damage the tree. The sticky gummy substance dries on the branches to form hard nodules which are picked by hand and are sorted according to colour and size (Fig. 9.1). Commercial samples commonly contain Acacia species other than Acacia senegal notably Acacia seyal. In Sudan the gum from Acacia senegal and seyal are referred to as hashab and talha respectively. The former is a pale to orange-brown solid which breaks with a glassy fracture and the latter is darker, more friable and is rarely found in lumps in export consignments. Hashab is undoubtedly the premier product but the lower-priced talha has found recent uses which have boosted its value. It is not possible to identify precisely the exact balance between these two products in the market-place since it is continually changing. Some typical grades of Sudanese gum available are listed in Table 9.1.

9.2

Supply and market trends

Sudan has traditionally been the main producer of gum arabic and supplies in the late 1960s were in excess of 60,000 tonnes p.a. Drought and political unrest in the 1970s and 1980s resulted in supplies dropping to a low of ~20,000 tonnes p.a. Nigeria and Chad are the other main producers with combined exports of ~10,000 tonnes p.a. The current estimate of total gum arabic production is 40ÿ50,000 tonnes p.a. Europe is the largest

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Fig. 9.1

Collecting gum arabic from Acacia senegal trees.

market for the gum and imports averaged ~30,000 tonnes p.a. in the early 1990s. France and the UK are the major importers (10,000 and 7,900 tonnes p.a. respectively over the period 1989–95) although a large part of this is re-exported. France showed an upward trend over this period while the UK trend was downward. Germany and Italy are the next biggest markets averaging 4,200 and 3,700 tonnes p.a. over the same period. Outside Europe, the biggest market is the US where imports totalled 10,000 tonnes in 1994. Japanese imports are around 2,000 tonnes p.a. The variability of supply over the past 20–30 years has led to dramatic fluctuations in price and in turn injected uncertainty into the user market. When supplies almost dried up in the 1970s the price increased from about $1,500 to $5,000 per tonne and then even to $8,000. It is almost impossible to evaluate the equivalence at today’s prices since inflation in commodity prices has been uneven and less than in manufactured goods. The

Gum arabic Table 9.1

157

Grades of Sudanese gum

Grade

Description

Hand-picked selected

The most expensive grade. Cleanest, lightest colour and in the form of large nodules.

Cleaned and sifted

The material that remains after hand-picked selected and siftings are removed. Comprises whole or broken lumps varying in colour from pale to dark amber.

Cleaned

The standard grade varying from light to dark amber. Contains siftings but dust removed.

Siftings

Fine particles remaining following sorting of the choicer grades. Contains some sand, bark and dirt.

Dust

Very fine particles collected after the cleaning process. Contains sand and dirt.

Red

Dark red particles

price stabilised in 1996 to around $5,000 per tonne but then overnight the Sudanese dropped their price to $2,500 per tonne which led to consternation and extreme problems for the industry’s processors, who were left holding stocks at the higher price. The price reduction was motivated by the inability of the Sudanese to move their stocks which were reported to be in the region of 42,000 tonnes at that time. The price dropped even further in 1998 to $1,800 per tonne. It is now the same in the US as it was in the 1970s and even cheaper in Europe. Inflation in Sudan has been rampant and hence the price in Sudanese pounds may look attractive locally but hides a dramatic decrease in revenue for the country. The low prices have had a severe effect in Chad and now the value of the trees for energy and other uses approaches the value of the gum. There is currently a great deal of uncertainty in the market-place making long-term planning difficult. The situation with talha is somewhat different. Hitherto it has been regarded as an inferior gum only to be used for a price advantage or when supplies of hashab were low. The 1996 price of $500 per tonne reflected this position. More recently, however, the functionality of talha in ‘health beverages’ as a fermentable fibre to maintain the wellbeing of the colon has given it a value in its own right. There has been a rush for talha so much so that it is now difficult to obtain supplies of the required quality. Sudan has depleted its stocks and it is believed does not intend to increase production. The Government policy is to maintain the hashab plantations only and to use the talha trees for charcoal production. There are indications that this policy might change in view of increasing demand. The price is now approaching that of hashab. The traditional product is Acacia seyal var. seyal but there is also another material, the so-called ‘white talha’ from Acacia seyal var. fistula which Chad is seeking to exploit. The future supplies of talha, however, are likely to come from Nigeria where it is referred to as ‘Nigerian No. 2’.

9.3

Manufacture

Following export to Europe and the US some grades are processed providing greater quality and convenience to the user but also increasing the price by $1,000 to $1,500 per tonne. Processing can involve mechanical grinding (kibbling) which breaks up the

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nodules into various specific sizes. One of the benefits of kibbled gum is that it dissolves at a much faster rate than lump gum. Spray-dried and roller-dried grades are also produced. These processes involve dissolving the gum in water with heating and stirring. The temperature is kept to a minimum in order to ensure that the gum is not denatured since this can have a deleterious effect on its functional properties. After removing insoluble material by decantation or filtering, the solution is pasteurised and then spray or roller dried. Spray drying involves spraying the solution into a stream of hot air. The water quickly evaporates and the dry powder, typically 50–100 microns, is separated from the air using a cyclone. During roller drying the solution is passed onto steamheated rollers and the water is evaporated off by a flow of air. The thickness of the gum film produced is controlled by adjusting the gap between the rollers. The film is scraped off the roll using a knife yielding flake-like particles several hundred microns in size. Spray-dried and roller-dried samples have an advantage over the raw and kibbled gum in that they are virtually free of microbial contamination and they dissolve much faster.

9.4

Regulatory aspects

Gum arabic has been evaluated by the Joint Expert Committee on Food Additives (JECFA) on several occasions between 1970 and 1998 resulting in eight changes in specifications. Surprisingly, however, none of these specifications were recommended for adoption by the Codex Alimentarius Commission. The specification changes made are summarised below. • FAO Food and Nutrition Paper No. 4 1978: ‘A 1 in 10 solution of the sample filtered through diatomaceous earth is slightly laevorotatory’. • FAO Food and Nutrition Paper No. 25 1982: No change. • FAO Food and Nutrition Paper No. 34 1986: The requirement of specific rotation is eliminated. • FAO Food and Nutrition Paper No. 49 1990: The specific rotation was re-introduced and ‘should be within ÿ26º and ÿ34º. Nitrogen must be between 0.27 and 0.39%’. Gum arabic is defined as ‘a dried exudation obtained from the stems and branches of Acacia senegal (L) Willdenow or closely related species’. (For the first time nitrogen and specific rotation limits were imposed and the word ‘closely’ introduced.) • FAO Food and Nutrition Paper No. 52 Add. 3 1995: Both nitrogen and specific rotation requirements were removed but the word ‘closely’ retained. Additionally tests were introduced to ensure that manose, xylose and galacturonic acid were absent thus eliminating non-Acacia gums. • FAO Food and Nutrition Paper No. 52 Add. 5 1997: The acceptance of Acacia seyal as a ‘closely related’ species was acknowledged. It is evident, therefore, that Acacia seyal has always been regarded as a component of commercial gum arabic apart from the time when the radical change was made to the specification in 1990 which was subsequently abandoned in 1995. The full specification that acknowledged this for the first time arose from the 49th JECFA meeting in 1997 and was thought to be the final definition (INS 414). This states as follows: Gum Arabic is a dried exudate obtained from the stems and branches of Acacia senegal (L) Willdenow or closely related species of Acacia (fam. Leguminosae). Acacia seyal is a closely related species. Gum arabic consists mainly of high

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159

molecular weight polysaccharides and their calcium, magnesium and potassium salts which on hydrolysis yield, arabinose, galactose, rhamnose and glucuronic acid. Items of commerce may contain extraneous materials such as sand and pieces of bark which must be removed before food use. Gum Arabic from Acacia seyal is sometimes referred to as talha. However, this specification was again changed at the 51st JECFA (1998). There are again important changes, as described in Food and Nutrition Paper 52 Add. 6 (1998). The important changes are summarised here. Synonyms: Gum arabic (Acacia senegal) Gum hashab, kordofan gum Gum arabic (Acacia seyal) Gum talha Acacia gum, arabic gum INS No 414 The geographical names have not previously been included, related only to the Sudan, and do not represent the other gum arabic producing countries. Definition: Gum arabic is a dried exudation obtained from the stems and branches of Acacia senegal (L) or Acacia seyal (fam. Leguminosae). Gums from other Acacia species are not included in these specifications. The references to ‘related species’ as in all other published specifications or ‘closely related species’ introduced by JECFA in 1990 have been deleted, on the misguided assumption that the producer developing countries could produce one species without any other ‘related species’ being included unwittingly by the local farmer. For such an eventuality the phrase ‘related species’ has always been included. Description: Gums from A. senegal and from A. seyal respectively, may be differentiated using immunological methods. Gum arabic (A. senegal) and gum arabic (A. seyal) are not necessarily technologically interchangeable. These new characteristics were presumably introduced to allow the two species that make up gum arabic to be distinguished and to acknowledge their different applications. The Final Conclusion? Once again the revised Gum Arabic Specification prepared at the 51st JECFA (1998) held in Geneva was finally referred for approval to the Codex Committee for Food Additives and Contaminants held in The Hague, in March 1999. A Working Group was convened to screen all the proposals. No definitive conclusion could be taken and the Report of the Working Group reflected the lack of consensus. JECFA were unwilling to take it back for further review because no new scientific information was available to them. Gum arabic, therefore, faced the prospect of being left out in the cold with neither approval of the current JECFA Specification nor its rejection – a most unsatisfactory situation for all concerned. Eventually in the Plenary Session with all countries present Gum arabic came up for consideration. The old arguments again surfaced, but as the discussion continued, apart from the Sudan, one producing African country after another supported the JECFA Specification. Many trade organisations too added their voices in favour, and then an almost unprecedented situation occurred – the Chairman gave the lead and proposed acceptance of the Specification in Category II (Recommended for Adoption after Editorial Changes, including Technical Revisions). The Editorial changes suggested were as follows:

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• Under ‘Synonyms’ delete Gum hashab, kordofan and Gum talha. • Under ‘Definition’ delete the last sentence (Gums from other Acacia species are not included in these specifications). • Under ‘Description’ delete fourth and fifth indent, i.e. sentences referring to immunological differentiation and technological interchangability. This was the proposal which was accepted and went to the Codex Alimentarius Commission at its 23rd Session in Rome, 28 June–3 July 1999 and adopted. It is thus a historic occasion. For the first time there is an approved Codex Alimentarius Advisory Specification for gum arabic (Annex 1) which establishes the definition as: Gum arabic is a dried exudation obtained from the stems and branches of Acacia senegal (L) or Acacia seyal (fam. Leguminosae). There are other regulatory definitions which are adhered to according to specific interests or geographical location. EU Gum Arabic Specification (E414) Acacia gum is a dried exudation obtained from the stems and branches of natural strains of Acacia senegal (L) Willdenow or closely related species of Acacia (Fam. Leguminosae). It consists mainly of high molecular weight polysaccharides and their calcium, magnesium and potassium salts, which on hydrolysis yield arabinose, galactose, rhamnose and glucuronic acid. Therefore, despite contrary proposals in various drafts, the EU has followed JECFA and not introduced specific rotation limits. However, in an unexpected and quite inexplicable innovation the Directive has introduced a molecular weight clause indicating that gum arabic should have a molecular weight of ca. 350,000. This appears inappropriate since in the scientific literature molecular weight values of between 200,000 to 800,000 have been reported (see, for example, Idris et al. (1998) Food Hydrocolloids 12, 379). European Pharmacopeia Definition: Acaciae Gummi (Acacia) Acacia is the air-hardened gummy exudate flowing naturally from or obtained by incision of the trunk and branches of Acacia senegal (L) Willdenow and other species of Acacia of African origin. The specification has retained ‘A 10% w/v solution is laevorotatory’. Implicitly, therefore, the definition acknowledges the acceptability of Acacia seyal within commercial Gum Arabic. United States Food Chemical Codex The Food Chemical Codex is an activity of the Food and Nutrition Board of the Institute of Medicine that is sponsored by the United States Food and Drug Administration. The gum is defined (INS 414) as:

A dried gummy exudation obtained fron the stems and branches of Acacia senegal (L) Willdenow or of related species of Acacia (Fam. Leguminosae). Unground Acacia occurs as white or yellowish white spheroidal tears of varying

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161

size or in angular fragments. It is also available commercially in the form of white to yellowish white flakes, granules or powder. 1g dissolves in 2ml of water forming a solution that flows readily and is acid to litmus. It is insoluble in alcohol. It should be noted that there is no specific rotation requirement in the current definition. United States Pharmacopeia and the National Formulary Acacia is the dried gummy exudate from the stems and branches of Acacia senegal (linne) or of related African species of Acacia (Fam. Leguminosae). This definition was reported in the USP 21st Revision, January 1st 1985 NF XVI ed. Official Monographs for USP XXI p. 1538. It remains the same in the current Official Monograph for NF 18 (USP2) 1996. In the 22nd Revision (November 15th 1991) the following was introduced: ‘Add the following to Specific rotation: between ÿ25º and ÿ35º, calculated on the anhydrous basis determined on a 1% w/v solution’. However, a subsequent supplement (7USP XXII–NF XVII 1992) removed the specific rotation requirement. USP and NF, therefore, now fall in line with the EU and JECFA.

9.5

Structure

The gums from Acacia senegal and Acacia seyal are complex polysaccharides and both contain a small amount of nitrogenous material that cannot be removed by purification. Their chemical compositions vary slightly with source, climate, season, age of the tree, etc., but typical analytical data for each are given in Table 9.2. The gums consist of the same sugar residues but Acacia seyal gum has lower rhamnose and glucuronic acid contents and higher arabinose and 4-O-methyl glucuronic acid contents than gum from Acacia senegal. Acacia seyal gum contains a lower proportion of nitrogen and the specific rotations are also very different. Determination of these latter parameters can provide a rapid means of differentiating between the two species. The amino acid compositions are similar (Table 9.3) with hydroxyproline and serine the major constituents. Both gums have complex molecular mass distributions that display similar features but the average molecular mass of gum from Acacia seyal is higher than that of Acacia senegal (Table 9.2). Typical molecular mass profiles of the two gums obtained by gel permeation chromatography using refractive index coupled with light scattering detection and UV absorbance (206nm) detection are presented in Figs 9.2(a) and 9.2(b) respectively. Refractive index is a sensitive measure of gum concentration and the Table 9.2

Characteristics of gum from Acacia senegal and Acacia seyal Acacia senegal

% galactose % arabinose % rhamnose % glucuronic acid 4-O-methyl glucuronic acid % nitrogen Specific rotation/degrees Average molecular mass (Mw)

44 27 13 14.5 1.5 0.36 ÿ30 380,000

Acacia seyal 38 46 4 6.5 5.5 0.15 +51 850,000

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Table 9.3 residues)

Hyp Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg

Amino acid composition of Acacia senegal and Acacia seyal gums (residues/1000 Acacia senegal

Acacia seyal

256 91 72 144 36 64 53 28 3 35 2 11 70 13 30 52 27 15

240 65 62 170 38 73 51 38 42 16 85 13 24 51 18 11

profiles indicate that the gums consist of two components, the main one (peak 1) representing ~90% of the total with a molecular mass of a few hundred thousand and the other (peak 2) which represents about 10% of the total with a molecular mass of several million. The UV absorbance profiles differ considerably and show three peaks. Two correspond to the peaks observed by refractive index but the intensities are different. This has been shown to be due to the presence of higher concentrations of proteinaceous material in the high molecular mass fraction. The third peak corresponds to protein rich material and represents only about 1% of the total mass. This fraction has a molecular mass of ~200,000. Most structural studies have been concerned with the gum from Acacia senegal. Carbohydrate analysis has indicated that the components of this gum corresponding to the three UV absorbance peaks all have a highly branched structure consisting of a 1,3 linked D-galactose core with extensive branching through 3- and 6-linked galactose and 3-linked arabinose. Rhamnose and glucuronic acid are positioned at the periphery of the molecules where they terminate some of the branches (Fig. 9.3). The main component, (peak 1), commonly contains < 1% protein. Material corresponding to peak 2, has protein content of ~10%. Since this fraction is readily degraded by proteolytic enzyme it has been reported to have a ‘wattle blossom-type’ structure where blocks of carbohydrate of molecular mass ~250,000 are linked to a common polypeptide chain (Fig. 9.4). Material corresponding to peak 3 has a lower glucuronic acid content than the other two fractions and has a reported protein content of 20–50%. Since this fraction cannot be degraded by proteolytic enzyme it is believed that the proteinaceous component is located within the centre of the molecules. Whereas the predominant amino acids in fractions corresponding to peaks 1 and 2 are hydroxyproline and serine, the predominant amino acids in the fraction corresponding to peak 3 are aspartic, serine, leucine and glycine. All three fractions interact with Yariv’s reagent and hence can all be classified as arabinogalactan– protein complexes (AGPs).

Gum arabic

163

Fig. 9.2 Molecular mass distribution of Acacia senegal and seyal gums obtained by gel permeation chromatography using (a) refractive index and multiangle laser light scattering detection, (b) UV absorbance at 206nm detection.

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Fig. 9.3 Possible structure of the carbohydrate component of gum from Acacia senegal. A = arabinosyl; filled circles = 3-linked galactose (galactose attached); open circle 6-linked galactose (galactose or glucuronic acid attached or end group); R1 = rhamnose-glucuronic acid; R2 = galactose-3arabinose; R3 = arabinose-3arabinose-3arabinose. (From Food Polysaccharides and their applications with kind permission of Marcel Dekker).

Fig. 9.4

9.6

Wattle blossom-type structure of the high molecular mass fraction of Acacia senegal gum.

Properties

Gum arabic readily dissolves in water to give clear solutions ranging in colour from very pale yellow to orange-brown and with a pH of ~4.5. The highly branched structure of Acacia senegal gum gives rise to compact molecules with a relatively small hydrodynamic volume and as a consequence gum solutions become viscous only at high concentrations as illustrated in Fig. 9.5. A comparison of the viscosity of the gum with xanthan gum and sodium carboxymethylcellulose, which are common thickening agents, is shown in Fig. 9.6. It is seen that even 30% gum arabic solutions have a lower viscosity than 1% xanthan gum and sodium carboxymethylcellulose at low shear rates. In addition, while gum arabic is Newtonian in behaviour with viscosity being shear rate independent, both xanthan gum and sodium carboxymethyl cellulose display nonNewtonian shear thinning characteristics. This is explained by the fact that the latter are linear molecules and intermolecular entanglements can readily occur while this is not the

Gum arabic

Fig. 9.5

Fig. 9.6

165

Viscosity of gum arabic as a function of concentration.

A comparison of the viscosity shear rate profiles of solutions of 1% xanthan gum, 1% sodium carboxymethyl cellulose and 30% gum arabic.

case for the highly compact, branched gum arabic molecules. The viscosity decreases in the presence of electrolytes due to charge screening and at low pH when the carboxyl groups become undissociated. The other major functional characteristic of gum arabic is its ability to act as an emulsifier for essential oils and flavours. It is now known that the protein-rich high molecular mass component adsorbs preferentially onto the surface of the oil droplets. It is envisaged that the hydrophobic polypeptide chains adsorb and anchor the molecules to the surface while the carbohydrate blocks inhibit flocculation and coalescence through electrostatic and steric repulsions. This is schematically illustrated in Fig. 9.7. Since only part of the gum is involved in the emulsification process, the concentration required to produce an emulsion is much higher than for pure proteins. For example, in order to

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Fig. 9.7

Schematic illustration of the stabilisation of oil droplets by gum arabic molecules.

produce a 20% orange oil emulsion then gum arabic concentrations of ~12% are required. Once formed the emulsions can remain stable for long periods of time (several months) with no evidence of coalescence occurring. Prolonged heating gum arabic solutions causes the proteinaceous components to precipitate out of solution thus influencing the gum’s emulsification properties.

9.7

Applications

9.7.1 Confectionery The major application of gum arabic is in the confectionery industry where it is used in a variety of products including gums, pastilles, marshmallows and toffees. The traditional wine gums incorporated gum arabic at concentrations of 40–55% and wine was used to add flavour. During the preparation the gum is dissolved in water keeping the temperature as low as possible (~60ºC) in order to avoid precipitation of the proteinaceous components which would give rise to a turbid solution. The gum is then added to a pre-boiled sugar/glucose solution (70%) followed by the flavourings and colours. After standing to allow air bubbles to rise, any surface scum is removed and the liquid deposited into starch trays which are placed in a stoving room for 4–6 days. The gums are then taken from the moulds, brushed to remove starch and often glazed with oil or wax. Softer gums or pastilles can be obtained by reducing the stoving time to 2–3 days. In recent times, because of gum shortages and price fluctuations, considerable efforts have been made to find replacements for gum arabic and nowadays pastilles are prepared using gum arabic at much lower concentrations in combination with other hydrocolloids, notably starch, maltodextrin, gelatin, pectin and agar. In these formulations demixing may occur due to incompatibility between the various hydrocolloids. The extent of demixing will depend on the rate of gel formation induced by the other hydrocolloids present and will consequently dictate the final texture obtained. In marshmallows the gum is used as a foam stabiliser while in toffees it is used to emulsify the fats present. Typical formulations are given in Tables 9.4 and 9.5. Gum arabic is also used to form a glaze on coated nuts and similar products.

Gum arabic Table 9.4

Typical formulation for marshmallows

Water Sugar Dextrose Albumen Gum arabic Gelatin Salt Table 9.5

167

39.0% 37.0% 19.0% 1.8% 2.4% 0.5% 0.3% Typical formulation for caramel-type products

Corn syrup Sweet condensed whole milk Granulated sugar Invert sugar Hydrogenated vegetable oil Salt Gum arabic

38.4% 34.4% 9.6% 9.6% 3.8% 0.2% 4.0%

9.7.2 Beverages Gum arabic is stable in acid conditions and is widely used as an emulsifier in the production of concentrated citrus and cola flavour oils for application in soft drinks. The gum is able to inhibit flocculation and coalescence of the oil droplets over several months and furthermore the emulsions remain stable for up to a year when diluted up to ~500 times with sweetened carbonated water prior to bottling. In the preparation of the emulsion a weighting agent is normally added to the oil in order to increase the density to match that of the final beverage and thus inhibit creaming. Typical weighting agents that are used, subject to legislation in various parts of the world, are glycerol ester of wood, gum damar and sucrose acetate isobutyrate (SAIB). SAIB is not normally used by itself but usually in conjunction with rosin or gum damar. The emulsion is prepared by adding the oil to the gum arabic solution under high speed mixing followed by homogenisation yielding a droplet size of ~1 micron. A typical formulation might contain 20% gum arabic, 10% flavour oil and 5% weighting agent while the final beverage might contain 0.1–0.2% concentrated emulsion, 10% sugar and 0.2% citric acid/colouring.

9.7.3 Flavour encapsulation Microencapsulation is commonly used to transform food flavours from volatile liquids to flowable powders that can be readily incorporated into dry food products such as soups and dessert mixes. The process also renders the flavour stable to oxidation. Encapsulation involves spray-drying an emulsion of the flavour oil which is produced using gum arabic as emulsifier. Nowadays maltodextrin is commonly mixed with the gum in order to reduce costs. The spray-dried particles formed are typically 10–200 microns in size and the retention of the volatile material, which is normally > 80%, depends on a number of variables including the inlet temperature of the spray dryer, the emulsion concentration and viscosity and the proportion of gum arabic to maltodextrin. Typical formulations are given in Table 9.6.

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Handbook of hydrocolloids Table 9.6 Formulation for flavour encapsulation Flavour Gum arabic Maltodextrin

9.8

7% 28% nil

10% 15% 25%

Bibliography

GLICKSMAN, M., ed. (1969) Gum Technology in the Food Industry Academic Press, New York. GLICKSMAN, M., ed. (1982, 1983, 1986) Food Hydrocolloids vols I, II, III, CRC Press, Boca Raton, Florida. IMESON, A., ed. (1992) Thickening and Gelling Agents for Food Blackie Academic and Professional Publishers,

Glasgow. NUSSINOVITCH, A.,

ed. (1997) Hydrocolloid Applications; gum technology in the food and other industries Blackie Academic and Professional, London. STEPHEN, A. M. ed. (1995) Food Polysaccharides Marcel Dekker, New York. WHISTLER, R. L. and BEMILLER, J. N., eds (1993) Industrial Gums; polysaccharides and their derivatives, 3rd edn, Academic Press, San Diego. WILLIAMS, P. A. and PHILLIPS, G. O. (1998) Gums and Stabilisers for the Food Industry 9 Royal Society of Chemistry, Cambridge UK. Special Publication No. 218.

10 Pectins C. D. May, Consultant

10.1

Introduction

Commercial pectins used as food additives are hetero-polysaccharides which contain at least 65% by weight of galacturonic acid-based units, which may be present as free acid, methyl ester or, in amidated pectins, acid amide1 (Fig. 10.1). They form a part of the wider class of pectic substances which are one of the major cell wall polysaccharides in land plants. The commercial materials normally contain added sugar to standardise performance.

10.2

Manufacture

Pectins are present in many fruits in variable amounts and qualities. The traditional use of pectin has been as a gelling agent, and this has largely dictated the types of fruit from which commercial grades can be manufactured. A major consideration is the availability of fruit by-products in sufficient quantity and quality. Before the development of a distinct pectin industry it was often the practice for jam makers to make a simple pectin extract from waste fruit material such as apple cores or surplus orange pith, but commercial production demands large quantities of available raw material. The history of the industry up to 1950 is described by Kertesz.1 Since that date, there has been a geographical shift of the production of pectin, driven to a large extent by difficulties with water supply and more especially effluent disposal in areas such as southern California, so that the major US plants producing citrus pectin, who had come to dominate that market, have been closed down by the companies concerned. The largest pectin plants today are either in Europe or in Latin America, and the expectation is that more of the industry may move to citrus-producing areas in future.

10.2.1 Raw materials Today the major sources are citrus peel, the residue from the extraction of citrus juice and oil, and apple pomace, the dried residue from the extraction of apple juice. Within the

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Fig. 10.1 Galacturonic acid, ester, and amide units found in pectins. Arrows indicate the potential for degradation by -elimination in the ester form.

commercially processed types of citrus, the peel from lemons or limes is preferred for most qualities of pectin, although orange peel is available in much larger quantities, and can be used for many applications. Citrus peel may be washed free from acidity and carefully dried to preserve the pectin quality, or may be processed directly in the wet state. Wet peel processing is particularly appropriate in the case of orange peel, but does require a large and consistent source of peel very near to the pectin plant. Pectin is very susceptible to degradation either by enzymes in the wet peel or by heat during drying and subsequent processing, and such loss of quality must always be controlled as far as possible. Pectin producers devote considerable resources to ensuring both the availability and quality of raw materials, and quality has a major effect on the types of pectin which can be economically produced.

10.2.2 Production processes Although various alternative processes have been patented in recent years, most pectin is produced by the extraction of the raw material with hot aqueous mineral acid. Each manufacturer has developed conditions which suit the major type of raw material processed in their plant, but the aim is always to produce a slurry which contains solid residue which can be easily separated by the chosen technology, and a liquid phase containing as high a concentration of high molecular weight pectin as possible, without generating excessive viscosity. The liquid extract may be treated to remove impurities, and clarified by removal of particulate matter, before proceeding to isolate a solid pectin. In principle, pure pectin may be isolated in various ways. The most commonly used method is to mix the concentrated extract with an organic solvent in which pectin is insoluble, but which will permit many of the impurities to remain in solution. International food standards permit the use of only methanol, ethanol, or isopropanol as the organic solvent. In this process, the clarified pectin extract is concentrated to about

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2% pectin, and mixed with sufficient alcohol to give a precipitate firm enough to be handled with the separation technology chosen by the manufacturer concerned, which may be either filtration or centrifugation. The pectin is separated as completely as possible from the mother liquor, and washed once or several times with further aqueous alcohol to remove salts and other impurities. The washing liquor may include sufficient food grade alkali to adjust the solution pH of the final pectin to the desired range. An alternative method, which is gradually going out of use, is based on the fact that basic aluminium salts form charge-transfer complexes with anionic polymers such as pectin. The aluminium chemistry involved is in fact complex, and involves the formation of polymerised aluminium oxy-ions which interact with the negatively charged pectin molecule, and careful control of all the conditions is required. The presence of excess citrate ion from a citrus raw material competes with the pectin and should be removed by washing the peel before the pectin is extracted. An advantage is that the pectin extract does not require concentration, and if conditions are right the precipitate flocculates and can be separated easily from the large volume of liquor. At this stage the precipitate is a greenish yellow due to the interaction of the aluminium ions with impurities present. The ‘yellow mass’ can then be pressed to a low moisture content, before being treated with a limited volume of aqueous alcoholic acid which leaches out the aluminium leaving purified pectin. Once the pectin isolated by either process has been separated from as much alcohol as possible, it is dried and ground to a fine powder. The pectin produced in this way will have a varying gelling power depending on the nature and quality of the raw material, and it will make a rapidly-setting gel under traditional jam-making conditions. The variable gel strength can be adjusted by blending one or more batches with sufficient sugar to give a standard performance. Other, more subtle, variations are minimised by selection of initial batches for cross-blending at this stage to ensure consistent performance. This pectin can be sold as such, or further modified to make it suitable for a wide range of applications.

10.3

The chemical nature of pectin

Pectins as defined for use in food are high molecular weight heteropolymers containing a majority (at least 65% by weight) of galacturonic acid units. The acid group may be free (or as a simple salt with sodium, potassium, calcium or ammonium) or naturally esterified with methanol. However, pectins are derived from the breakdown of more complex protopectins which are present in the plant tissue, and also contain a range of neutral sugars, including rhamnose, galactose, arabinose and lesser amounts of other sugars. These sugar units are present in a non-random structure, which consists of blocks of differing character retaining fragments of the original plant cell wall structure. The use of purified enzymes has shown that pectin extracted under very mild conditions contains both linear blocks (smooth regions) consisting of homopolygalacturonic acid, and highly branched blocks (hairy regions) which themselves contain several types of structures. Figure 10.2 illustrates the results of studies by the Wageningen group initiated by Prof. Walter Pilnik, and including Voragen, de Vries, Schols, and others. This work was summarised in the Pilnik Lecture delivered by Schols in 1997.2 It is not at present clear exactly how this complex structure and its variations influence the functional performance of commercial pectins, but the practical implications of certain basic aspects of structure are better understood.

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Fig. 10.2 Hypothetical structure of apple pectin showing I xylogalacturonan region, II region with arabinan side chains, III rhamnogalacturonan region making up the ‘hairy region’. (From H. Schols et al. ‘Structural Features of Native and Commercially Extracted Pectins’, in Gums and Stabilisers for the Food Industry 9 ed P. A. Williams and G. O. Phillips, RSC Cambridge, 1998, by permission of the authors.)

The regions of the pectin molecule which contain largely galacturonic acid units consist of a mixture of methyl ester, free acid and salt derivatives of the carboxyl group of the acid. Because commercial pectins are extracted under hot acidic conditions, many of the regions containing a high proportion of neutral sugars are hydrolysed, leaving mostly the more acid-stable galacturonate blocks. In certain pectins, such as those from sugar beet and potato, a proportion of the hydroxyl groups will also be acetylated. It has been known for a long time that the properties of pectin are dependent on pH, and on the percentage of acid group present in the form of ester (degree of esterification).

10.4

Commercial pectin: properties, modification and function

The pectin described above is normally from around 67–73% esterified. Apple pectin can, with great care, be extracted with up to 80% esterification. Pectin is readily degraded by a -elimination mechanism at ambient temperature or above at neutral or alkaline pH values. The ester groups can be hydrolysed under either alkaline or acidic conditions, or by pectin esterases. Commercially, acidic treatment is most commonly used, producing pectins with around 60% of ester groups which are ‘slow setting’. Under identical conditions of 65% total sugar solids by refractometry and a pH of say 3.1, the gel will take much longer to set. The setting of these gels is both time and temperature dependent,

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and the setting temperature depends on the rate of cooling. Slow-setting pectins permit gels to be prepared at higher sugar contents, valuable for sugar confectionery, biscuit jams, and so on. Because of the higher charge density on the slow-set pectin molecules, there is also a change in the pH requirements for gelation towards a lower pH in gels of otherwise similar composition. Further de-esterification to below 50% esterification produces a range of ‘low methoxyl’ pectins. These show a markedly greater reactivity towards calcium ions, which will cause gelation under suitable conditions of soluble solids and pH. Conditions for effective gelation depend on a balance of several factors, including soluble solids content, pH, calcium and pectin concentrations, and the presence of sequestrants. Amidated pectins (mostly of the low methoxyl type) are produced by reaction of suitable high methoxyl pectins with ammonia. The reaction is normally carried out in an aqueous alcohol slurry of the pectin at ambient or lower temperature. The process requires careful control of the relative rates of de-esterification and amidation, whilst minimising the rate of polymer chain degradation.

10.4.1 Gelation properties of pectins Because pectin is a charged hydrocolloid, it is sensitive to variations in pH and to a greater or lesser extent to the nature and quantity of cations present in the system. Gelation may be considered as a state between solubility and precipitation of a polymer, and therefore the nature of the solvent is also significant. Gelation of high methoxyl pectins High methoxyl pectins will gel only in the presence of sugars or other co-solutes, and at a sufficiently low pH, so that the acid groups in the polymer are not completely ionised. Both gel strength and setting temperature are influenced by these factors. In a system with sucrose as the sweetener, at around 65% soluble solids, typical of high sugar jams and preserves, high methoxyl pectins gel at up to pH3.4 (rapid set pectin) or 3.2 (slow set pectin). As the pH is reduced, gel strength and setting temperature will increase (Fig. 10.3) up to the point at which the setting temperature approaches the temperature at which the gel is deposited. Below this pH, pectin tends to pre-gel, and the resulting nonhomogeneous gel is weaker and more subject to syneresis. However, if the gel mixture is prepared at higher pH, and acidified immediately before or on depositing, the gel strength is maintained to low pH values. The gelation of high methoxyl pectins is also time-dependent, and setting temperatures will therefore depend on the rate of cooling, being higher with slower cooling. Very rapid cooling under shear can be used to produce a thick heavy texture useful in some industrial applications of fruit products. Changing the nature of the sugars present has a noticeable effect on the performance of pectin. For example, the replacement of a substantial amount of sugar by glucose syrup leads to increased setting temperature with a corresponding increase in the optimum pH for gelation, but some loss in maximum gel strength. Other sugars such as maltose may show a similar effect. Increasing the concentration of sugars increases both setting temperature and optimum pH. In making confectionery jellies, at 75–80% soluble solids, a slow set pectin would be used at a pH of 3.4–3.6 to give a suitable depositing time, enabling a batch to be deposited over 15–20 minutes or more.

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Fig. 10.3

(a) Variation of gel strength of high methoxyl pectins with pH in a 65% sucrose gel (relative values) (b) Variation of gel setting temperature in the same system.

Gelation of low methoxyl pectins The gelation of low methoxyl pectins is governed mainly by the interaction between the pectin and calcium ions. For this reason, the availability of calcium ions is important, and this is commonly governed by sequestrants either naturally present (e.g. citrate and other organic acid ions from fruit or milk) or added (commonly food grade di- or polyphosphates). Reactivity to calcium is governed by the proportion and arrangement of carboxyl groups in the pectin chain. Reactivity increases with decreasing degree of esterification, and is greater but less controllable if the arrangement of acid groups is

Fig. 10.4 Variation of relative gel strength of low methoxyl pectin gels (conventional and amidated) with added calcium at 30% added sucrose and pH 3.0 with a citric acid/sodium citrate buffer system.

Pectins

Fig. 10.5

175

The range of commercial non-amidated pectins with some typical applications.

less random, with blocks of de-esterified galacturonate units. Amide groups have a moderating influence, and permit gelation over a wider range of calcium concentrations (Fig. 10.4). Gelation is favoured by increased soluble solids, but decreased by increasing pH, or by increasing the level of sequestrant. However, a certain level of a sequestrant such as citrate is essential to produce a practically workable gel system. With correct formulation, low methoxyl pectins can gel over a wide range of soluble solids (10–80%), and in either acidic or less acid-tasting products, at a pH of 3.0 to above 5.0. 10.4.2 Availability of pectin types Pectin manufacturers generally offer a wide range of pectin types for different applications (Fig. 10.5).

10.5

Nutritional and safety aspects

Manufacturers will provide detailed nutrition data for labelling purposes on request. Pure pectin is essentially a soluble dietary fibre, but the commercial product will contain limited amounts of protein, sodium and calcium, and sugar or dextrose for standardisation. The amount of carbohydrate to be declared in nutrition labelling depends on national legislation, as soluble fibre may be either included, or totally excluded. This also affects the declaration of energy value. Pectins may contain 1–2% of protein, but if legislation requires that protein is declared as N 6.25 (or other factor) it is arguable whether the amide nitrogen should be included in the calculation. The levels of most other nutrients are not significant when the use level of pectin in the final product is considered, except in the case of blends with, e.g., sodium, potassium or calcium salts.

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If fibre is to be declared, the figure will depend on the analytical method accepted locally. Where the AOAC method is specified, the content of pure pectin, after deducting moisture and ash contents, can be taken as fibre. The Englyst method gives lower results, typically around 65% of the AOAC value.

10.5.1 Health and safety characteristics Pectin is a fine organic powder, and like flour, starch, and similar carbohydrate materials, has the potential to cause a dust explosion. It is therefore important to observe good housekeeping practices such as collecting up spilled material, and minimising dusting by careful handling. A lesser but more common hazard is spillage of pectin solution or of powder pectin onto a wet floor, creating the risk of slips and falls. Pectin is not a particular environmental hazard, but does have a significant BOD, and large spillages should be contained and disposed of carefully. Pectin is a component of the normal diet, and an approved food additive, and ingestion of pectin at reasonable levels is safe. As with any water-soluble gum, it is inadvisable to consume large amounts of dry pectin which could swell and possibly risk obstruction of the gullet.

10.6

Uses and applications

In all pectin applications, the action of the pectin is very dependent on the exact conditions in the product, pH, ionic strength and composition, the proportion of sweeteners and their nature, and, where fruit is present, the amount and nature of the pectin provided by the fruit. It is therefore always wise to test any change in formulation, including a new season or source of fruit, on a small (saucepan) scale before embarking on full-scale manufacture.

10.6.1 Dissolving pectin In most pectin applications it is essential to ensure that the pectin is dissolved before gelling conditions are reached. Pectin will not dissolve when near gelling conditions, and high methoxyl pectins in particular will not dissolve in sugar solutions of more than 20– 25%. As with most gums, it is vital to disperse the solid particles before they partially dissolve and stick together. This may be achieved either with a high shear mixing system (batch or in line) or by mixing the pectin with several times its weight of sugar, and stirring vigorously. Occasionally, the pectin may be dispersed in a sugar syrup and the mixture diluted with stirring to achieve a similar result.

10.6.2 Optimising pectin formulations Pectin formulations will typically require adjusting to particular production conditions. The factors described above which influence gelation must always be kept in mind. In most practical situations, the soluble solids level and fruit content (if any) are established as part of a product brief, and cannot be modified. There is always an optimum set of conditions between lack of set on the one hand and pre-gelation on the other, but it is possible to mistake some pre-gel conditions for lack of set because processing may result

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in a smooth syrup rather than a broken gel. If a gel is not obtained, and neither lowering the pH nor increasing the pectin content causes gelation to occur, this should be suspected, especially if the pectin level is already high. If small changes in pectin content or pH do not result in improved performance, changing the pectin for a slightly different type should be considered. If this is not effective, it is wise to consult the pectin supplier who should be able to advise, and will treat detailed information in confidence if requested. There are many different pectin types, and selection for a particular application requires considerable experience.

10.6.3 High sugar jams and preserves The earliest application for pectin was in fruit jams with 60–70% total soluble solids and a pH in the range 3.0–3.3. For this application, a high methoxyl pectin can be used. The composition of jams is often closely regulated, and the relevant regulations should be checked. In Europe, there are also specific labelling requirements in addition to the general Food Labelling Regulations. Depending on the nature of the fruit, the fruit content, and the desired soluble solids content, differing amounts and types of pectin will be required. Table 10.1 gives an indication of the proportion of pectin required for a jam at around 65% total soluble solids as measured by refractometer. The type of pectin will be determined by the type of product and the process to be used. Jams made by the traditional open pan cooking method will require a rapid setting pectin, whilst those made by vacuum cooking at a lower temperature require a slower setting pectin to avoid pre-setting. Clear jellies are usually made with a slow or extra slow set pectin, to allow any small bubbles of air to rise before the gel sets in the final container. If there is a problem with floating fruit, it may be useful to use a proportion of low methoxyl pectin to increase hot viscosity. A typical traditional jam recipe is given in Formulation 10.1. The recipe for orange marmalade in Formulation 10.2 illustrates the use of a mixture of high and low methoxyl pectins to prevent fruit floating and give a soft, spreadable, texture.

Table 10.1

Typical pectin requirement for traditional jam at 65% soluble solids Fruit content %

Fruit type

35

45

60

0.30–0.39%

0.22–0.28%

0.11–0.16%

0.23–0.29%

0.15–0.22%

0.07–0.11%

0.14–0.21%

0.08–0.14%

0.0–0.07%

‘Low pectin’ e.g. strawberry, peach, raspberry, pineapple ‘Medium pectin’ e.g. apricot, blackberry, marmalade ‘High pectin’ e.g. apple, damson, gooseberry, plum, quince, redcurrant

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Formulation 10.1

Traditional raspberry jam

Ingredients

Weight (g)

A

Rapid set pectin Sucrose

2.2 10

B

Water

50

C

Raspberries Sucrose Water

450 610 50

Final batch weight pH (50% solution at 20ºC) Soluble solids (approximately)

1 kg 3.0–3.2 67%

Preparation 1. 2. 3. 4.

Dry mix ingredients A and dissolve in water B, using a suitable high shear/speed mixer. Heat ingredients C to the boil while stirring. Add the pectin solution and boil down to 1015g. Cool to 85ºC and deposit into jars.

Note: The pH should be maintained in the range 3.0–3.2. Depending on the type of fruit it may be necessary to add a dilute solution of citric acid. If this is required it should be added slowly with vigorous mixing at the end of the boiling process to avoid pre-gelation. Formulation 10.2

Soft set orange marmalade

Ingredients

Weight (g)

A

Rapid set pectin Low methoxyl pectin Sucrose

1.0 0.5 10

B

Water

100

C

Orange pulp and peel Sucrose Water

200 640 150

D

Citric acid monohydrate (50% weight/vol.)

1.5 ml

Final batch weight pH (50% solution at 20ºC) Soluble solids (approximately)

1 kg 3.0–3.2 67%

Preparation 1. 2. 3. 4.

Dry mix ingredients A and dissolve in water B, using a suitable high shear/speed mixer. Heat ingredients C to the boil while stirring. Add the pectin solution and boil down to 1015g. While stirring add ingredients D, cool to 85ºC and deposit into jars.

10.6.4 Low sugar jams and jellies Low sugar jams and jellies cannot be made with high methoxyl pectins, and are usually best prepared with amidated low methoxyl pectins. However, in Europe, only nonamidated pectins may be used if an ‘organic’ claim is to be made for the product. With

Pectins Formulation 10.3

179

Reduced sugar strawberry jam

Ingredients

Weight (g)

A

Amidated pectin, medium set Sucrose

6 30

B

Water

250

C

Strawberries Sucrose

550 325

D

Citric acid monohydrate (50% w/v)

4 ml

E

Potassium sorbate (20% w/v)

5 ml

Final batch weight pH (as is at 20ºC) Soluble solids (approximately)

1 kg 3.2–3.4 43%

Preparation 1. 2. 3. 4.

Dry mix ingredients A and dissolve in water B using a suitable high shear/speed mixer. Gently heat ingredients C to the boil. Add the pectin solution and boil down to 1020g. With stirring add the citric acid D, followed by the potassium sorbate E. Cool to 85ºC and deposit.

low sugar products, it is particularly important to use good quality fruit in generous quantity to provide an acceptable product. The type of pectin used should be matched to the product, and, in general, lower solids require the use of a ‘high reactivity’ or ‘fast setting’ low methoxyl pectin. The recipe in Formulation 10.3 illustrates a typical ‘middle of the road’ reduced sugar jam. Local regulations and requirements will dictate the preservative to be used, if any.

10.6.5 Industrial fruit products This heading covers a varied and expanding range of products, from traditional bakery and biscuit jams to a range of fruit bases and toppings many of which have to be tailored to a specific end use. For example, fruit bases prepared specifically for fruit yoghurts need to be easy to handle in bulk without fruit separation, but may either be required to mix with the yoghurt, or to be deposited as a separate layer without forming a skin at the interface. In some cases there may be a requirement for the pectin to add extra body to the finished mixed yoghurt.

10.6.6 Bakery products For bakery jams various special properties may be required, but bake stability is one of the common requirements. For example, in open jam tarts baked with the filling, the jam must flow to give a smooth glossy surface but must not boil out of the pastry case. In such a complex field it is not possible to give more than a few typical recipes, and pectin manufacturers expect to provide technical assistance in formulating recipes for specific requirements. The following recipes (Formulations 10.4–10.6) provide a few illustrations of the many possibilities for pectins in this area.

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Formulation 10.4

Bake resistant jam

Ingredients

Weight (g)

A

Medium rapid set pectin Sucrose

5 20

B

Water

230

C

Strawberries 63DE glucose syrup (82% soluble solids) Sucrose

300 250

Citric acid monohydrate (50% weight/volume)

1 ml

D

Final batch weight pH (50% solution at 20ºC) Soluble solids (approximately)

340

1 kg 3.0–3.2 60%

Preparation 1. Dry mix ingredients A and dissolve in water B using a suitable high shear/speed mixer. 2. Heat ingredients C to the boil, add the pectin solution and return to the boil and boil down to 1020g while stirring. 3. Add the citric acid D, while stirring. 4. Cool with gentle stirring to 70ºC and deposit as required. Note: A bakery jam that will withstand baking and is suitable as a pie or tart filling, can be produced using medium rapid set pectin. Formulation 10.5

Heat-reversible biscuit or wafer filling

Ingredients

Weight (g)

A

Phosphate-buffered amidated pectin Sucrose

17 50

B

Water (70ºC) Citric acid monohydrate

350 1.3

C

Sucrose 63DE glucose syrup (82% soluble solids)

330 535

Colour and flavour

As required

D

Final batch weight pH (50% solution at 20ºC) Soluble solids (approximately)

1 kg 4.3–4.5 84%

Preparation 1. 2. 3. 4.

Dry mix ingredients A and dissolve in hot water B using a suitable high shear/speed mixer. Heat the pectin solution and ingredients C to the boil and boil down to 1020g. Add colour and flavour if required. Cool to 70ºC. Depending on the type of equipment used, spread the filling between 60–70ºC.

Note: This filling may be packed into suitable containers and melted as required by heating to 70–75ºC.

Pectins Formulation 10.6

Flan glazing jelly (without fruit pulp)

Ingredients

Weight (g)

A

Phosphate-buffered amidated pectin Sucrose

15 50

B

Water (70ºC)

350

C

Water Citric acid monohydrate (50% weight/volume) Sucrose 42DE glucose syrup (82% soluble solids)

50

Colour and flavour

As required

D

181

Final batch weight pH (on 50% solution) Soluble solids (approximately) Setting temperature (approximately)

4.5 ml 400 200

1 kg 3.3–3.4 63% 40ºC

Preparation 1. 2. 3. 4. 5. 6.

Dry mix ingredients A and dissolve in water B, using a suitable high shear/speed mixer. Heat together ingredients C and boil down to 75–76% soluble solids (refractometer). Stirring thoroughly, pour the hot pectin solution into the boiling batch. Boil down to 1010g and remove from heat. Stirring slowly, cool to 65–70ºC, and add required colour and flavour. Fill into containers and cool to at least 40–45ºC.

If the glaze is to be used in an undiluted form, gently mash and liquefy the glaze by heating to 95ºC. The glaze may then be applied at any temperature down to 60ºC. If the glaze is to be used in a dilute form, gently mash the glaze with up to 60% water and heat to 95ºC. The diluted glaze may then be applied at any temperature down to 30ºC. Any unused glaze can be kept and melted back when required without any loss in quality.

In contrast to the non-melting behaviour of the recipe in Formulation 10.4, it is equally possible to use a pectin system to make fully themally reversible gels at a range of soluble solids levels, as in Formulation 10.6 for a glazing jelly based on an amidated pectin combined with phosphate salts which control both the availability of calcium and the pH of the gel. The use of pectin in bakery applications is not restricted to high sugar products, and the recipe (Formulation 10.7) for a bakery pie filling illustrates how resistance to baking can be maintained down to only 30% soluble solids.

10.6.7 Fruit bases The requirements for pumpability and absence of fruit separation require a degree of restructuring after shear, usually best provided by an (amidated) low methoxyl pectin. The exact properties of these bases need to be tailored to the equipment and systems in use by both the fruit processor and the end user, as well as those demanded by the final

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Formulation 10.7

Apple pie filling

Ingredients

Weight (g)

A

Pectin type 170 Sucrose

14 40

B

Water (70ºC)

250

C

Diced apple Sucrose

500 200

D

Calcium lactate pentahydrate Water (100ºC)

1.5 50

Final batch weight pH (as is at 20ºC) Soluble solids (approximately)

1 kg 3.1–3.3 30%

Preparation 1. Dry mix ingredients A and dissolve in hot water B using a suitable high shear/speed mixer. To aid dissolving place the mixer so as to obtain maximum turbulence. Gradually add the dry mix ensuring addition is complete before thickening slows agitation. 2. Heat the pectin solution, add ingredients C and bring to the boil. 3. Prepare the hot calcium lactate solution and add to the boiling mix while stirring continuously. 4. Reduce to 1010g, remove from the heat and cool to 85ºC and deposit as required. Formulation 10.8

Low sugar fruit base

Ingredients

Weight (g)

A

Pectin (low methoxyl or amidated) Sucrose

9 40

B

Water

300

C

Strawberries Sucrose Water Citric acid monohydrate (50% weight/volume) Sodium citrate dihydrate (20% weight/volume)

500 210 70

Final batch weight pH (as is at 20ºC) Soluble solids (approximately) Filling temperature (approximately)

As required As required 1 kg 3.3–3.5 31% 30ºC

Preparation 1. Dry mix ingredients A and dissolve in hot water B using a suitable high shear/speed mixer. 2. Weigh ingredients C and the pectin solution into a pan and heat to 90ºC. 3. Maintain temperature at 90ºC for 10 minutes to pasteurise, or heat until the batch weight has been reduced to 1020g. 4. Cool the fruit preparation while stirring to 30ºC to obtain a thickened rather than a gelled texture. This may be achieved by in line cooling under shear.

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product, and recipes can only be a guide to what is possible – fruit contents, soluble solids levels, and flow properties can be adjusted to meet a wide range of requirements but in some cases a custom blend of pectin with other additives may be required. This may contain a pectin, one or more phosphate sequestrants, and a calcium source such as a calcium orthophosphate. In order to obtain the correct characteristics, a number of factors can be adjusted. The amount of total citrate will control both the acid taste and the binding of the calcium ion present. An increase will increase the perceived acidity, but reduce the body of the base. The pH can be adjusted by altering the balance of these ingredients. If the base is too liquid, either a faster setting, more reactive, pectin can be used, or calcium can be added as a dilute solution of calcium chloride or lactate to the finished product before cooling. Various modifications to the formulation are possible to produce a base for layer desserts or to add extra thickness to a mixed fruit yoghurt (post-dosage thickening) by interaction between the pectin from the fruit base and the calcium in the yoghurt. Similar formulations may be used as toppings for gateaux and cheesecakes, including also caramel, butterscotch and other non-acid flavours.

10.6.8 Dairy products Pectin can have two distinct functions in dairy products and dairy analogues such as those prepared from soya. High methoxyl pectins can act as protein dispersion stabilisers at reduced pH as in yoghurt or milk/fruit juice drinks. Low methoxyl pectins behave quite differently, and can gel either milk or more acid products by interaction with calcium. Acid milk stabilisation Pectin is an effective protective colloid for casein particles at a pH of 3.9–4.1, typical of yoghurt. It is important to homogenise the yoghurt to break up protein aggregates in the presence of the pectin, before heat treatment is carried out. The amount of pectin required will be a function of the amount of homogenisation carried out, and this will also affect the product viscosity. Surplus pectin over the amount required for stabilisation of the dispersion will increase the viscosity of the final product, which may or may not be desirable. Typically, about 0.4% of a specially selected and standardised pectin will be added either as a solution or dispersed in sugar or a syrup, and the product then homogenised. Heat treatment may, in some cases, be followed by further homogenisation, often at a lower pressure, to ensure maximum stability. The resulting drink is free from chalkiness and sedimentation. Similar advantages are possible for blends of milk and fruit juices, enabling a milkbased fruit drink with a true fruit flavour and acidity. Either liquid milk or milk powder can be used, as in Formulation 10.9. The principle can be extended to carbonated milk drinks, and other products, including drinks based on whey, which can be stabilised against precipitation of the soluble whey proteins. Milk gels and desserts Low methoxyl amidated pectins can be used to gel milk by directly dispersing pectin powder, preferably mixed with several times its weight of sugar, in cold milk, and heating with stirring to boiling to dissolve the pectin. The amount of pectin required will be from

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Formulation 10.9

Milk/fruit juice drink

Ingredients

Weight (g)

A

Pectin (dairy stabiliser grade) Sucrose

4 10

B

Water

152

C

Skim milk powder Sucrose

25 80

D

Water

615

E

Fruit Juice Sucrose Citric acid monohydrate (50% w/v)

100 10 4 ml

Final batch weight pH (as is at 20ºC) Soluble solids (approximately)

1 kg 3.9–4.1 15%

Preparation 1. Dry mix ingredients A and dissolve in water B using a suitable high shear/speed mixer. 2. Dry mix ingredients C and dissolve in water D. 3. Mix the pectin solution into the milk solution for 2–3 minutes using a suitable high shear/speed mixer. 4. With continuous mixing add ingredients E, and continue mixing for a further minute. Homogenise as required. 5. Pasteurise as necessary. Note: The pH must be in the range 3.9–4.1 to obtain stability to heat treatment, outside this range the stability will not be as great and more pectin will be required. The pH can be adjusted by altering the level of fruit juice or by adding a dilute solution of acid or citrate as required.

0.6–0.9%, and the texture of the gel can range from firm and brittle to very soft and creamy, depending on the pectin type chosen. It is also possible to formulate a fruit syrup which, on adding to cold milk, will produce a lightly gelled fruit dessert (Formulation 10.10).

10.6.9 Other dessert products Pectin gels can be used as an alternative to gelatine in fruit desserts and trifles (Formulation 10.11). Pectin gels can be deposited and set very rapidly so that other components such as sponge or cream can be added after a short cooling stage, enabling the complete dessert to be assembled on line. They give an attractive light texture with excellent flavour release. Whipped cream or protein whipping agents can be added to produce mousse-type products (Formulation 10.12).

10.6.10 Sugar confectionery Pectin confectionery jellies can be divided into two types: tender, fruity jellies made with high methoxyl pectin (Formulation 10.13), and elastic jellies based on low methoxyl

Pectins Formulation 10.10

185

Syrup for milk dessert

Ingredients

Weight (g)

A

Amidated pectin Sucrose

15 80

B

Water

400

C

Raspberries Water Citric acid monohydrate (50% w/v) Sodium citrate dihydrate (20% w/v) Sucrose

200 300 4 ml 20 ml 80

Final batch weight pH (as is at 20ºC) Soluble solids (approximately)

1 kg 4.0–4.2 20%

Preparation 1. Dry mix ingredients A and dissolve in water B using a suitable high shear/speed mixer. To aid dissolving, place the mixer so as to obtain maximum turbulence. Gradually add the dry mix ensuring addition is complete before thickening slows agitation. 2. Warm ingredients C, add the pectin solution and heat to the boil while stirring. 3. Boil down to 1020g, remove from the heat, cool and deposit. 4. To prepare the dessert, take equal parts of fruit syrup and milk. Mix the milk into the fruit syrup for 15–30 seconds. The dessert may be eaten within 5 minutes of preparation or may be stored in the fridge until required. Note: It may be necessary to adjust the amount of citric acid or sodium citrate used.

Formulation 10.11

Water-based dessert jelly

Ingredients

Weight (g)

A

Pectin type 3000 Sucrose

13 40

B

Water Citric acid monohydrate

600 2.5

C

Sucrose

240

D

Water (100ºC) Calcium lactate pentahydrate

200 1.8

E

Colour and flavour

As required

Final batch weight pH (as is at 20ºC) Soluble solids (approximately)

1 kg 3.5–3.7 30%

Preparation 1. Dry mix ingredients A and dissolve in water B using a suitable high shear/speed mixer. 2. Heat the pectin solution to the boil and add the sucrose C.

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3. Pre-dissolve the calcium lactate in the hot water D, and stir into the pectin solution, while maintaining a full rolling boil. 4. Boil down to 1020g and remove from the heat and stir in ingredients E. 5. Deposit as required. Notes 1. The texture of the jelly can be altered by changing the pectin/calcium ratio, i.e. a high pectin to a low calcium level will result in an elastic gel, whereas if more calcium is added the resultant gel will become more brittle. 2. It is important to add the calcium lactate as a hot dilute solution and with efficient agitation to avoid the formation of jelly lumps (fisheyes). Formulation 10.12

Chocolate mousse

Ingredients

Weight (g)

Chocolate base A

Pectin type 2000 Sucrose

10 50

B

Water (70ºC)

330

C

Cocoa powder (5% lecithin) Sucrose

20 100

Cream base D

Double cream (47% fat)

Final batch weight pH (as is at 20ºC) Soluble solids (approximately)

500 1 kg 6.0–6.2 15%

Preparation 1. Dry mix ingredients A and dissolve in water B using a suitable high shear/speed mixer. 2. Add ingredients C to the pectin solution with efficient mixing, and continue mixing until the sucrose has dissolved. 3. Whip the cream D until aerated and firm, or the desired over run. 4. Gently mix the cream and chocolate syrup together until homogenous and deposit as required.

pectins blended with sequestering phosphate buffers, suitable for traditional turkish delight flavours, and also mint and other non-acid flavours (Formulation 10.14). The latter approach can also be used to add structure and thixotropy to caramel and similar fillings. It is essential to include a proportion of glucose syrup to prevent crystallisation of the sugar.

10.6.11 Other food applications Many of the above formulations may be adapted to produce condiment jellies, glazes and marinades for chilled and frozen recipe dishes, and for other similar uses. Pectin/sugar solutions can be used to coat confectionery items, either chocolate or sugar coated, by standard panning techniques.

Pectins Formulation 10.13

Fruit-flavoured confectionery jelly

Ingredients

Weight (g)

A

Pectin (Slow set) Sucrose

8.8 40

B

Water (70ºC) Citric acid monohydrate (50% weight/volume) Potassium citrate monohydrate (40% weight/volume)

325

Sucrose 42DE glucose syrup (82% soluble solids)

470

C

D E

187

2.8 ml 6.25 ml

300

Citric acid monohydrate (50% weight/volume)

6 ml

Colour and flavour

As required

Final batch weight pH (50% solution at 20ºC) Soluble solids (approximately)

1 kg 3.3–3.6 78%

Preparation 1. Dry mix ingredients A and dissolve in hot ingredients B using a suitable high shear/speed mixer. 2. Bring the pectin solution to the boil and add ingredients C (the sucrose followed by the glucose syrup). 3. Boil rapidly to 1020g, and thoroughly mix in the citric acid D. 4. Remove from the heat, mix in the colour and flavour and deposit into moulds. Depositing must be completed before the batch begins to set – if it is held at 90ºC within 20 minutes of adding the acid.

Formulation 10.14

‘Turkish delight’ jelly

Ingredients

Weight (g)

A

Phosphate-buffered amidated pectin Sucrose

20 60

B

Water (70ºC) Citric acid monohydrate (50% weight/volume)

300

C

0.5 ml

42DE glucose syrup (82% soluble solids)

300

D

Sucrose

440

E

Colour and flavour

As required

Final batch weight pH (10% solution at 20ºC) Soluble solids (approximately)

1 kg 4.6–4.8 76%

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Preparation 1. Dry mix ingredients A and dissolve in ingredients B using a suitable high shear/speed mixer. 2. Heat the pectin solution to the boil. 3. Add the glucose syrup C and return to the boil, followed by the sucrose D. Continue boiling until the weight has been reduced to 1020g. Add colour and flavour, if required, and pour into moulds. Notes: This formulation will set at about 50ºC. Slight reformulation will reduce the setting temperature sufficiently to permit depositing into pre-formed chocolate shells at around 30ºC. It may also be used for other non-acid flavours.

10.7

Legal status

Pectin is generally regarded as one of the safest and most acceptable of food additives, and this is recognised by Acceptable Daily Intake (ADI) levels of ‘not specified’ by both the Joint Food Experts Committee (JECFA) for Codex Alimentarius purposes, and the Scientific Committee for Food (SCF) in the European Union, and by Generally Regarded as Safe (GRAS) status in United States legislation. Similar status has been awarded in most other jurisdictions. In general, there are few restrictions on the use of pectin in most foods, but most authorities prescribe restricted lists or the absence of food additives in a limited number of basic food categories, and pectin may or may not be listed in these cases. The Codex and US specifications include both amidated and non-amidated pectins. In the EU, there are separate specifications for E440(ii) and E440(i) respectively, but permitted uses are similar except in organic foods, where only E440(i), non-amidated pectin, is permitted.

10.8 1. 2.

References KERTESZ, Z. I. (1951) The Pectic Substances, New SCHOLS, H. A., ROS, J. M., DASS, P. J. H., BAKX, E. J.

York, Interscience Publishers Inc. and VORAGEN, A. G. J. (1998) ‘Structural Features of Native and Commercially Extrcated Pectins’, Gums and Stabilisers for the Food Industry 9, Wrexham, The Royal Society of Chemistry.

11 Milk proteins M. P. Ennis and D. M. Mulvihill, University College, Cork

11.1

Introduction

Dried milk protein-enriched products produced from bovine (cow) milk are considered to be high esteem food ingredients due to their high nutritional value. In formulated food products these protein enriched ingredients are used to bind and emulsify fat, bind and entrap water and entrap and stabilise air, and these physico-chemical and functional properties are important in the modification of the textural and rheological characteristics of the formulated foods and in contributing to product stability and sensory appeal. In this chapter methods for the commercial production of dehydrated bovine milk proteinenriched products are outlined and the functional properties of these products and their applications in foods are described.

11.2

The milk protein system

Normal bovine milk is a highly complex system that contains approximately 3.5% by weight protein (Table 11.1); the protein is traditionally divided into two main fractions based on solubility. The caseins, which comprise about 80% of the total nitrogen in milk, are insoluble at their isoelectric points (~pH4.6) at temperatures > 8ºC and precipitate from milk under these conditions, while 20% of the total nitrogen remains soluble in the serum, about 15% being whey proteins and the remainder being non-protein nitrogeneous components. While most of the whey proteins are also insoluble at their isoelectric points (~pH5) at very low ionic strength, they are soluble at this pH in the ionic environment of milk. The highly heterogeneous casein fraction comprises four principal primary proteins (gene products), namely s1-, s2-, -, -caseins, the -caseins (proteolytically derived from -casein) and several minor proteins and peptides (Fig. 11.1). The s1- and caseins contain no cysteine or cystine while s2- and -casein each possess two halfcystine residues (Table 11.2) that normally exist as homogeneous disulphide bonds. The high prolyl content of caseins tends to prohibit the formation of secondary structure and the protein molecules are small, amphipathic, randomly coiled and relatively

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Table 11.1 Composition of bovine milk (from Swaisgood, 1992;36 Zall, 199237 and Creamer and MacGibbon, 19964) Component Total protein Caseins

Whey proteins

Concentration g/l 30–35.8 9–15 9–11 3–4 3–4 2–4 0.7–1.5 0.6–1.0 0.1–0.4 0.6–1.8 37–50 47–49.6 7.1 ~860

s1-casein -casein -casein s2-casein -lactoglobulin -lactalbumin Igs BSA Proteose peptones

Fat Lactose Ash Water

unstructured. Genetic polymorphism among the caseins resulting in differences in amino acid contents, different degrees of phosphorylation, and variability in glycosylation of casein (Figure 11.1) contribute to variability in the protein net charge, hydrophilicity and metal binding and thus influence the behaviour of the milks during manufacturing processes such as renneting and gelation. The caseins are prone to association due to regions of high hydrophobicity and the charge distribution arising from the amino acid sequence, phosphorylation and glycosylation. In milk the caseins are present in the form of large, roughly spherical colloidal associations, or micelles, of molecular weights in the region of 108 Da, which can be separated from the molecularly dispersed whey proteins by ultracentrifugation. Micelles also contain colloidal calcium phosphate, magnesium, sodium, potassium and citrate. The micelles are believed to be composed of submicelles containing variable amounts of the caseins and arranged in such a way that -casein is largely situated at the micelle surface. Hydrogen bonding, hydrophobic interactions and electrostatic interactions are all important in maintaining micelle structure. Binding of calcium to charged regions of the proteins modulates hydrophobic interactions between proteins and between submicelles, and colloidal calcium phosphate salt bridges contribute to micelle stability. In native milk the micelles are prevented from aggregating by both electrostatic and steric repulsion due to the -casein ‘hairy coat’ layer. Proteolytic removal of the ‘hairy coat’ by renneting or ethanol-induced collapse of the coat destabilises the micelles and calcium-mediated aggregation may occur depending on conditions of temperature, pH and ionic environment. Table 11.2 Protein s1-casein s2-casein -casein -casein

Some physico-chemical characteristics of the caseins Number of residues

mol P/ mol protein

Isoionic pH

Mol. wt. (kDa)

Number of prolines

Number of half-cys

199 207 209 169

8 10–13 5 1

4.94 5.45–5.23 5.14 5.61

23.6 25.2–25.4 24 19

17 10 35 20

0 2 0 2

Fig. 11.1

Distribution of proteins in bovine milk.

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Table 11.3 Some physico-chemical characteristics of casein micelles (after McMahon and Brown, 198438) Characteristic

Average value

Diameter Surface area Volume Mass Density (hydrated) Water content (hydrated) Hydration Voluminosity Zeta potential (at 25ºC) Particle weight (hydrated) Number of monomers (av MW 25,000)

130–160 nm 8.0 10ÿ10 cm2 2.1 10ÿ15 cm3 2.2 10ÿ15 g 1.0632 gcmÿ3 63% 3.7 g H2O/g protein 4.4 cm3gÿ1 ÿ18.7 0.3 mV 1.3 109 Da 5000

Limited dissociation of casein from micelles may be induced by cooling or heat treatment. Removal of calcium from the micelle by sequestering salts leads to dissociation of caseins and ultimately disintegration of the micelles, depending on the conditions used. Acidification solubilises the colloidal calcium phosphate, disrupts the micelle structure and reduces the charge on the proteins causing aggregation and precipitation of the caseins. Some characteristics of casein micelles are given in Table 11.3. The whey protein fraction is also highly heterogeneous (Figure 11.1) and includes the principal whey protein -lactoglobulin, -lactalbumin, blood serum albumin, immunoglobulins, -casein-derived proteose peptones, numerous minor proteins including lactoperoxidase and lactotransferrin, and various enzymes. The monomeric lactoglobulin contains two intramolecular disulphide cross-links and one free SH group. Numerous genetic variants are known, one of which is glycosylated. Monomeric lactalbumin is a calcium metalloprotein that has four intramolecular disulphide crosslinks and also exists as several genetic variants that may have minor glycosylated forms. The genetic variants present in milks, and the variability in their degree of glycosylation, affect the functional behaviour of the milks during processing. Some characteristics of the major whey proteins are summarised in Table 11.4. Caseins are extremely heat-stable proteins, whereas the whey proteins denature on heating at temperatures ~70ºC. At its normal pH (~6.7), milk may be heated at 140ºC for 20 min. before coagulation occurs; however, on heating milk at > 72ºC, whey proteins denature and interact with casein to form a complex. Some of the differences between caseins and whey proteins form the basis of industrial methods for casein and whey protein isolation; however, caseins and whey proteins can also be isolated together in Table 11.4

Some physico-chemical characteristics of the major whey proteins

Protein -lactoglobulin -lactalbumin BSA Ig Proteose peptones

Number of residues

mol P/ mol protein

Isoionic point

Mol. wt. (kDa)

Number of prolines

Number of half-cys

162 123 582 variable variable

0 0 0

5.2 4.2–4.5 5.3

18 14 66 150–900 4.1–40.8

8 2 28

5 8 35

Milk proteins

193

various high-protein products, referred to as co-precipitates, milk protein concentrates or total milk proteins. Detailed descriptions of the milk protein system are given in Fox (1989),1 Fox (1992),2 Holt (1992)3 and Creamer and MacGibbon (1996).4

11.3

Manufacture of milk protein products

11.3.1 Casein and caseinate manufacture Methods for the manufacture of caseins and caseinates have been reviewed by Muller (1971, 1982),5, 6 Mulvihill (1989, 1992)7, 8 and Fox and Mulvihill (1990)9 and are summarised in Fig. 11.2. Casein is manufactured from skim milk to minimise possible flavour defects arising from deterioration of lipids in the dried casein products. Casein micelles are destabilised by acidifying the milk to pH ~ 4.6 by either (i) injection of mineral acid (typically 1–2 M HCl) into the milk at ~30ºC, (ii) mixing milk at ~10ºC with sufficient cation exchange resin in the hydrogen form to allow exchange of cations in the milk for the H+ resulting in a milk acidified to pH ~ 2.0, separation of the acidified milk and resin and subsequently mixing this acidified milk with untreated milk to achieve the desired pH, or (iii) inoculation of milk with starter cultures and incubation at 30ºC to produce lactic acid from lactose naturally present in the milk, whereupon the caseins

Fig. 11.2 Industrial preparation of casein-based protein products.

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coagulate as the pI of the proteins is reached. Alternatively, casein micelles are destabilised at the natural pH of milk using any of a number of proteolytic enzyme preparations such as calf rennet or increasingly, genetically engineered chymosin or rennet substitutes such as microbial proteinases. Cleavage of the -casein to para-casein and soluble glycomacropeptides occurs and the modified casein micelles become susceptible to the calcium present in the serum phase of the milk and coagulate at temperatures above 20ºC. Continued coagulation of the destabilised micelles to form a curd which may be easily separated from the whey is facilitated by a process referred to as ‘cooking’ which involves increasing the temperature to 50–60ºC. The curd is then separated from the whey using vibratory, moving or stationary inclined nylon or stainless steel mesh screens, or polyester fabric screens in a cascade arrangement, or using mechanical devices such as horizontal bowl centrifuges or roller presses. After separation of the curd from the whey, the curd is washed to remove residual constituents such as lactose, salts and whey proteins, mechanically dewatered and dried to a moisture content of < 12% using pneumatic ring driers, fluidised bed driers or attrition driers. Tempering and blending of casein particles to achieve uniform moisture content is followed by grinding or milling to the required particle size. Mineral acid casein is produced by method (i), lactic acid casein by method (iii) and rennet casein by the proteolytic coagulation method. Acid caseins are not soluble when redispersed in water. Spray-dried sodium caseinate, the most commonly used water-soluble casein used in foods, is prepared by solubilising acid casein with NaOH. Wet acid casein curd mixed with water at 40ºC to a solids content of about 25% (or alternatively, dry acid casein in a slurry with water) is milled in a colloid mill and NaOH (2.5 M) mixed with the slurry to give a final pH of 6.6–6.8. The viscous slurry is then vigorously mixed and heated to ~75ºC in a series of vats to complete solubilisation and then further heated to 95ºC. The pH of the solution is adjusted with NaOH if necessary to give a caseinate of the required pH and the sodium caseinate solution is then spray dried. Holding time at high temperature is minimised to limit Maillard browning of the casein and the exposure time to high pH during dissolving minimised to prevent lysinoalanine formation and the production of off-flavours. Other caseinates may be produced by using different manufacturing methods or by employing alternative bases for neutralisation of the acid casein. Roller-dried sodium caseinate can be prepared by mixing moist curd with an alkaline sodium salt and feeding this onto the drum of the drier. Granular sodium caseinate may be produced by reacting low moisture (40% water) acid casein curd with sodium carbonate with agitation and drying in pneumatic ring driers to give a product of high bulk density and improved dispersability compared to spray- or roller-dried sodium caseinate. Potassium or ammonium caseinates may be produced by substituting KOH or NH4OH for NaOH and by substituting trisodium or tripotassium citrate for NaOH citrated caseinate can be made using the spray-drying process. Granular ammonium caseinate may also be prepared by exposing dry acid casein to ammonia gas in a fluidised bed reactor. Calcium caseinate can be made by mixing milled soft casein curd with a 10% Ca(OH)2 slurry to the required pH, agitating at low temperature until dispersion is complete and then heating the dispersion to 70ºC followed by spray drying. In an extrusion process casein at 50% solids from a dewatering device was fed into an extruder, and 25% NaOH added to form a viscous sodium caseinate. Caseinate was also prepared by feeding dry acid casein into the extruder and reacting it with 20–40% NaOH at temperatures up to 120ºC. The caseinates were subsequently roller dried and ground to a fine powder.10 Compositional standards for caseins and caseinates are given in Table 11.5.

Table 11.5

Compositions of caseins and casein-derived milk protein products EU regulation 2921/90 EU Acid casein

Annex I

USDA

EU Rennet casein

Annex Annex II I

EU Caseinates

Codex Alimentarius Stan A-18-1995

Edible dry casein Rennet Acid Caseinates (acid) casein casein

Annex II

Annex I

Annex II

Annex III

Extra Standard grade grade

FIL-IDF 72:1974

FIL-IDF 45:1969

Caseinate

Acid casein

Extra grade

First grade

Extra Standard grade grade

Protein (%, min)

–

–

–

–

88.00

88.00

85.00

95 dry basis

90 dry basis

84 dry basis

90 dry basis

88 dry basis

90 dry basis

88 dry basis

95 dry basis

90 dry basis

Moisture (%, max)

12.00

10.00

12.00

8.00

6.00

6.00

6.00

10

12

12

12

8

6.0

8.0

12

12

Fat (%, max)

1.75

1.50

1.00

1.00

–

–

1.50

1.5

2

2.0

2.0

2

1.5 dry basis

1.5 dry basis

1.7 dry basis

2.25 dry basis

Ash (%, min)

–

–

7.50

7.50

–

–

–

–

–

7.5

-

–

–

–

–

–

Ash (%, max)

–

–

–

–

–

–

6.50

2.2

2.2

–

2.5

–

–

–

–

–

Max fat and ash

–

–

–

–

6.00

6.00

–

–

–

–

–

–

–

–

–

–

Lactose (%, max)

–

–

–

–

–

–

1.00

1

1

1.0

1.0

1.0

0.5

1.0

0.20

1

Max free acid

0.30

0.20

–

–

–

–

–

0.20

0.27

–

0.27

–

–

–

0.20

0.27

Note: Products may also be subject to microbiological testing including standard plate count, coliforms, salmonella, thermophiles, yeasts and moulds.

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11.3.2 Fractionation of caseins Industrial scale fractionation of caseins may be desirable for a number of reasons. Human milk contains - and -caseins but no -casein, making -casein an attractive ingredient for infant formulae. The high surface activity of -casein makes the protein a desirable emulsifier or foaming agent. As -casein is responsible for stabilising micelles, it may find application as a stabiliser in certain milk products. Following appropriate proteolysis caseins may give rise to biologically active peptides such as immunomodulating peptides, caseinomorphins from -casein, platelet-modifying peptides from -casein and angiotensin converting enzyme inhibitor from s1-casein.11 Commercial preparation of these peptides will require purified proteins. A number of methods for fractionating casein into -casein-rich and s-/-casein-rich fractions with potential for industrial scale application have been proposed. Essentially all of these methods are based on the influence of temperature on the association characteristics of the individual caseins. In a micellar casein system such as milk and in a dispersion of calcium caseinate and a solution of sodium caseinate, the individual casein molecules associate via a range of forces, including hydrophobic interactions. The strength of the hydrophobic interactions are dependent on temperature and the hydrophobicity of the protein molecules and at low temperatures (< ~ 5ºC) -casein molecules dissociate from s-/-casein complexes and exist in solution as monomers. Renneting of calcium caseinate at 4ºC to leave -casein in solution as monomers while s- and para--caseins coagulate, isolation of -casein by microfiltration of calcium caseinate at 5ºC, purification of -casein from whole casein at 4ºC and pH4.2–4.6, and ultrafiltration of a dilute sodium caseinate solution to produce a -casein-rich permeate and an s-/-casein-rich retentate have been described.8

11.3.3 Production of whey protein-enriched products Methods for the recovery of proteins from whey have been reviewed by Marshall (1982),12 Matthews (1984),13 IDF (1987),14 Morr (1989),15 Mulvihill (1992)8 and Mulvihill and Grufferty (1997).16 These methods are summarised in Fig. 11.3 and are outlined below. Whey is the liquid fraction of milk that remains following removal of fat and casein during the manufacture of cheese or acid and rennet casein. Sweet whey (minimum pH 5.6) is obtained from the manufacture of cheese or rennet casein while acid whey (maximum pH 5.1) is obtained from the manufacture of acid casein. Acid whey has a higher mineral/ash content than sweet whey, and if starter bacteria produced the acid by fermentation of lactose, the lactose concentration is reduced. Whey and whey protein-enriched solutions are normally pasteurised using minimum temperature and holding times and maintained at low temperature to minimise microbial spoilage and physico-chemical deterioration of the proteins and other whey constituents that would affect the functional and organoleptic properties of the resulting protein-enriched products. Compositions of typical whey-derived milk protein products are given in Table 11.6.

11.3.4 Whey powders and modified whey powders Whole whey powders containing less than 15% protein are produced by concentrating whey by evaporation alone or in combination with reverse osmosis followed by spray drying. Special methods are used to produce non hygroscopic, non-caking wettable whey powders. Following concentration to 50–60% solids concentrates are cooled to 30ºC,

Milk proteins

Fig. 11.3

197

Industrial isolation of protein products from whey.

seeded with finely ground -lactose monohydrate or well-crystallised whey powder, held for several hours and then cooled to 10ºC to crystallise lactose as -lactose monohydrate which is less hygroscopic than -lactose. The concentrate may then be dried by spraydrying or, more commonly, by multistage processes for improved functionality of the powders. In two-stage drying the pre-crystallised concentrate is spray dried at low temperature and post-crystallisation and drying takes place in a vibrating fluidised bed; in a three-stage process the drying process is modified. Another three-stage process involves atomisation, with primary drying, post-crystallisation and two drying stages on a conveyor belt system. The two- and three-stage processes produce large agglomerates of low bulk density that are non-hygroscopic and readily hydratable. The mother liquor obtained as a by-product of lactose manufacture may be concentrated and spray dried as a delactosed whey protein concentrate powder containing ~30% protein. Delactosed whey powder has a high mineral content (up to 25%) that may restrict its use in certain food applications, and affect its flavour and nutritional qualities. Demineralisation by reverse osmosis, electrodialysis or ion-exchange and/or lactose crystallisation to reduce the lactose and/or mineral concentration of whey is used to produce modified whey powders such as demineralised and demineralised-delactosed

Table 11.6

Compositions of typical whey-derived milk protein products USDA

Codex Alimentarius Stan A-151995

Codex Alimentarius Stan A-151995

Armor proteines

Armor proteines

Armor proteines

Dry whey

Acid whey

Whey powder

WPC

WPC

WPI

WPC

Foamarmor 375

Oragel HG 80

Protarmor 905

UF WPC 35

Hi-gel 45

WPC 30

US extra grade Protein (min) Moisture (max) Fat (max) Ash (max) Lactose (min)

Dairygold Dairygold Co-op Co-op Society Society

Glanbia plc Glanbia plc

WPC

DENA GmbH

WPI

Whey protein hydrolysate Hydrolac 80-2

11

10

11

37

80

89

34.0

44.0

30.5

90.0

68

5.0

5.0

5.0

5

6

5

4.5

4.5

4.0

5.0

6

1.50

2

2

1

7

1

4.0

4.0

4.0

0.5

1

–

15.0

9.5

8

7

2.5

–

6

61.0

61.0

49 (by difference)

0.0 (by difference)

1

Typically 6.7 Typically 40.3

6.5

–

Typically 6.5 Typically 50.5

53.0

1.0

–

Note: Products may also be subject to microbiological testing including standard plate count, coliforms, salmonella, thermophiles, yeasts and moulds.

Milk proteins

199

whey powders which contain 15–35% protein. By using a combination of processes the mineral profile of whey products can be carefully controlled.

11.3.5 Whey protein concentrate (WPC) production WPCs of varying protein concentrations are manufactured under mild conditions of pH and temperature by ultrafiltration (UF), a physico-chemical separation technique in which whey flows under pressure over asymmetric microporous membranes that facilitate the separation of small molecules such as lactose, salts and water as a permeate from whey proteins, fat globules and suspended solids, which are concentrated relative to other solutes in the retentate. In diafiltration (DF), dilution of retentate with water and repeated UF is used to increase the removal of membrane-permeable molecules. Following UF/DF the retentate is normally cooled to 4ºC and stored until sufficient volume is accumulated for spray drying. Pasteurisation or even UHT treatment of the retentate may also be necessary as bacteria and spores in the whey are also concentrated during UF/DF. Whey is commonly pre-treated prior to UF/DF processing by methods involving adjustments to temperature and/or pH, addition of calcium or calcium complexing agents and either quiescent standing, centrifugation or microfiltration to dissolve colloidal calcium phosphate and/or to remove insoluble cheese curd or casein fines, milkfat and calcium lipophosphoprotein complexes. These pre-treatments modify the properties of the whey protein concentrates, resulting in low fat products with improved functionality in some applications.

11.3.6 Whey protein isolate (WPI) production At pH values lower than their isoelectric point (~pH4.6), amphoteric whey proteins have a net positive charge and behave as cations while at pH values above their isoelectric point, the proteins have a net negative charge and behave as anions allowing fractionation by ion exchange processes. In one cation ionic exchange process whey is adjusted to pH < 4.6 with acid and stirred with a cellulose-based ion exchanger to allow protein adsorption. Lactose and other non-adsorbed materials are filtered off with the water and the resin is resuspended in water and the pH adjusted to > 5.5 with alkali to release the proteins from the ion exchanger. The solution of proteins is separated from the resin by filtration, concentrated by ultrafiltration and evaporation, and spray dried as WPI containing ~95% protein. In other processes ion exchangers in fixed-bed column reactors are used to achieve fractionation. Acidified whey at pH < 4.6 is applied to the Spherosil S cation exchanger to allow protein adsorption, lactose and other non-adsorbed solutes are eluted with water, following which the pH is raised by addition of alkali to elute adsorbed proteins. The protein-rich eluate is concentrated by UF and evaporation and spray dried to produce WPI. Sweet whey (pH > 4.6) is applied to the Spherosil QMA anion exchanger to permit adsorption of negatively charged protein molecules. Following elution of non-protein materials, the proteins are released by lowering the pH and the released proteins concentrated and spray dried to produce WPI. The products are characterised by high protein and low lactose and lipid concentrations and have good functionality. However, the need to concentrate and purify the dilute protein-containing eluate by UF, evaporation and drying, the excessive time requirement for conducting each fractionation cycle, the large volumes of washing and regeneration solutions used and microbial contamination of the reactor are major problems associated with these ion exchange processes.

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11.3.7 Lactalbumin production The globular whey proteins readily denature on heating to adopt more random structures that expose sulphydryl and hydrophobic groups facilitating aggregation and precipitation of the denatured proteins to extents dependent on heating temperature and holding time, pH and concentration of calcium. Commercial precipitation conditions depend on whey type and the desired final product characteristics and whey may be preconcentrated and/or demineralised prior to precipitation. The precipitated protein is recovered and washed to reduce mineral and lactose contents before drying using any of several types of drier to give a product referred to as lactalbumin (which should not be confused with the protein -lactalbumin). Lactalbumin containing up to 90% protein on a dry weight basis may be recovered, depending on precipitation pH and degree of washing.

11.3.8 Fractionation of whey proteins The compositions of bovine milk and human milk differ significantly. As human milk does not contain -lactoglobulin, which is in fact the most allergenic of the bovine milk proteins to the human infant, -lactalbumin appears to be more appropriate for use in preparing ‘humanised’ infant formulae. There is thus considerable commercial incentive for fractionating -lactalbumin and -lactoglobulin. Numerous methods with potential for commercial scale fractionation of whey proteins have been reviewed by Maubois et al. (1987),17 Mulvihill (1992)8 and Mulvihill and Grufferty (1997).16 Mild heat treatments of a whey concentrate or a clarified whey under controlled pH and ionic conditions or demineralisation of whey concentrate under controlled pH conditions can be used to effect selective reversible precipitation of -lactalbumin or -lactoglobulin enriched whey protein fractions. Following separation of the precipitate from the lactoglobulin or -lactalbumin enriched solutions, the precipitate is resolubilised by water addition and pH adjustment and then dried while the soluble protein is further concentrated by ultrafiltration/diafiltration before drying. On acidification of whey to pH < 5.0 -lactalbumin loses its calcium and is transformed into apo--lactalbumin. The apoprotein aggregates on heating to ~55ºC and can be separated from the soluble proteins by centrifugation, filtration or microfiltration. Both -lactalbumin and -lactoglobulin are insoluble in pure water at their isoelectric points; -lactoglobulin requires a higher ionic strength for solubility than -lactalbumin. This characteristic may be used to fractionate the proteins by acidifying UF-concentrated whey to pH4.65 and demineralising by electrodialysis to < 0.023% ash, whereupon lactoglobulin precipitates and may be recovered by centrifugation. Ion exchangers used to recover WPI may also be used to fractionate whey proteins. All the proteins in the whey are initially adsorbed on the resin but on continued passage of whey through the exchanger -lactoglobulin, which has a higher affinity for the resin than the other proteins, displaces bound -lactalbumin and serum albumin in an eluent rich in these proteins; highly purified -lactoglobulin is obtained by eluting the protein-saturated column using 0.1M HCl. Lactoperoxidase (LPO), a broad specificity peroxidase present at high levels in bovine milk but very low levels in human milk, is involved in antibacterial activity and as such is of commercial interest for applications such as cold sterilisation of milk, protection against mastitis and as a supplement for calf or piglet feeds to protect against enteritis.11 LPO is cationic at neutral pH and can be isolated from either milk or whey by industrial scale cation exchange chromatography. Lactotransferrin (Lf) binds iron strongly and is

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important in iron absorption and protection against enteric infection in neonates.11 As human milk contains considerably higher levels of Lf than bovine milk there is interest in supplementing bovine milk-based infant formulae with the protein. Like LPO, Lf is cationic at neutral pH and so adsorbs to the cation exchanger and can be eluted either together with LPO, or separately. Immunoglobulins (Ig, or antibodies), a class of proteins involved in the protection of mammals against infection, are present in mammary secretions. There is considerable interest in the use of Igs to enrich ruminant feeds and in the preparation of formulae for pre-term infants.11 Milk immunoglobulin concentrates may be prepared by ultrafiltration/diafiltration of acid whey from colostrum and early lactation milk of immunised cows, avoiding temperatures greater than 56ºC to prevent inactivation of the Igs. The glycomacropeptide (GMP) derived from -casein on renneting and present in cheese wheys contains no Phe, Tyr, Trp, Lys or Cys residues, making it suitable for the nutrition of patients with phenylketonuria. One production method involves passing whey through an ion exchanger, the GMP is in the non-adsorbed fraction which is desalted and concentrated. Another method involves selective adsorption of GMP on an ion exchange resin and recovery by desorption using dilute acids or salt solutions. UF combined with heat processing, and heating delactosed whey to induce protein precipitation and subsequent recovery of GMP from the supernatant by ethanol precipitation have also been used to prepare GMP.

11.3.9 Miscellaneous methods of casein and co-precipitate manufacture Whey proteins can be co-precipitated with casein by first heating milk, at its natural pH, to temperatures that denature the whey proteins and induce their complexation with casein, followed by precipitation of the milk protein complex by acidification to pH4.6 or by addition of CaCl2 combined with variable levels of acidification. Such products, referred to as high-, medium- or low-calcium casein-whey protein co-precipitates based on calcium content, generally exhibit poor solubility. Methods for preparing similar products with good solubility have been described. Total milk protein (TMP) was prepared by heating skim milk to 40–70ºC, alkalising to pH9.5–10.5 and holding for 3 min. followed by acidification to pH3.5 and holding for 5 min. before isoelectric precipitation at pH4.7. Soluble lacto-protein (SLP) was prepared by adjusting the pH of skim milk to 7–7.5, heating to 80–145ºC for periods of several seconds to 20 min., cooling to 4–45ºC and isoelectric precipitation of the protein. The curd was separated, washed, dispersed in water, dissolved at pH6.7 and spray-dried. Methods for separating casein micelles from all the other constituents of skim milk utilising membrane filtration have been developed at laboratory level but have not yet been scaled up for industrial use.

11.3.10 Production of milk protein concentrate Skim milk may be processed by UF/DF to yield milk protein concentrates (MPC) having protein contents up to ~80% and in which the casein is in a similar, micellar, form to that found in milk while the whey proteins are also reported to be in their native form. Since protein-bound minerals are retained the ash contents of these products are relatively high.

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Functional properties of milk protein products

As the utilisation of many milk protein products in foods is dependent on their physicochemical and functional properties, a brief overview of their major functional characteristics is included here. Fox and Mulvihill (1983),18 Kinsella (1984),19 De Wit (1989a),20 Mulvihill and Fox (1989)21 and Mulvihill (1992)8 provide more extensive reviews on these topics.

11.4.1 Solubility Acid casein is completely insoluble when dispersed in water as the pH of the dispersion is close to caseins isoelectric pH, i.e. pH4.0–5.0; when the pH of the dispersion is raised to values > 5.5 by addition of monovalent cation base the casein is converted to a completely soluble cationic (Na, K, NH3) salt. Solutions containing 10–15% of these latter caseinates can be easily prepared at pH6.0–7.0. At pH values < 3.5, casein is also soluble but at this pH the solution is more viscous than at neutral pH values and gel-like systems are formed. Rennet casein is also insoluble in water at pH ~7.0, but can be solubilised either by raising the pH above ~9.0 or by adding calcium chelators such as food-grade polyphosphates and/or citrates. Sodium salt forms of conventional co-precipitates are also largely insoluble at pH6.0– 7.0, but the sodium salt forms of casein-whey protein co-precipitates prepared from milk heated at alkaline pH values (SLP and TMP) possess solubility characteristics similar to caseinates. Calcium caseinates and medium and high calcium co-precipitates form coarse colloidal dispersions rather than solutions. Whey proteins in their native globular conformation are soluble at low ionic strength over the entire pH range encountered in food applications. Their solubility decreases at high salt concentrations due to salting out and they may also become insoluble following thermal denaturation at temperatures above 70ºC. The level of denaturation and subsequent insolubility at pH7.0 and at pH4.6 (an index of the extent of denaturation caused by processing and storage of protein-rich whey products) depends on heating temperature and time, and whey pH and ionic calcium concentration on heating.

11.4.2 Gelation and coagulation As already stated, milk undergoes gelation when subjected to a number of treatments and usually casein is the gelling component involved. Limited proteolysis of milk to hydrolyse the micelle-stabilising -casein produces para--casein-containing micelles that coagulate at the concentration of Ca2+ in the milk serum. Casein micelles may also be destabilised to form gels or precipitates on mixing equal volumes of milk and 80%, v/ v, ethanol. Acid induced gelation or coagulation of milk produces acid gels that may or may not expel whey (synerese), depending on the pre-heat treatment of the milk. Preheating to temperatures greater than the denaturation temperature of the whey proteins (usually > 85ºC) reduces syneresis in fermented milks while milk used for the production of acid cheeses and caseins is heated as little as possible to promote whey expulsion. The viscosity of casein is much higher at low pH (2.5–3.5) than at neutral pH and gellike structures are formed at > 5% protein at temperatures below 40ºC. Calcium caseinate is the only milk protein system reported to exhibit reversible thermal gelation. Concentrated calcium caseinate dispersions (> 15% protein) gel on heating to 50–

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60ºC, on cooling the gel slowly liquefies but reforms on heating. Gelation temperature increases with protein concentration from 15–20% and with pH in the range 5.2–6.0. Thermal sensitivity is generally undesirable in the preparation of soluble whey protein-enriched products, however, the property can be exploited for production of thermal gels from whey proteins, which have excellent thermal gelling properties. Characteristics of the whey protein product such as method of production, the extent of whey protein denaturation during manufacture, the contents of protein, total ash, selected minerals and other non-protein components all influence the minimum protein concentration and heating regime required for thermal gelation and characteristics of the gel formed, such as opacity, strength and elasticity or brittleness. Solution conditions such as pH, ionic species present, other non-protein components added and the presence of reducing agents also influence the gelation characteristics. WPCs and WPIs possessing different gelling properties can be obtained by careful selection of whey type and manipulation of processing conditions during manufacture.

11.4.3 Hydration properties Water binding or hydration is an important functional property of dairy proteins in food applications. The level of hydration depends on the particular products; reported hydration values for casein micelles range from 1.4 to 6.4g H2O/g, for casein and caseinate samples range from 0.7 to 3.8g H2O/g and for individual native whey proteins range from 0.32–0.60g H2O/g. However, when whey protein solutions of sufficient protein content and suitable solution conditions (pH, ions, etc.) are heated, gels are formed and the water holding capacity of such gels make significant contributions to the texture and rheology of a number of processed foods. Reported values for the water sorption capacity of several milk protein products in a model flour dough system range from 0.96–3.45g H2O/g of product.

11.4.4 Viscosity Caseinates form highly viscous solutions at concentrations > ~ 15% owing to hydration, swelling and polymer-polymer interactions. The viscosity of solutions containing > ~ 20% protein is so high that processing is difficult even at high temperatures. The viscosity of sodium caseinate increases logarithmically with increasing concentration, while there is a linear relationship between log viscosity and the reciprocal of absolute temperature. Sodium caseinate viscosity is also very pH-dependent, with minimum viscosity occurring at ~pH 7.0. The viscosity of casein is much higher at low pH (~2.5– 3.5) than at neutral pH, and gel-like structures are formed at > 5% protein at temperatures below 40ºC. Caseinates exhibit pseudoplastic rheological behaviour and are thixotropic at high shear rates. The cation present also has a significant effect on the viscosity of caseinate. The viscosity of caseinate containing 1% Ca shows unusual temperature dependence as it decreases sharply in a curvilinear fashion with increasing temperature from 30–38ºC, then remains constant up to ~57ºC, above which the solution gels at pH5.4 but not at higher pH values. The relationship between viscosity and temperature depends on protein concentration, pH and [Ca2+]. Low levels of added calcium increase the viscosity of sodium caseinate above pH7.0 but below this pH and at sufficient calcium addition micelle formation tends to decrease the viscosity. Manufacturing conditions also affect the viscosity of casein/caseinates. Excessive heating of milk prior to casein manufacture or of casein curd during drying ultimately

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leads to increased caseinate viscosity. Precipitation at pH values lower than normal (e.g. ~3.8) and especially at higher pH values (e.g. ~5.05) also increases the viscosity of caseinates. Roller dried caseinates generally exhibit higher viscosities than spray dried caseinates. Solubilised conventional co-precipitates are more viscous than sodium caseinate and their viscosity increases with increasing calcium concentration, while solutions of total milk protein have viscosities intermediate between those of sodium caseinate and conventional co-precipitates. Solutions of non-denatured whey proteins are much less viscous than caseinate solutions. They exhibit minimum viscosity around their isoelectric point (pH4.5) and relative to water, their viscosity decreases between 30–65ºC, but increases at higher temperatures as a result of thermal denaturation of the globular proteins. WPC solutions containing 4–12% protein were reported to exhibit Newtonian flow while at higher concentrations flow became more pseudoplastic and at 18–20% yield values were observed.

11.4.5 Surface active properties Milk proteins are strongly amphipathic and exhibit good surface active properties: they adsorb readily at surfaces/interfaces decreasing surface tension and forming surface/ interfacial films with variable rheological properties. The order of surface activity reported for the individual milk proteins is -casein > monodispersed casein micelles > serum albumin > -lactalbumin > s-casein = -casein > -lactoglobulin > euglobulins. Sodium caseinate depresses interfacial tension more effectively than whey protein, blood plasma, gelatin or soy protein as it diffuses more quickly to an interface and on reaching the interface adsorbs more quickly than the other proteins. Partial heat denaturation of whey proteins enhances their surface activity. Hydrolysis of sodium caseinate by plasmin (to produce -caseins and proteose peptones) greatly increases its surface activity. The

2 - and 3 -caseins are small, very hydrophobic peptides and thus are very surface active. Surface films of sodium caseinate or -casein are much more flexible and less viscoelastic at both oil/water and air/water interfaces than films of -lactoglobulin, lactalbumin or bovine serum albumin.

11.4.6 Emulsifying and foaming properties Milk protein products in general, and caseinates especially, are very good fat emulsifiers and are widely used in emulsifying applications in foods. Sodium caseinate stabilised soybean oil emulsions, prepared in a valve homogeniser, exhibited lower creaming stabilities than similar emulsions stabilised by either WPC or soy isolate. Highly dispersed sodium, ammonium and low-calcium caseinates showed higher emulsifying capacities than more aggregated high-calcium caseinate and ultracentrifugal (micellar) caseins, while the emulsions formed using the latter were more stable than those stabilised by the highly dispersed caseinates. For all the proteins studied the fat surface area formed on emulsification increased (i.e. globule size decreased) with increased power input during emulsification and the extent of the increase was inversely related to the degree of aggregation of the emulsifying caseins/caseinates. The emulsions formed using aggregated caseins/caseinates had greater protein loads at the interface (mg/m2) than for the dispersed caseinates and protein load was directly related to emulsion stability. Caseinates generally produce foams with higher overruns but lower stability

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205

than foams produced using egg white or WPC and WPI. Whey protein-enriched products are widely used in foaming applications in foods and factors such as protein concentration, level of denaturation, ionic environment, pre-heat treatment and especially the presence of lipids in the WPC or WPI influence whipping properties.

11.5

Food uses of milk protein products

Details of many of the food uses of milk protein products are proprietary to milk protein product producers and food processors and are not reported in the literature. However, reviews on the food uses of milk proteins include Southward and Goldman (1978),22 International Dairy Federation (1982),23 Southward and Walker (1982),24 Otten (1985),25 Zadow (1986),26 Hugunin (1987),27 De Wit (1989b),28 Southward (1986, 1989),29,30 Mulvihill (1992)8 and Mangino (1992).31 Milk protein products have also seen applications as diverse as animal feed ingredients, plastics and industrial glues30 but these uses are not considered here. The following are brief outlines of some reported food applications of milk protein products.

11.5.1 Bakery products Milk proteins cannot replace wheat gluten to any great extent in bakery products. Caseins are particularly rich in lysine and so make excellent nutritional supplements for cereals, which are deficient in lysine. Casein/caseinates are added to breakfast cereals, milk biscuits, protein-enriched bread and biscuits, high-protein bread and cookies as a nutritional supplement and to frozen baked cakes and cookies as an emulsifier and to improve texture. The type of casein/caseinate must be carefully chosen to be compatible with the particular bakery application. Co-precipitates are used in pastry glaze to improve colour; in milk biscuits, cake mixes for diabetics, high-protein biscuits and cookies as a nutritional supplement and in fortified bread to improve dough consistency (due to water binding by the milk proteins), sensoric properties and to increase volume and yield. However, some milk fraction has been described as loaf volume-depressing.32 Depression of loaf volume by whole whey protein products was associated with proteose peptones since fortification of dough with 1% UF-WPC caused little loaf volume depression. The concentration of whey lipids during UF also contributes to good baking characteristics. Whey protein shows economic and nutritional advantages as a replacement for eggs in cake manufacture. However, simply replacing whole eggs by WPC in madeira-type cakes results in poor quality cakes although better results are obtained if the fat and WPC are pre-emulsified. Various WPCs have been used in products like muffins and croissants to increase their nutritional value. Bakery applications of milk protein products are outlined in Table 11.7.

11.5.2 Dairy products Milk protein products are used to supplement the protein content in conventionally processed dairy products and in the manufacture of imitation dairy products (Table 11.8). Caseins, vegetable fat, salts and water are used to make imitation cheeses (cheese analogues), which result in significant cost-saving, compared to the use of natural cheese, when used in pizza, lasagne and sauces and on burgers, grilled sandwiches, macaroni, etc. The important functional properties of casein in this application include fat and water

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Table 11.7

Application of milk protein products in baked products

Milk protein product

Application

Effect/property

High-calcium co-precipitate

Pastry glaze, cake mix for diabetics, milk biscuits, cookies Cookies Breakfast cereal, high-protein bread Milk biscuits, biscuits Frozen baked cake, protein-enriched milk biscuits Shortening Pie filling Powdered friable fat High-protein biscuit, fortified bread

Colour, shine, nutrition, cake volume, texture, appearance Nutrition, texture, appearance

Low-calcium co-precipitate Casein Calcium caseinate Sodium caseinate

Co-precipitate

Lactic casein Acid casein

Cookies Non-fat dry milk substitute

WPC

Cookies, muffins, croissants Bread Cake

Nutrition Nutrition Texture, emulsifier, nutrition Fat encapsulation Stabiliser Fat stabiliser Dough consistency, sensory, increase volume/yield, fast fermentation Nutrition, texture, appearance Structure building in dough, nutrition, flavour Water retention, colour, emulsifier, nutrition Dough formation Fat binding, heat setting

binding, texture enhancing, melting properties, stringiness and shredding ability. Rennet caseins, acid caseins and caseinates are used most commonly for cheese analogues although co-precipitates also have potential in this area. Sodium caseinate in powdered coffee creamers (which also contain vegetable fat, carbohydrate and emulsifiers/stabilisers) acts as an emulsifier/fat encapsulator and whitener, imparts body and flavour and promotes resistance to feathering (i.e. coagulation of cream in hot coffee solutions). These creamers are cheaper, have a longer shelf life and, requiring no refrigeration, are more convenient to use than fresh coffee creams. Sodium caseinate is used to reduce syneresis and increase gel firmness in yoghurts, and is added to milk shakes for its emulsifying and foaming properties. Caseins/caseinates, vegetable fat and carbohydrate, e.g. corn syrup, are the principal ingredients used in the manufacture of low cost imitation milk products that contain no lactose, to which some people are intolerant. Sodium caseinate is also used as an emulsifying and fat encapsulating agent in the manufacture of high-fat powders for use as shortenings in baking or cooking. Dry whipping fats or whipping creams contain casein products while a number of butter-like dairy spreads are manufactured using milk and/or vegetable fat and various casein products. In these applications casein acts mainly as an emulsifier and in the case of dairy spreads, it also enhances texture and flavour. Whey protein products are used in yoghurts and cheeses to improve the yield, nutritional value and consistency. Yoghurt viscosity and stability are improved by replacing skim milk solids with WPC. Up to 20% of the casein in Quarg can be replaced by thermally-modified WPC to improve the yield and nutritional value. The use of sweet

Milk proteins Table 11.8

207

Application of milk protein products in dairy-type products

Milk protein product

Application

Effect/property

Calcium caseinate

Processed cheese spread, imitation Mozzarella cheese, imitation cream cheese

Spreadability, stretch, browning, emulsifier

Sodium caseinate

Coffee creamer, UHT cream, nondairy creamer, imitation sour cream, cultured cream

Emulsifier, whitener, texture, body, resistance to feathering, sensory characteristics, water binding, viscosity, flavour Reduce syneresis, increase gel firmness, stabiliser, consistency Emulsifier, nutrition, foaming properties, increase yield Stretch, browning, emulsifier

Yoghurt, fruit yoghurt Imitation milk, milk shakes, cheese milk Imitation Mozzarella cheese, imitation cream cheese Ice cream products Dairy-based spreads, butter-type spread, butter powder Whipping composition for drinking

Consistency, flavour, foaming, water binding, aroma Texture, emulsion stabiliser Foaming

Co-precipitate

Fat-reduced milk Spread-type dairy products Cultured milk product

Nutrition Texture Increase biological value

Potassium caseinate

Fat-reduced milk

Nutrition

Hydrolysed casein

Ice cream Yoghurt

Overrun Growth media for starter

Acid casein

Simulated cheese, cheese analogue, imitation milk

Meltability, stringiness, cost, texture, meltability, nutrition, stability

Rennet casein

Processed cheese, Mozzarella substitute, cheese analogue, cheese-like spread

Emulsifier, meltability, stringiness on melting, texture, flavour

WPC

Soft serve ice cream Cheese products

Overrun, cost, Water and fat binding, cost, emulsification Reduce lactose Casein/caseinate replacer

Yoghurt Coffee whiteners Whey proteins

Yoghurt, Quarg, Ricotta

Yield, nutrition, consistency, curd cohesiveness

UF-WPC in Ricotta cheese manufacture increases the cohesiveness of the curd. Emulsions prepared using heat-denatured whey proteins and fat are used as a protein base for formulated cream cheeses and cream cheese spreads. Sliceable and squeezeable cheese-type products, based on the emulsifying and gelling properties of whey proteins, are produced by heat treatment of skim milk and WPC solids dispersed in an emulsion of milk fat in WPC. Whey protein concentrates are also used in cheese filling and dips as they complement the cheese flavour and result in a soft product.

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11.5.3 Beverages Casein products are used for their whipping and foaming properties or as stabilisers in drinking chocolate, effervescent drinks and beverages (Table 11.9). Sodium caseinate is used as an emulsifier and stabiliser in cream liqueurs, which typically contain cream, sodium caseinate, added sugar, ethanol, and trisodium citrate to prevent calcium-induced age gelation; it is also used to a lesser extent in other aperitifs. Casein products have also been used as fining agents, to decrease colour and astringency and to aid in clarification in the wine and beer industries. WPCs may be added to fruit juices, soft drinks or milk-based beverages to produce highly nutritious products often marketed as ‘sports drinks’. For use in soft drinks, defatted WPC with a low ash content, good solubility at pH 3.0 and a bland flavour are required. The WPC must also be resistant to physical deterioration or flavour changes during product storage and must not interact with flavour components and thereby mask the typical flavour of the drink. WPCs and WPIs are added to milk-like flavoured drinks to impart viscosity, body and colloidal stability and they are included as protein supplements in high protein powdered athlete-targeted flavoured beverages and in frozen juice concentrates.

11.5.4 Dessert-type products Sodium caseinate is used in ice cream substitutes and frozen desserts to improve whipping properties, body and texture and to act as a stabiliser and also finds use in mousses, instant puddings and whipped toppings for similar reasons and because it acts as an emulsifier and film-former (Table 11.10). In the manufacture of whipped toppings the basic ingredients of vegetable fat, sugar, protein (sodium caseinate), emulsifier, stabilisers and water are blended at 38–46ºC and the mixture is pasteurised and homogenised and then either cooled rapidly to below freezing point or spray dried. In ice cream manufacture, part of the skim milk solids can be replaced by whey powder, and even more may be replaced by Table 11.9

Application of milk protein products in beverages

Milk protein product

Application

Effect/property

Casein

Beer, wine

Clarification, colour removal, stabiliser, palatability, colour stability, removal of phenolic compounds Stabiliser

Effervescent lemonade ingredient Potassium caseinate

White wine

Removal of tannins and phenolic compounds, taste, colour removal

Sodium caseinate

Apple juice Cream liqueur, alcoholic creamcontaining beverage, wine aperitif Soluble tea products Drinking chocolate

Colour removal Emulsification

Casein hydrolysate

Non-alcoholic fruit beverage

Whippability, foaming

WPC

Citrus-based beverages, soft drinks Hot cocoa beverages, chocolate drink

Flavour, nutrition, solubility Foamer, cost, colloidal stability

Prevention of ‘tea cream’ Stabilisation

Milk proteins Table 11.10

209

Application of milk protein products in dessert-type products

Milk protein product

Application

Effect/property

Sodium caseinate

Whipped dessert, whipping fat, whipped topping, mousse, ice cream frozen dessert, frozen puddings, instant dessert/pudding bases

Whippability, fat encapsulation, overrun, powder flow properties, replace milk solids, emulsifier, stabiliser, flavour, texture

Caseinate

Spongy dessert

Whippability, aeration

Hydrolysed sodium caseinate

Whipped topping

Freeze-thaw stability

WPC

Fruit jellies and jams Frappes, whipped toppings, frozen desserts/puddings Flan-style desserts, custards

Flavour Overrun, cost, whippability Gelling ability

using delactosed, demineralised whey powder or UF-WPC with no adverse effect on flavour, texture or appearance. WPC has also been used in frozen juice bars and in compound coatings, especially chocolate coatings, for frozen desserts.

11.5.5 Pasta products Milk protein products may be incorporated into the flour base for pasta manufacture to improve nutritional quality and texture (Table 11.11). Products fortified by addition of sodium or calcium caseinate, low calcium co-precipitate or WPC prior to extrusion include macaroni and pasta. Enrichment of pasta flours with non-denatured whey protein products results in firmer cooked noodles which are also more freeze-thaw stable and suitable for microwave cooking. Imitation pasta-type products containing substantial proportions of milk protein have also been manufactured.

11.5.6 Confectionery Whey proteins are suitable for use in aerated candy mixtures and are incorporated as a frappe, a highly aerated sugar syrup containing the whipping protein (Table 11.12). Table 11.11

Application of milk protein products in pasta products

Milk protein product

Application

Effect/property

Sodium caseinate

Protein enriched pasta

Nutrition, consistency, binder, texture, taste, appearance

Calcium caseinate

Enriched wheat macaroni, high protein pasta

Nutrition, texture

Casein

Enriched, fortified macaroni

Nutrition, texture

Soluble low-calcium co-precipitate

Imitation rice

Nutrition, texture

WPC

Pasta

Flour replacer, nutrition

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Table 11.12

Application of milk protein products in confectionery products

Milk protein product

Application

Effect/property

Sodium caseinate

High protein chocolate snack, confectionery bars, Aerated cake icing

Nutrition, storage stability, flavour, aeration, body, mouthfeel, texture

Hydrolysed sodium caseinate

Coated confectionery product

Nutrition

Calcium caseinate

Aerated cake icing

Aeration, body, mouthfeel, texture

Hydrolysed casein

Aerated confection

Whippability

Co-precipitate

Protein-rich chewable bar

Nutrition, texture

Hydrolysed whey protein

Aerated confection

Whippability

WPC

Protein-rich chewable bar Frappe, meringue

Nutrition, texture Whippability, foam stability

Whey protein

Coated confectionery product

Nutrition

Caseins are used in toffee, caramel, fudge and other confections as they form a firm, resilient, chewy matrix on heating and they contribute water binding and aid emulsification. WPCs are less useful in these products as they produce a softer coagulum and the high lactose content may cause crystallisation during storage. Casein hydrolysates may replace egg albumen as foaming agents in marshmallow and nougat as they confer stability up to high cooking temperatures as well as good flavour and browning properties. Use of WPC or WPI as a replacer of egg white in the manufacture of meringues produces acceptable products only when defatted products are used; in contrast the manufacture of acceptable sponge cakes requires fat-containing WPCs.

11.5.7 Meat products In comminuted meat products caseins release meat proteins for gel formation and water binding and thus contribute to fat emulsification, water binding and improved consistency. Sodium caseinate is a common additive in meat applications although various co-precipitates have also been used (Table 11.13). Up to 20% of the meat protein in frankfurters and luncheon rolls may be replaced by whey proteins, which are used to prepare pre-emulsions of part of the fat and support network formation, by gelation, during subsequent cooking. Soluble, low viscosity WPCs may be used in injection brines to fortify whole meat products such as cooked hams. Injection of fresh and cured meats with milk protein solution increases yield.

11.5.8 Dietary and medical applications Milk protein products are used extensively in special dietary preparations for the ill or convalescing, for malnourished children and for people on weight-reducing diets (Table 11.14). Modified low-mineral whey powders are used to produce ‘humanised’ infant formulae with a whey protein-to-casein ratio resembling that of human milk. Whey protein hydrolysates have been used in hypoallergic, peptide-based formulae. Fractiona-

Milk proteins Table 11.13

211

Application of milk protein products in meat products

Milk protein product

Application

Effect/property

Sodium caseinate

Liver sausage, sausage, blood, patties, low fat meat paste

Binder, decolouriser, emulsifier

Potassium caseinate

Low fat meat paste

Emulsifier

Sodium salt of co-precipitate

Pate´ sausage

Nutrition, sensory, cost

WPC

Meat products

Cost, improved performance, water and fat binding, water solubility at low viscosity

Table 11.14

Application of milk protein products in dietary and medical applications

Milk protein product

Application

Effect/property

Sodium caseinate

Candy for space feeding, Carnation SlenderÕ, enriched dairy drink for infants, meat replacement

Nutrition

Calcium caseinate

Bakery products for diabetics Meat replacement, infant dietary food

Flour substitute Nutrition

Casein

Water dispersible protein, special dietary foodstuffs Toothpaste

Nutrition, texture

Acid casein

Low-sodium infant formula

Nutrition

Co-precipitate

Carbohydrate-free and low-lactose infant food

Nutrition

High-calcium co-precipitate

Cake mix for diabetics

Nutrition, cake volume

WPC

Geriatric/hospital/liquid diets Infant formulae

Nutrition Nutrition, digestability

Prevent caries

tion of whey proteins allows the formulation of infant formulae that possess whey protein compositions more closely resembling that of human milk, -casein and -lactalbumin enriched protein fraction together with lactotransferrin are being considered as ingredients for the production of the next generation of more ‘humanised’ infant formulae. Milk protein hydrolysates are used for intravenous nutrition for patients suffering from protein metabolism disorders, intestinal disorders, and for post-operative patients. Caseins are used in special preparations to enhance athletic performance and have been incorporated into formula diets for space feeding. Caseinates and co-precipitates are used in low-lactose formulae for lactose-intolerant infants while selected caseinates are used in the production of infant foods where a specific mineral balance is required, e.g., low-sodium infant formulae for children with specific renal problems. Casein hydrolysates are used in specialised foods for premature infants and in formulae for

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infants suffering from various intestinal disorders, while a special casein hydrolysate, low in phenylalanine, has been prepared for use in formulae for feeding infants with phenylketonuria. Casein products are also added to various foods and drinks as a nutritional supplement. Special casein preparations have been used as food for patients suffering from cancer, pancreatic disorders or anaemia. Specific peptide-based drugs have been produced from -casein including -caseinomorphins, and tetra- to heptapeptides which can regulate sleep, hunger or insulin secretion. Sulphonated glycopeptides prepared from casein have been used for the treatment of gastric ulcers, and the use of casein in toothpaste is claimed to prevent dental caries.

11.5.9 Convenience foods Applications of milk protein products in convenience foods are outlined in Table 11.15. Whey/caseinate blends are used as whitening agents in gravy mixes. Whey solids are included in dehydrated soup mixes and sauces to impart a dairy flavour, to enhance other flavours and to provide emulsifying and stabilising effects. Caseinates are used as emulsifying agents and to control viscosity in canned cream soups and sauces and in the preparation of dry emulsions for use in dehydrated cream soups and sauces. Sauces and gravies containing whey proteins are reportedly less prone to cook-on to utensil walls, require minimum agitation and are stable to freeze-thaw cycling. Caseinate-whey protein blends are used as cheap replacements for skim milk powders in some convenience foods. Whey protein products may replace egg yolk in salad dressing and modified whey protein-based products potentially able to replace lipids in a variety of convenience foods have been developed. Milk protein products have been proposed as texture, stability and flavour enhancers in microwaveable foods.

Table 11.15

Application of milk protein products in convenience foods

Milk protein product

Application

Effect/property

Sodium caseinate

Dry cream product for sauces, soups, nut substitute, imitation potato skin shells

Emulsification, nutrition, texture

Caseinate

Nut-like food Gravy mix

Film formation Whitening agent

Casein

Synthetic caviar

Texture

Hydrolysed casein

Whipping mixture

Whippability

Co-precipitate

Potato soup with rice Vegetable cutlets

Emulsification, nutrition, texture

WPC

Salad dressing, egg replacer

Viscosity, mouthfeel, emulsification Cost, extender Cost

Quiches, egg replacer Cream-based soups Whey solids

Gravy mix, dehydrated soup mix

Whitener, flavour, emulsifier, stabiliser

Milk proteins Table 11.16

213

Application of milk protein products in textured products

Milk protein product

Application

Effect/property

Casein

Puffed food

Emulsification, texture, nutrition

Acid casein

Extruded milk protein product Foamed snack bar

Texture

Potassium caseinate

Fine bread, biscuits

Texture

Sodium caseinate

Sucroglyceride for baking

Texture, handling

Rennet casein

Dietary fibre snack

Texture

Co-precipitate

Dietary fibre snack

Texture

Whey protein

Dietary fibre snack

Texture

WPC

Surimi

Texture, cost

11.5.10 Textured products Rewetted acid caseins or acidified rennet casein or co-precipitate, mixed with carbonates or bicarbonates of alkali metals or alkali earth metals, can be extruded to produce puffed snack foods while caseinates can be co-extruded with wheat flour to produce proteinenriched snack-type food products (Table 11.16). Fibrous meat-like structures can be formed from caseins, using fibre spinning techniques, and can be used as extenders in comminuted meats. If whey proteins are co-spun with the casein, fibres stronger than those containing casein alone are produced. Meat-like structure can also be formed from casein or co-precipitates by renneting followed by a combination of heat treatment and extrusion or working. Microwave heating of whey protein solutions results in simultaneous expansion and gelation to give textured products with potential for use in comminuted meats. WPCs are proposed as cost effective replacers for beef plasma protein or potato starch in the modification of surimi texture.33 11.5.11 Films and coatings Films formed from caseins/caseinates may be water soluble or water insoluble depending on the pH conditions used in their preparation, while the water vapour permeability of the film depends on the type of casein/caseinate used. Thermally induced disulphide crosslinking was found to be necessary when making films using WPCs and WPIs. The WPCbased films were excellent gas barriers, while the water vapour permeability of films could be reduced by incorporating lipids. Tensile strengths of the films were similar to synthetic films, and were enhanced by enzymatic polymerisation using e.g. transglutaminase. The films were generally flavourless, and transparent to translucent depending on protein source. Calcium-caseinate based emulsions applied to fruit and vegetables were used to reduce moisture loss. The potential for the use of milk proteins in films and coatings in food applications has been discussed by Chen (1995).34

11.6

Future developments

Although a large range of milk protein products are presently recovered from milk it is likely that this range will be extended in the future. Methods for fractionation of whey to

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produce individual proteins on a commercial scale are at early stages of development, while commercial scale methods for the production and isolation of biologically active peptides derived from caseins remain to be developed. Tailoring of milk protein products to meet specific functional requirements for individual applications will become more important. It is likely that enzymatic and physical modification methods will be applied to confer new physico-chemical and functional properties to milk protein products. The use of milk protein products in emerging technologies such as the manufacture of edible films34 and microencapsulation35 of ingredients will benefit from further development. Use of biologically active proteins and peptides in dietary, pharmaceutical and medical products has potential to become more widespread as consumers preference for a healthy lifestyle grows.

11.7

Sources of further information

For more detailed discussions of milk proteins, their manufacture, functional properties and uses the following are recommended: 39

FOX, P. F. (ed.) Developments in Dairy Chemistry – 1. London, Applied Science Publishers, 1982. 40 FOX, P. F. (ed.) Developments in Dairy Chemistry – 4. London, Elsevier Applied Science Publishers, 1989. 41 ZADOW, J. G. (ed.) Whey and Lactose Processing. London, Elsevier Applied Science Publishers, 1992. 42 CHANDAN, R. Dairy-based ingredients. St. Paul, Minnesota, Eagan Press, 1997.

Further information regarding regulatory requirements for milk protein products (and milk products in general) may be obtained from the following: American Dairy Products Institute 300 West Washington Street, Suite 400 Chicago, Illinois USA Fax + 1 312 782 5299

USDA/AMS Dairy Standardisation Branch Room 2750-South Building PO Box 96456 Washington, DC USA Fax + 1 202 720 2643

Food and Agriculture Organisation of the United Nations (Codex Alimentarius) Viale delle Terme di Caracalla 00100-Rome Italy Fax + 39 06 5705 4593

International Dairy Federation 41, Square Vergote 1030 Brussels Belgium Fax + 32 2733 0413

Information on milk protein products may be obtained from the following manufacturers and suppliers: Armor Proteines Le Pont 35460 Saint Brice en Cogles France Fax + 33 2 99 97 7991

Dairygold Food Ingredients West-end Mallow Co. Cork Republic of Ireland Fax + 353 (0) 22 21279

Milk proteins

215

Kerry Ingredients Ireland Tralee Road Listowel Co. Kerry Republic of Ireland Fax + 353 (0) 68 21562

Glanbia (formerly Avonmore-Waterford Group) Ballyragget Co. Kilkenny Republic of Ireland Fax + 353 (0) 33268

Golden Vale Plc. Charleville Co. Cork Republic of Ireland Fax + 353 (0) 63 35001

Swiss Milk Company Ltd. 6281 Hochdorf Switzerland Fax + 41 41 910 1313

DMV International PO Box 13 5460 BA Veghel The Netherlands Fax + 31 413 362 656

Carbery Food Ingredients Ballineen Co. Cork Republic of Ireland Fax + 353 (0) 23 47541

MD Foods Ingredients Head Office Skanderborgvej 277 DK-8260 Viby J. Denmark Fax + 45 8628 1838

New Zealand Dairy Board PO Box 417 Wellington New Zealand Fax + 64 4471 8600

Century Foods International PO Box 257 919 Hoeschler Drive Sparta, Wisconsin 54656 USA Fax + 1 608 269 1910

DENA GmbH Villa Flora Oberkasseler Str. 26 D-40545 Dusseldorf Germany Fax + 49 211 55 55 83

11.8 1. 2. 3. 4. 5. 6. 7.

References

ed. (1989) ‘The milk protein system’ in Developments in Dairy Chemistry – 4. London, Elsevier Applied Science Publishers, pp. 1–53. FOX, P. F. ed. (1992) Advanced Dairy Chemistry – volume 1 London, Elsevier Applied Science Publishers. HOLT, C. (1992) ‘Structure and stability of bovine casein micelles’ Adv. Protein Chem. 43 63–151. CREAMER, L. K. and MACGIBBON, A. K. H. (1996) ‘Some recent advances in the basic chemistry of milk proteins and lipids’ International Dairy J. 6 539–68. MULLER, L. L. (1971) ‘Manufacture and uses of casein and co-precipitate’ Dairy Science Abstracts 33 659–74. MULLER, L. L. (1982) ‘Manufacture of caseins, caseinates and co-precipitates’ in Developments in Dairy Chemistry – 1. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 315–37. MULVIHILL, D. M. (1989) ‘Caseins and caseinates: manufacture’ in Developments in Dairy Chemistry – 4. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 97–130. FOX, P. F.

216 8.

9. 10.

11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21.

22. 23. 24.

25. 26.

27. 28. 29.

30. 31. 32. 33. 34. 35.

Handbook of hydrocolloids (1992) ‘Production, functional properties and utilization of milk protein products’ in Advanced Dairy Chemistry – volume 1. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 369–404. FOX, P. F. and MULVIHILL, D. M. (1990) ‘Casein’ in Food Gels. P. Harris, ed. London, Elsevier Applied Science Publishers, pp. 121–74. MILLAUER, C., WIEDMANN, W. M. and STROBEL, E. (1984) ‘A new caseinate manufacturing process for high concentration caseinate’ in Thermal processing and quality of foods. P. Zeuthen, I. C. Cheftel, M. Eriksson, M. Jul, H. Leniger, P. Linko, G. Valera and G. Vos, ed. London, Elsevier Applied Science Publishers, pp. 137–44. MULVIHILL, D. M. and FOX, P. F. (1994) ‘Developments in the production of milk proteins’ in New and developing sources of food proteins. B. J. F. Hudson, ed. London, Chapmann and Hall, pp. 1–30. MARSHALL, K. R. (1982) ‘Industrial isolation of milk proteins: whey protein’ in Developments in Dairy Chemistry – 1. P. F. Fox, ed. London, Applied Science Publishers, pp. 339–73. MATTHEWS, M. E. (1984) ‘Whey protein recovery processes and products’ J. Dairy Sci. 67 2680–92. INTERNATIONAL DAIRY FEDERATION (1987) ‘Trends in whey utilisation’ International Dairy Federation Bulletin 212, Brussels, Belgium. MORR, C. V. (1989) ‘Whey proteins: manufacture’ in Developments in Dairy Chemistry – 4. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 245–84. MULVIHILL, D. M. and GRUFFERTY, M. B. (1997) ‘Production of whey-protein-enriched products’ in Food Proteins and Lipids. S. Damodaran, ed. New York, Plenum Press, pp. 77–93. MAUBOIS, J. L., PIERRE, A., FAUQUANT, J. and PIOT, M. (1987) ‘Industrial fractionation of main whey proteins’ International Dairy Federation Bulletin 212, Brussels, Belgium, pp. 154–9. FOX, P. F. and MULVIHILL, D. M. (1983) ‘Functional properties of caseins, caseinates and co-precipitates’ in Proceedings of IDF Symposium: Physico-chemical aspects of dehydrated protein-rich milk products. Helsingor, Denmark. International Dairy Federation, pp. 188–259. KINSELLA, J. E. (1984) ‘Milk proteins: physicochemical and functional properties’ CRC Crit. Rev. Food Sci. Nutr. 21 197–262. DE WIT, J. N. (1989a) ‘Functional properties of whey proteins’ in Developments in Dairy Chemistry – 4. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 285–322. MULVIHILL, D. M. and FOX, P. F. (1989) ‘Physicochemical and functional properties of milk proteins’ in Developments in Dairy Chemistry – 4. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 131–72. SOUTHWARD, C. R. and GOLDMAN, A. (1978) ‘Co-precipitates and their application in food products. II. Some properties and applications’ N. Z. J. of Dairy Sci. Technol. 13 97–105. INTERNATIONAL DAIRY FEDERATION (1982) ‘Dairy ingredients in food products’ International Dairy Federation Bulletin 147, Brussels, Belgium. SOUTHWARD, C. R. and WALKER, N. J. (1982) ‘Casein, caseinates and milk protein co-precipitates’ in CRC Handbook of Processing and Utilization in Agriculture, Volume 1. Animal Products. I. A. Wolf, ed. Boca Raton, Florida, CRC Press Inc., pp. 445–552. OTTEN, M. G. (1985) ‘Whey protein concentrate: past, present and future’ in Proceedings of IDF symposium: New dairy products via new technology. Atlanta, Georgia, International Dairy Federation, pp. 107–15. ZADOW, J. G. (1986) ‘Utilization of milk components: whey’ in Modern Dairy Technology, Volume 1. Advances in Milk Processing. R. K. Robinson, ed. London, Elsevier Applied Science Publishers, pp. 273– 316. HUGUNIN, A. G. (1987) ‘Applications of UF whey protein: developing new markets’ International Dairy Federation Bulletin 212, Brussels, Belgium, pp. 135–44. DE WIT, J. N. (1989b) ‘The use of whey protein products’ in Developments in Dairy Chemistry – 4. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 323–46. SOUTHWARD, C. R. (1986) ‘Utilization of milk components: casein’ in Modern Dairy Technology, Volume 1. Advances in Milk Processing. R. K. Robinson, ed. London, Elsevier Applied Science Publishers, pp. 317–68. SOUTHWARD, C. R. (1989) ‘Use of casein and caseinates’ in Developments in Dairy Chemistry – 4. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 173–244. MANGINO, M. E. (1992) ‘Properties of whey protein concentrates’ in Whey and Lactose Processing. J. G. Zadow, ed. London, Elsevier Applied Science Publishers, pp. 231–70. ERDOGDU-ARNOCZKY, N., CZUCHAJOWSKA, Z. and POMERANZ Y. (1996) ‘Functionality of whey and casein in fermentations and in breadbaking by fixed and optimised procedures’ Cereal Chem. 73 (3) 309–16. HSU, C. K. and KOLBE, E. (1996) ‘The market potential of whey protein concentrate as a functional ingredient in surimi seafoods’ J. Dairy Sci. 79 2146–51. CHEN, H. (1995) ‘Functional properties and applications of edible films made of milk proteins’ J. Dairy Sci. 78 2563–83. ROSENBERG, M. and YOUNG, S. L. (1993) ‘Whey proteins as microencapsulating agents. Microencapsulation of anhydrous milkfat – structure evaluation’ Food Structure 12 31–41. MULVIHILL, D. M.

Milk proteins 36. 37. 38. 39. 40. 41. 42.

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(1992) ‘Chemistry of the caseins’ in Advanced Dairy Chemistry – volume 1. P. F. Fox, ed. London, Elsevier Applied Science Publishers, pp. 63–110. ZALL, R. R. (1992) ‘Sources and composition of whey and permeate’ in Whey and Lactose Processing. J. G. Zadow, ed. London, Elsevier Applied Science Publishers, pp. 1–72. MCMAHON, D. J. and BROWN, R. J. (1984) ‘Composition, structure and integrity of casein micelles: a review’ J Dairy Sci. 67 499–512. FOX, P. F. ed. (1982) Developments in Dairy Chemistry – 1. London, Applied Science Publishers. FOX, P. F. ed. (1989) Developments in Dairy Chemistry – 4. London, Elsevier Applied Science Publishers. ZADOW, J. G. ed. (1992) Whey and Lactose Processing. London, Elsevier Applied Science Publishers. CHANDAN, R. (1997) Dairy-based ingredients. St. Paul, Minnesota, Eagan Press. SWAISGOOD, H. E.

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12 Cellulosics J. C. F. Murray, Hercules Limited, Reigate

12.1

Introduction

Cellulose is probably the most abundant organic substance existing in nature and is the major constituent of most land plants. It is the starting material for a wide range of modifications with uses both in the food industry, and an even greater variety of uses outside this sector. Cellulosics, as used in this chapter, covers the range of modified celluloses generally approved as food additives. These are methyl cellulose E461, hydroxypropyl cellulose E463, hydroxypropyl methyl cellulose E464, methyl ethyl cellulose E465, and sodium carboxymethyl cellulose E466 which is frequently called simply carboxymethyl cellulose and also known as cellulose gum. The respective abbreviations mc, hpc, hpmc, mec and cmc are widely used. Properties of modified celluloses such as hydroxyethyl cellulose, which do not have approval for food additive use, are not included in this chapter. The common feature of all of these additives is that they are hydrocolloids derived from cellulose raw material by chemical modification. Since there are many points which are common to this range of additives, and to avoid repetition, where appropriate topics will be covered as a class rather than as the individual additives.

12.2

Manufacture

12.2.1 Raw material The raw material for modified celluloses is cellulose pulp, which in turn is produced from wood pulp from specified species or from cotton linters. Cotton linters are the short fibres from the cotton ball, which are too short to be suitable for use in thread and weaving. The polymer chain length of cellulose varies with the different raw materials and hence the polymer length and the resultant viscosity required in the final product will govern the selection of the raw material.

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12.2.2 Manufacturing process In general terms, cellulose pulp is dispersed in alkali solution to form alkali cellulose and is then treated with appropriate reagents, under tightly controlled conditions, to substitute the anhydroglucose monomers of the cellulose chain. The substitution is at the hydroxyl groups and the substitution reagents are as follows: • • • • •

methyl cellulose – chloromethane hydroxypropyl cellulose – propylene oxide methyl hydroxypropyl cellulose – mixed substituents as above methylethyl cellulose – chloromethane and chloroethane mixed substituents carboxymethyl cellulose – monochloracetic acid.

The two stages of the reactions can be summarised as follows: 1. 2.

Cellulose + Alkali + Water Alkali cellulose + R–X Alkali cellulose + R–CH(O)CH2 Alkali cellulose + X–R–COOH

! ! ! !

Alkali cellulose Alkyl cellulose Hydroxyalkyl cellulose Carboxyalkyl cellulose

The substitution reaction is followed by purification and washing stages to remove byproducts and to achieve the purity levels specified for food additives.

12.3

Structure

The structure of the cellulose molecule is shown in Fig. 12.1. It is shown as a polymer chain composed of two repeating anhydroglucose units ( -glucopyranose residues) joined through 1,4 glucosidic linkages. In this structure, n is the number of anhydroglucose units or the degree of polymerisation. Each anhydroglucose unit contains three hydroxyl groups, which in theory can be substituted. The average number of hydroxyl groups substituted per anhydroglucose unit is known as the degree of substitution (ds). Without exception, the ds required to produce desirable properties is much below the theoretical maximum. In the example in Fig. 12.2, carboxymethyl cellulose with ds 1.0 is shown.

12.4

Properties

12.4.1 General There are three main factors, which influence the properties of modified celluloses. These are first, and most importantly, the type of substitution of the cellulose, secondly, the

Fig. 12.1

Structure of cellulose.

Cellulosics

Fig. 12.2

221

Idealised unit structure of cellulose gum, with a ds of 1.0.

average chain length or degree of polymerisation of the cellulose molecules (dp) and thirdly, the degree of substitution of the chain. Additionally, the particle size of the hydrocolloid may be varied. Particle size and powder bulk density affect the dissolving characteristics of the product. Granular material is less prone to clumping or balling but takes longer to dissolve. Fine powdered material can give very rapid hydration, but does not disperse so easily and good stirring or blending techniques are necessary. Degree of polymerisation is a measure of the chain length of the polymer. Increasing dp very rapidly increases the viscosity of the modified cellulose in solution, although the viscosities of two differently substituted modified celluloses of comparable dp will not necessarily be comparable. In general the modified celluloses give neutral-flavoured, odourless and colourless clear solutions. It should be noted that all modified celluloses, in powder or even granular form, are capable of absorbing water from the atmosphere. It is therefore desirable to store these products in airtight packs.

12.4.2 Methyl cellulose and hydroxypropyl methyl cellulose The properties of these two hydrocolloids are very similar and will be covered together. Mc and hpmc are both soluble in cold water to give solutions with a wide range of viscosity, which is dependent on both dp and ds. These solutions show reasonable viscosity stability over the range pH3–11. More important, however, is the behaviour of solutions on heating since the solution will change into a gel once the temperature of the solution has been raised above a point known as the incipient gel temperature (igt). The igt varies from 52ºC for mc, to a range of 63–80ºC for hpmc types with increasing degree of hydroxypropyl substitution increasing the igt. These gels are reversible on cooling although there is a pronounced hysteresis between heating and cooling. Both polymers are good film formers and also exhibit some surface activity. Commercially mc and hpmc are distinguished by viscosity in 2% aqueous solutions, and also by the ds.

12.4.3 Hydroxypropyl cellulose Hpc is also soluble in cold water and again a range of viscosity can be obtained dependent on dp. Hpc becomes insoluble at temperatures above approximately 45ºC but unlike mc and hpmc, no gel is formed. Hpc is unusual in food hydrocolloids in that it is soluble in ethanol and mixtures of ethanol and water. However, the most interesting properties of hpc are probably its good film formation and its high surface activity compared to most other hydrocolloids. Commercially, the various grades of hpc for food use are differentiated by viscosity.

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12.4.4 Methylethyl cellulose In common with mc or hpmc, mec is also soluble in cold water and forms gels on heating, albeit weak gels, above the igp. These properties alone would not justify great interest in mec as a food additive, rather it is its surface activity and consequent excellent performance as a whipping aid, particularly in the presence of protein, which are of technical use.

12.4.5 Carboxymethyl cellulose General Cmc is soluble in both hot and cold water to give clear and colourless solutions with neutral flavour. As with other modified celluloses, the solution viscosity depends on dp, but it is possible to produce 1% aqueous solutions with viscosity of 5,000 mPas at ambient temperatures. These solutions do show a reversible reduction of viscosity on heating but in food systems do not gel either alone or with other hydrocolloids. The rate of viscosity build-up is obviously dependent on dp, particle size and to some extent on ds. With suitable fine grind powders an extremely rapid viscosity development can be obtained. A maximum degree of substitution of 1.5 is permitted in a recent amendment to EU legislation, but more typically ds is in the range 0.6–0.95 for food applications. ds, together with the uniformity of substitution, affects the rheology of the solution. Solutions of lower ds are thixotropic, whereas higher ds tends to pseudoplasticity. Uniformity of substitution favours pseudoplastic rheology, and solutions of such types give a particularly ‘smooth’ mouthfeel. Commercially, cmc types are distinguished by viscosity, by particle size and to a more limited extent by ds and special solution characteristics. It is necessary to check the concentration of the solutions for which viscosities are specified, as there is not a single standard value for concentration. Interaction of cmc with proteins Cmc is an ionic polymer and this allows the formation of complexes with soluble proteins such as casein and soy at, or around, the isoelectric region of the protein. Although the effect on the system is primarily dependent on pH, it is also dependent on the composition and concentration of the protein, temperature, and the concentration and type of the cmc. At pH less than 3.0 or higher than 6.0, cmc reacts in the cold with the proteins in milk to form a complex, which can be removed as a precipitate. In the pH range approximately 3.0–5.5, a stable complex is formed. At the maximum of stability the viscosity is abnormally high compared to the individual components. A representation of the effects of pH on the viscosity of a solution of cmc and casein is shown in Fig. 12.3. The system containing the cmc and casein complex is relatively shear sensitive, and the viscosity decreases under agitation. The complex is heat stable and little viscosity decrease is observed on heating. The casein is denatured to a much smaller extent than would be the case in the absence of cmc.

12.5

Applications

12.5.1 Methyl cellulose and hydroxypropyl methyl cellulose The major applications of these two hydrocolloids are in the fields of binding and shape

Cellulosics

Fig. 12.3

223

Viscosity effect of CMC–casein complex at varying pH.

retention, film formation and barrier properties, and avoidance of boil-out and bursting at higher temperatures. The thermogellation properties of mc and hpmc can be used to bind and to give shape retention to products where the ingredients themselves do not have particularly good biding properties. This includes such categories as reformed vegetable products such as potato croquettes and waffles, onion rings and the whole range of shaped soya protein and similar vegetarian products. These have poor binding properties and a tendency to disintegrate on heating due to the disruptive effects of steam formed during heating. Inclusion of hpmc, or better, mc means that on heating above the igp the hydrocolloid will gel and bind the ingredients of the product. Because the gelation is thermoreversible and the gel has reverted to solution form at temperatures above normal eating temperatures, no alteration in texture from gelation is observed by the consumer. Two examples of applications of this type are given in Formulations 12.1 and 12.2. In each of these cases the hydrocolloid is added to the cold mix in order that it may hydrate. Neither mc nor hpmc will hydrate in hot water above the igp, a property that can be used to ensure good dispersion of the gum if it is necessary to produce solutions and good stirring is not available. The use of thermogelation properties to inhibit boil-out in is shown in Formulation 12.3. This is a bakery filling, but the principle is valid for sauces and other fillings where boil-out needs to be avoided. In this case the dry ingredients are blended and mixed cold with the water to permit satisfactory solution of the methyl cellulose, and after standing 60 minutes the filling is baked. In cases where the sauce or filling is heat-treated prior to storage, care must be taken in the choice of addition point of the mc or hpmc, otherwise the dissolved hydrocolloid in the product will gel in the heat-processing equipment. A

224

Handbook of hydrocolloids Formulation 12.1 Potato croquettes Ingredients Mashed potato Potato flake Salt BenecelÕ hpmc type MP852 Water

Composition (%) 79 11 1 0.5 to 100

Formulation 12.2 Soya burgers Ingredients Soya protein Vegetable fat Starch Potato flour Benecel type M043 methyl cellulose Dried onion Salt Seasonings and flavours Water

Composition (%) 21 15 2 2 2 1.5 1 0.5 to 100

Formulation 12.3 Bake-stable filling Ingredients Sugar Pregelatinised modified starch Skimmed milk powder Whole milk powder Benecel type M043 methyl cellulose Disodium phosphate Flavour and colour Water

Composition (%) 13 5 4 3.5 0.5 0.15 to taste to 100

solution to this problem is to disperse the hydrocolloid in the hot product, where it will not dissolve until the heating stages are complete and the product is cooled below the igp. The thermogellation properties will then be exhibited at the next heating cycle. The same thermogelling binding properties are utilised in batter and coating mixes. Low or medium-low viscosity grades are used at levels of up to 1% to improve adhesion and also to reduce oil or fat absorption. This application could also be said to utilise film formation properties, and film formation also contributes to the reported use of these additives in providing reduced oil uptake when deep frying such products as seafood and potatoes. Coating is normally effected by dipping or spraying a solution of the cellulosic, followed by drying.

12.5.2 Hydroxypropyl cellulose To an extent, hpc is still a product waiting for applications in the food industry. The good surface activity of hpc is exploited in use of lower viscosity grades of hpc in toppings for

Cellulosics Formulation 12.4

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Topping for whipping

Ingredients Vegetable oils or fats Milk protein KlucelÕ hydroxypropyl cellulose type GF Emulsifiers Sugar or glucose syrup Salt, flavour and colour Water

Composition (%) 20–35 1–4 0.2–0.3 0.4–1.0 6–20 to taste to 100

whipping or dispensing from aerosol cans. Toppings stabilised with hpc retain the whipped structure at high ambient temperatures and in this respect are considered superior to other hydrocolloids. Use levels are typically 0.2–0.3% of the topping. A recipe example is given in Formulation 10.4. Hpc is soluble in ethanol and gives clear solutions in aqueous ethanol when the ethanol concentration is under approximately 50%. This gives potential for variations in viscosity or mouthfeel in a wide range of alcoholic beverages. It also has good filmforming properties and these films exhibit good flexibility, good oil and air barrier properties and a lack of tackiness. These properties have been examined in the pharmaceutical industry and may have potential in the speciality confectionery sector.

12.5.3 Methyl ethyl cellulose The major use of mec has been in foam formation and stabilisation. Solutions of mec can be whipped to produce a fine foam with an over-run comparable to egg white. The solutions can be re-whipped even if the foam is allowed to return to liquid after standing. Importantly, mec foams are compatible to many common food ingredients including egg white and fat. This has made it suitable for toppings, mousses, batters and the like.

12.5.4 Carboxymethyl cellulose General Since viscosity production is the primary property of cmc, this review will start with applications where viscosity is the major property required. In such cases it is normal to use high viscosity grades of cmc, partly for economic reasons as cmc prices are based more on the quantity of the gum than on the amount of viscosity developed. Additionally, the lower concentrations of gums required with high viscosity grades, compared to medium or lower viscosity grades, produce more acceptable and less gummy mouthfeel. A range of granulometries is available. Although the standard particle size will be suitable in many applications, the coarser particles will be preferred when it is necessary to make up solutions with poor mixing equipment, and the thirty minutes stirring necessary to dissolve this particle is not a problem. On the other hand, extra fine powders will be necessary for vending mixes and other instant applications. In these applications cmc will normally give a more rapid viscosity build up than guar gum Instant products With instant products such as vending drinks and powdered drink mixes fine grind cmc is used to provide the required rapid build-up of viscosity. If high viscosities are required

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Handbook of hydrocolloids Formulation 12.5 Instant fruit drink powder Ingredients Fine sugar Citric acid Sodium citrate Clouding agent BlanoseÕ cmc type 7HXF Flavour and colour Powder Water Drink

% of powder

% of drink

89.45 6.50 2.45 0.80 0.80 to taste 100

10.55 0.75 0.30 0.10 0.10 88.2 100

Formulation 12.6 Instant chocolate drink Ingredients Skimmed milk powder Sugar Cocoa powder Blanose cmc type 7H3SXF Total powder Water for 5:1 dilution

% of powder

% of drink

53.5 36.0 9.75 0.75 100.0

8.9 6.0 1.6 0.13 16.6 83.4

and consequently high cmc concentrations are used, there may be a perception of a ‘gummy’ mouthfeel. This can be reduced or eliminated by use of evenly substituted types with smooth flow properties, such as BlanoseÕ cmc type 7H3SXF. Some examples of cmc use in both cold and hot instant products are given in Formulations 12.5 and 12.6, illustrating that cmc has both hot and cold viscosity. The first formulation shows use of the high viscosity type Blanose cmc 7HXF, but this could be substituted by the Blanose cmc type 9M31XF at approximately twice the use level. Frozen products The viscosity produced by cmc contributes to the stabilisation of frozen products such as ice cream, water ices and ripples. Benefits observed include the conservation of texture and inhibition of ice crystal formation, a slow meltdown and an improved resistance to dripping. Where products such as water ices have a low pH it may be desirable to use a cmc which tolerates acidic conditions without loss of viscosity, such as Blanose cmc type 7HOF (Formulation 12.7). Cmc can also be used as the primary stabiliser in ice cream to control ice crystal size and growth during freeing and storage, to provide a smooth eating texture and to provide heat shock resistance (Formulation 12.8). When compared to locust bean gum or guar gum, there is a higher over-run which means that cmc is particularly suitable for use in soft serve products. Use of cmc tends to produce whey separation, but this can be avoided by use of carrageenan together with cmc in a ratio of about 1:6. Table sauces and dressings The thickening effects of cmc can be used in products such as salad dressings and tomato sauces. Properties of cmc which make it suitable for these applications include its rapid solubility in both hot or cold water, its good water binding and good tolerance to low pH

Cellulosics Formulation 12.7

Water ice or ripple

Ingredients

Water ice (%)

Sucrose Dextrose Blanose cmc type 7HOF Fruit juice Citric acid Flavour and colour Water Formulation 12.8

25 7 0.25

Ripple syrup (%) 30

0.4 to taste to 100

0.75 40 0.3 to taste to 100

Ice cream

Ingredients

Composition (%)

Milk fats Milk solids non fat Sucrose Glucose syrup Emulsifier Blanose cmc type 7HXFMA GenulactaÕ carrageenan type L-100 Formulation 12.9

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12 11 12.5 4 0.1 0.17 0.03

Ketchup

Ingredients Tomato concentrate (28%) Sugar Salt Citric acid and vinegar (to pH 3.7–3.9) Blanose cmc type 7HXFMA Blanose cmc type 7HOF Preservative Water

Compositions (%) 40.0 20.0 1.5 7.5

40.0 20.0 1.5 7.5

1.1 0.2 44.7

0.9 0.2 44.9

Note: If tomato concentrate levels are increased to 40%, then the cmc levels can be halved to around 0.5%.

levels. In salad dressings, typical use levels would be 1.0% Blanose cmc type 7HOF when oil contents are 30% and 0.75% when oil contents are increased to 50%. Tomato sauce or tomato ketchup is a further application for cmc, and is particularly interesting because variation of the cmc type can be used to vary the structure of the product. In Formulation 12.9, type 7HOF can be used for a long-structured ketchup, while type 7HXFMA will provide a much pulpier and shorter textured product. Soft drinks The viscosifying properties of cmc are widely used in ready-to-consume soft drinks. Additional body or mouthfeel can be provided to low calorie drinks sweetened with intense sweeteners, to more closely match the mouthfeel obtained with sucrose or glucose syrup.

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Use levels are typically 0.025–0.5% of the drink. Cmc is widely used to suspend fruit pulp and to inhibit ‘neck ringing’ by flavour oils in fruit squashes and other dilutable soft drinks and in ready-to-consume drinks containing fruit pulp. This has become more necessary as manufacturers have substituted intense sweeteners for a part of the bulk sweeteners. Generally, the concentration of Blanose cmc type 7HOF will be 0.05–0.1% of the fruit squash or ready-to-consume drink depending on the exact composition and density of the drink. Medium viscosity cmc types such as Blanose type 9M31F are used at approximately twice the addition level. Since cmc develops its viscosity more effectively if dissolved in water before addition of either acids or bulk sweeteners, it is good practice to produce a 1% stock solution of high viscosity types which can then be added as required. Bakery products A further application where viscosity development is required is in cake mixes and batter mixes. Incorporation of cmc can improve the volume yield of certain doughs as a result of its viscosity drop during baking and improves the suspension and distribution of ingredients such as dried fruit. Additional water is required in comparison to recipes without cmc, and so results in increased yields and improved moistness, particularly after storage. The fine grind types will normally be used in bakery mix concepts. In this case not only is the granulometry more compatible with flour, but also the fine grind allows a better water absorption in competition with other components such flour and sugar (Formulation 12.10). Some speciality cmc types have particularly good water binding properties, although their solution properties may be comparatively poor. This excellent water binding is utilised in baked products, including breads and morning goods, to either increase yield or to retard staling and hence improve consumer acceptability and prolong shelf life. The effect is compatible with other bread additives and improvers. Use of AquasorbÕ cmc type A-500 at levels of 0.5–1.5%, and normally 0.5–1.0%, of flour weight is recommended in this type of application. The water binding effect of these cmc types can also be used to reduce the fat absorption of doughnuts during cooking. The improved water binding is believed to inhibit fat absorption. Use levels of up to 0.3% are quoted. Low pH milk products Due to its ionic nature, cmc can react with soluble proteins to form a complex at or around the isoelectric point of the protein. In a sour milk medium at pH3.8–5.0, cmc reacts with casein to form a soluble complex, stable to heat treatment and to storage. In practice this effect is best utilised in the higher part of this pH range since at pH below about 4.2 there tends to be too great variation in viscosity with even small changes in pH. Formulation 12.10

Fruit cake mix

Ingredients Flour Sugar Fat Baking powder Blanose cmc type 7H4XF Eggs Water (additional to control)

Composition (%) 37 31 31 0.6 0.3 31 5

Cellulosics Formulation 12.11

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Buttermilk drink pH4.5–4.6

Ingredients

Composition (%)

Buttermilk Sugar Blanose cmc type 7HOF

89.6 10 0.3–0.4

Preparation 1. 2. 3. 4. 5.

Blend the sugar and cmc and add this blend gradually into the buttermilk while stirring rapidly. Continue stirring for 15 minutes. Heat up to 70ºC in a heat exchanger. Homogenise to 150 to 200 bar. Pasteurise at 85ºC for 15 seconds. Cool to 20ºC and fill aseptically if extended shelf life is required.

Formulation 12.12

Milk orange juice beverage at pH4.5–4.6

Ingredients Whole milk Orange juice (11º Brix) Sugar Blanose cmc type 7HOF

Composition (%) 53.8 41.3 4.7 0.15–0.20

Preparation 1. 2. 3. 4. 5. 6. 7. 8.

Blend the cmc and sugar and add the blend to the cold milk with stirring. Stir until the cmc is fully dissolved (15 minutes with good agitation). Slowly add the orange juice, cooled to 10ºC or lower, to the milk while stirring well. Continue to stir for at least five minutes after the end of juice addition. Heat to 70ºC in a heat exchanger. Homogenise at 150–200 bar. Pasteurise at 85ºC for 15 seconds. Cool to 20ºC and fill.

At pH levels 4.3–3.8 pectin is frequently the preferred stabiliser, although even at these levels cmc is sometimes used if economy is of prime importance. Examples of low pH milk preparations are given in Formulations 12.11 and 12.12.

12.6

Regulatory status

12.6.1 Names and serial numbers In the member states of the European Union, use of the modified celluloses described in this chapter is permitted by Directive 95/2/EC on food additives other than colours and sweeteners. National legislation in the individual member states implements this directive. The EU directive has assigned names and serial numbers (E numbers) to these additives as follows:

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E461 E463 E464 E465 E466

methyl cellulose hydroxypropyl cellulose hydroxypropyl methyl cellulose methyl ethyl cellulose carboxymethyl cellulose or sodium carboxymethyl cellulose.

It is hoped that in the future the style ‘modified cellulose’, which is already permitted in other jurisdictions, will also be permitted in EU member states. The directive sets specifications for these additives when they are to be used in food applications. These specifications cover such matters as purity, permitted degree of substitution, molecular weights and moisture levels for each individual additive, but detailed discussion of specifications is outside the scope of this chapter. Food industry users of modified celluloses should be aware that in general there is a greater use of these products in other classes of industry, where specifications are to lower standards of purity, and checks should always be made that food specification material is being used. For example, cmc for food use has a minimum purity level of 99.5% cmc, whereas technical grades are commonly sold at only 98.0% purity or lower.

12.6.2 Permitted use levels All these additives are placed in Annex I of the directive, which permits their use at ‘quantum satis’ level in products other than those where there are specific regulations on composition. These specific exceptions are listed in Annexes VI, VII and VIII, but these limited exceptions will not be detailed. The term ‘quantum satis’ means that no maximum level of the additive in or on a food is specified but in or on a food the additive may be used in accordance with good manufacturing practice at a level not higher than is necessary to achieve the intended purpose and provided that such use does not mislead the consumer. Outside Europe, use of modified celluloses is also permitted under purity criteria set by the Food and Agriculture Organisation and the Wold Health Organisation (FAO/ WHO) and the US Food Chemicals Codex. In the United States use of modified celluloses is allowed in a wide range of foods, and this is true in many other countries. However, space does not permit a detailed review of the legislative situation in every country, and specialised advice should always be sought in case of doubt. The Joint FAO/ WHO Expert Committee on Food Additives (JECFA) assigned in 1990 an Acceptable Daily Intake (ADI) ‘not specified’ to the modified celluloses.

12.6.3 Ingredient declarations In EU member states there is an approved manner to label foodstuffs where an additive is used and has a technological effect in the foodstuff. The additive should be described on the foodstuff package ingredient label as ‘thickener’ or ‘stabiliser’ or ‘gelling agent’ as appropriate, followed by the serial number (E number) or the approved name as given in Section 12.6.1. There is no legal requirement to include processing agents in the ingredient declaration of a foodstuff.

13 Tragacanth and karaya Wang Weiping, Arthur Branwell & Co. Limited, Essex

13.1

Gum tragacanth

13.1.1 Introduction Gum tragacanth has been known and used for thousands of years. It is defined by the Food Chemical Codex as the ‘dried gummy exudation obtained from Astragalus gummifer Labillardiere or other Asiatic species of Astragalus (Fam. Leguminosae)’.1 In the European Pharmacopoeia gum tragacanth is defined as ‘the air-hardened gummy exudates, flowing naturally or obtained by incision from the trunk and branches of Astragalus gummifer Labillardiere and certain other species of Astragalus from Western Asia’.2 Although the Astragalus genus comprises more than 2000 species,3 most commercially traded gum tragacanth is obtained from the two species, Astragalus gummifer Labill. and A. microcephalus Willd. The plants are small, low bushy perennial shrubs having a large tap root along with branches, and grow wildly in the dry deserts and mountainous regions of South West Asia, from Pakistan to Greece, and in particular, in Iran and Turkey. Plants develop a mass of gum in the centre of the root, which swells in the summer heat. If the stem is slit, soft gum is exuded. The plants are tapped for commercial gum collections, usually with a knife by making careful longitudinal incisions in the tap root and the bark of the branches. The gum exudes readily from these cuts in the form of ‘ribbons’or ‘flakes’ which become brittle on drying. The plants require an abundance of water during the growing season, but need a dry climate during collecting time that extends from July to September for both ribbons and flakes. Gum production is labour-intensive and is carried out in remote, hostile areas of Iran and Turkey. Iran, the largest exporter, is the best commercial source and supplies the highest quality of the gum. In general, Turkish tragacanth is considered of inferior quality. This doubtless reflects the fact that different Astragalus species yield the gum in the different locations.4 Villagers in gum production areas gather the gum, which is brought to village centres and thence to specialised traders who hand sort, grade, pack and arrange export. After collection

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from villages, the gum is cleaned and selected by hand into five ‘ribbon’ (superior quality), thus named after their long, curved shape about 50–100mm in length, 10–20mm wide, and many ‘flake’ grades, which are of inferior quality as well as of darker colour. The best quality gum tragacanth is tasteless, whitish in colour and translucent in appearance, giving an aqueous solution of high viscosity, free from sand or bark. The lower grades are generally more strongly coloured than the higher grades. The ‘ribbon’ types of gum tragacanth, which represents the best commercial grades, are obtained from a shrub, slightly different from that which yields ‘flakes’. It is impossible to obtain both types (ribbon and flake) from the same shrub. It is, in fact, very unusual to find both types of shrub growing in the same locality. The best quality gum is obtained from tapping rather than from natural exudation. Gum tragacanth has been used as a stabiliser, emulsifier and thickener in food, pharmaceutical, cosmetic industries and in technical applications for many years. The gum swells and dissolves in cold water to form a solution of very high viscosity. More outstanding characteristics, however, are its high degree of stability under strong acid conditions and good emulsification property. Demand for gum tragacanth fell greatly during the 1980s, from several thousand tonnes to several hundred tonnes per year nowadays. The main reason is competition from xanthan gum, a fermentation-derived product developed in the USA in the 1970s and was finally approved for food use in 1980. In addition, during this period, interventions by the Iranian government at origin effected a sharp rise in export price, which was already increasing due to higher labour costs, because of more attractive alternative employment opportunities becoming available to the village communities. The high inflation rates in Turkey had the same effects. Xanthan gum replaces the functionality of gum tragacanth in many of its more traditional applications, but at a more cost-effective and stable price. Xanthan gum has the added advantages of constant quality and virtual sterility as a result of the manufacturing process.5 However, due to certain outstanding and unique properties, there are certain applications in which gum tragacanth cannot be replaced successfully by xanthan or any other gums.

13.1.2 Manufacture Gum tragacanth is collected and sorted by hand into various grades of ‘ribbon’ or flake at origin according to local grading systems based on colour, length of ribbon or size of flake. The Iranian system is more clearly defined than from other original sources. Under this system, it gives Ribbon No. 1, 2, 3, 4 and 5; Mixed ribbons and Flakes No. 25, 26, 27, 28, 31, 55, 101, 102; Nubs and Siftings.6 The most commonly used Iranian qualities are ribbon 1 and 4, mixed ribbon and flakes 27, 28 and 55. The gum that gives high viscosity, good colour in solution and low microbiological limits is regarded as indicating high quality. It is important, therefore, that cleanliness is maintained through the entire harvesting operation. Processing is normally carried out by importers/processors in Europe and the USA after purchasing materials based on approval of pre-shipment samples. The exudate is pulverised to a fine mesh powder mechanically. During this powdering, selective sifting, aspirating and density-table separating are used to remove foreign matter. The preferred medium for conveying the gum is clean, filtered air, which acts to cool the powder and to prevent viscosity loss. The final powder is blended to ensure uniformity of colour and viscosity. The gum is further processed to conform to sterility standards when required and to assure customer satisfaction of a product to fit their exact needs.

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13.1.3 Structure The chemistry of gum tragacanth from Astragalus microcephalus, Astragalus gummifer and Astragalus kurdicus has been studied and considerable variations among the species identified.7, 8 This explains the variability shown by commercial consignments of gum tragacanth which are liable to be mixtures of gum from different Astragalus species. Gum tragacanth is a highly branched, heterogeneous hydrophilic carbohydrate polymer. Methoxyl groups are also presented. It has a molecular weight of about 840,000 ˚ by 19A ˚ for a flake type of non-degraded gum.9 dalton, and an elongated shape of 4500A It is a complex, slightly acidic polysaccharide bounded with small proportions of protein, and with trace amounts of starch and cellulosic material present. Calcium, magnesium and potassium are the associated cations.10 It is suggested several chains may aggregate parallel to the long axis in the structure.11 After acid hydrolysis, gum tragacanth commonly produces sugars of D-galacturonic acid, D-galactose, L-fucose (6-deoxy-L-galactose), D-xylose, L-arabinose, L-rhamnose. The exact proportion of each sugar varies between, and in gums from different locations. Using commercial Iranian samples of gum tragacanth, Anderson and Grant found that gum with higher viscosity contained high proportions of fucose, xylose, galacturonic acid and methoxyl groups, and low proportions of arabinose and nitrogenous fractions. The samples with lower viscosity showed a lower content of galacturonic acid and methoxyl group, but contained more arabinose and galactose.12 Chemically, gum tragacanth consists of two fractions. One fraction, termed ‘Tragacanthic acid’ or ‘Bassorin’ which represents 60–70% of the total gum, though insoluble in water, has the capacity to swell and form a gel. Another small fraction, termed ‘Tragacanthin’ is soluble in water to give a colloidal, hydrosol solution. Bassorin probably yields tragacanthin on demethoxylation. The water-swellable component ‘tragacanthic acid’ is an acid fraction, which upon acid hydrolysis yields D-xylose, L-fucose, D-galacturonic acid, D-galactose and a very small amount of L-rhamnose. This acid portion of the molecule is associated with calcium, magnesium and potassium cations. The property of gum tragacanth is largely dependent on this major component, with its high molecular weight and rodlike molecular shape. The partial structure of tragacanthic acid polymer is shown in Fig. 13.1.11 D-galacturonic acid repeating units form the main chain by 1,4-linkages and short side chains consisting of D-xylose residue connected to the main chain by 1,3-linkages.13 L-fucose may be replaced by D-galactose.14 Tragacanthin is a neutral polysaccharide component. It is a highly branched arabinogalactan in which L-arabinose is the preponderant sugar. It is believed the structure consists of a core of repeated D-galactose residues to which highly ramified chains of L-arabinose residues are attached and has a spheroidal molecular shape.11,15 The majority of the interior chains of D-galactose are connected by 1,6-linkage, and the

Fig. 13.1 Partial structure of tragacanthic acid.

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small portions by 1,3-linkages. The L-arabinose residues are mutually joined by 1,2-, 1,3, and 1,5-linkages. The structure also contains small proportions of D-galacturonic acid and L-rhamnose residues whose modes of linkage are not clear. Both fractions contain small proportions of proteinaceous materials with fairly similar amino acid compositions. The methoxyl group content is enriched, however, in the soluble fraction.7 The neutral fractions, arabinogalactan, may be separated from the tragacanthic acid polymer in an aqueous solution with the addition of ethanol. The arabinogalactan is soluble in ethanol at concentrations as high as 70%, while the tragacanthic acid will precipitate.16

13.1.4 Technical data Gum powder made from ribbon is white to light yellow in colour, is odourless and has an insipid, mucilaginous taste. The flakes vary from yellow to brown to give cream to tan powders in colour. Both ribbon and flake gums are available in a variety of particle sizes and viscosities depending on the end use. A typical product specification of a commercial gum tragacanth powder is shown below: Appearance: Loss on drying: Ash: Acid insoluble ash: Viscosity 1% in water: Particle size: Heavy metals: Pb: As: Microbiology:

Off white to creamy coloured fine powder 12% maximum1 3.0% maximum 0.3% maximum 800 150 cps2 90% minimum pass 150 mesh BSS 10 ppm maximum 3 ppm maximum Salmonella/E.Coli

Absent

Notes: 1 105ºC–60 minutes 2 Basis Brookfield Viscometer, Spindle No.3 20 rpm @ 25ºC 24 hours (Specification of Luxara 2662 gum tragacanth, courtesy of Arthur Branwell & Co Ltd.) Solubility Gum tragacanth swells rapidly in either cold or hot water to form a viscous colloidal solution, which acts as a protective colloid and stabiliser. While it is insoluble in alcohol and other organic solvents, the gum can tolerate small amounts of alcohol or glycol. The gum solution is fairly stable over a wide pH range down to extremely acidic conditions at about pH 2. Viscosity The viscosity is the most important factor in evaluating tragacanth and is regarded as a measure of its quality as well as a guide to its behaviour as a suspending agent, stabiliser, or emulsifier. The viscosity of 1% solutions may range from about 100–3500 cps depending on the grade. Ribbon types give a higher viscosity than flake types. The best quality of ribbon type gum tragacanth shows up to 3500 cps (1.0%, 25ºC, 24 hrs, 20 rpm by Brookfield viscometer). The solution viscosity reaches a maximum in 24 hrs at 25ºC, 8 hrs at 40ºC and 2 hrs at 50ºC. Though fine powdered gum has shorter hydration time than coarse powder, good

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dispersion is needed to avoid the formation of aggregates. The maximum initial viscosity of tragacanth solutions is at pH8, but maximum stable viscosity is at about pH5.16 The viscosity is quite stable over a wide pH range from 2–10, particularly for the flake type of the gum.17 The addition of strong mineral and organic acids causes some drop in viscosity. Divalent and trivalent cations can also cause a viscosity drop or may result in precipitation, depending on the metal ion type and concentration. Rheological properties Tragacanth solutions show pseudoplastic behaviour typical of most gums. The apparent viscosity decreases as the shear rate increases and is reversible, with the original viscosity returning upon the reduction of the shear rate. Pseudoplastic properties have an effect on the pouring and texture of the finished products. Acid stability Tragacanth solutions are naturally slightly acidic. A 1% solution has a pH of 5–6, depending on the grade of gum used. The viscosity is most stable at pH4–8, but with very good stability at both the higher pH and at the lower end of pH2. Tragacanth is one of the most acid-resistant gums, and is chosen for this characteristic for use under conditions of high acidity. However, when acids are used in the system, if possible acids should not be added until the gum has had time to fully hydrate. Surface activity Gum tragacanth has well defined surface activity properties and produces a rapid lowering of the surface tension of water at low concentration, less than 0.25%.18 Flake types of tragacanth (lower viscosity) are superior to the ribbon types (higher viscosity) for the reduction of surface tension and interfacial tension effects. Stauffer reported at 1% concentration, the ribbon type gave 61.7 dynes/cm surface tension value compared with the value of 52.5 dynes/cm given by the flake type.19 Emulsification ability Gum tragacanth, regarded as a bifunctional emulsifier, is a most efficient natural emulsifier for acidic oil-in-water emulsions. It is not only thickening the aqueous phase but also lowering the interfacial tension between oil and water. It has a reported hydrophilic lipophilic balance (HLB) value of 11.9,20 but it is believed HLB values run from 11–13.9 depending on the grades because flake types have lower interfacial tension between oil and water than ribbon types.21 Heat stability Elevated temperatures may also affect viscosity through a thinning effect on the solution. Upon cooling, however, the solutions tend to revert to nearly their original viscosity. Prolonged heating can degrade the gum and reduce viscosity permanently. Compatibility Tragacanth is compatible with other hydrocolloids as well as carbohydrates, most proteins and fats. There is an interesting interaction, however, between gum tragacanth and gum arabic, which results in an unusual viscosity reduction. A combination of gum tragacanth with gum arabic has been used to produce superior, thin, pourable, smooth emulsions with oils, which also have a long shelf life.

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Preservatives Tragacanth solutions are less sensitive to microbial attacks and have longer shelf life without loss of viscosity in comparison with other plant hydrocolloids. When preservatives are needed, glycerol or propylene glycol at 94ml/litre serve as excellent preservatives in many emulsions. Sorbic acid, benzoic acid or sodium benzoate at less than 0.1% concentration are effective when used below pH6. A combination 0.17% methyl and 0.03% propyl parahydroxybenzoate is effective at pH3–9. Benzoic acid esters are also effective for maintaining solution properties throughout product preparation and shelf life.17

13.1.5 Uses and applications Solution preparation: Like most cold-water soluble hydrocolloids, powdered gum tragacanth has a tendency to lump when first wet with water. The surface of these lumps solvate, forming a barrier which prevents wetting in the interior of the lump. The key to rapid preparation of gum solutions is uniform dispersion. Depending on user’s formulation, this may be accomplished by any of the following methods: • Slowly adding the gum to the vortex of vigorously agitated water. • Dry blending the gum with at least five to ten times its weight of any dry non-swelling, powdered material, such as sugar, before adding to the water • Dispersing the gum in alcohol, glycerine or propylene glycol, or other water-miscible liquid that will not swell the gum before adding to the water; or dispersing the gum in a non-miscible liquid, such as vegetable or mineral oil, before adding to the water. • Feeding the gum through a funnel into an aspirator where it is dispersed by water flowing at high velocity. Tragacanth solution slowly reaches its peak viscosity in cold water after standing overnight. Particle size affects the rate of hydration, the coarser the mesh size, the slower the rate of hydration. Temperature of preparation and concentration also have an effect on viscosity. Applications: Gum tragacanth has been used as stabiliser, thickener, emulsifier and suspending agent in various applications based on its high viscosity at low concentrations, good suspending action, unusually high stability to heat and acidity, effective emulsifying properties. It also has pourable, creamy mouthfeel and good flavour release properties and extremely long shelf life. Food applications In general, gum tragacanth is used in salad dressings, condiments, sauces, bakery emulsions, fillings and toppings, confectionery, soft drinks, jellies, deserts, flavours and spices. 1.

Pourable salad dressings and sauces: Tragacanth is widely used as a thickener and stabiliser in many pourable-type creamy salad dressings to thicken the water phase and prevent the oil droplets from coalescing. For similar reasons, it is used in relish sauces, condiment bases, sweet pickles, liquors, mayonnaise, mustard sauce, barbecue sauce and many other low pH products to give creamier, more natural looking dressings with long shelf life and good refrigerator stability.

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2.

3.

4.

5. 6.

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In low calorie salad dressings, where if the oil content is about 1–5% a high level of the gum (0.5–1.2%) is used to stabilise the emulsion; where if oil is not used at all, the mouthfeel and body of oil can be simulated by using gum tragacanth.22 Good acid stability, natural emulsifying property as well as long shelf life, make gum tragacanth very useful on condiment and sauce type products where vinegar and oil are essential ingredients. Normally at use level of 0.4–0.8%, depending on oil contents. Oil and flavour emulsions: In combination with gum arabic, tragacanth produces a superior citrus oil flavour emulsion and other acidic oil-in-water type emulsions. It is used in fish oil emulsions to emulsify fat-soluble vitamins such as A, D, E with acid flavours and other nutrient supplements. Use level is about 0.8–1.2%. Ice creams, ices and sherbets: At use level of 0.2–0.5%, tragacanth can be used very effectively in ice cream as a stabiliser to satisfy the requirements of smooth body and texture. It maintains these qualities during storage by minimising the formation of crystals induced by fluctuations of freezer temperatures. When used in water ices, ice pops and sherbets, gum tragacanth effectively prevents the migration of syrups or colour in the ice matrix during storage at a use level about 0.5%. The gum can be used in low calorie milkshake-type food to suspend solid and emulsify the immiscible fats in the system, which gives the end product a mouthfeel similar to milkshake made with increased solids. Tragacanth improves the shelf life and reduces syneresis when used as a cold process stabiliser for meringues, where air-in-liquid emulsion stability is required. Bakery fillings: Gum tragacanth has been used to stabilise bakery fillings and toppings, which contain fruit, fruit pure´es and flavours, to give a shiny and transparent appearance and a creamy mouthfeel as well as a good shelf life. In frozen pie fillings, tragacanth provides clarity and brilliance in conjunction with starch as thickener. Soft drinks: In citrus beverages, tragacanth acts as a thickening agent to impart good mouthfeel and stability due to its good acid resistance. Confectionery and icings: Tragacanth has been used in candy cream centres containing natural fruit or acid as a thickener because of its effective resistance to hydrolysis by food acid. It has been used as a binder in the cold-press process and in the extrusion process for making candy cigarettes and lozenges, where it gives a smooth texture and emulsifies any flavour oils or fats. Gum tragacanth can be used in chewing gum formulations in combination with gelatin to give a chewy and cohesive texture. It can be used as a binder in fruit tablets, gum drops and pastilles to provide a desired consistency, mouthfeel and good flavour release. Tragacanth is widely used in icings as an emulsifier and water binder to maintain the pliability and avoid cracks in the products. It also gives smooth texture and creamy taste for the prodcuts.17

Pharmaceutical and cosmetic applications Tragacanth is used in a wide variety of medicinal emulsions, jellies, syrups, ointments, lotions, and creams as a thickener, emulsifier or lubricant by increasing the viscosity of the external phase and suspending the active ingredients from settling out. Tragacanth is used at a level of 0.4–0.8% to suspend oil globules and insoluble matters uniformly throughout the preparation of fat-soluble vitamins, minerals, steroid glycosides

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and cod liver oil emulsions to facilitate the absorption of poorly soluble substances. It can be used in low-calorie elixirs and syrups. Gum tragacanth was used as an emulsion stabiliser in dermatological cream and lotions. Tragacanth can be used with glycerin and water to form a thick paste, which is a useful excipient to bind pill masses as a tablet binder. It was used as a basis of jelly lubricants for catheters and surgical instruments. Gum tragacanth is demulcent and was employed in pharyngits by allowing a piece of the gum to hydrate slowly in the mouth. It was also used in medicinal troches for this demulcent effect.23 Gum tragacanth is used in some oral-intake suspensions to suspend solids and liquids in water-based syrups and medicaments for oral ingestion. At 1.5–2% use level with a humectant gum tragacanth acts as a suspending agent in jellies and toothpastes to form creamy, brilliant products due to its non-gelling characteristic to permit good squeezing and spreading. Its long shelf life and film forming properties make it useful in hair lotions, hand lotions and creams. It can be used as a constituent of glycerol toilet creams. Tragacanth can also be used as a base for skin medication preparations containing gelatin, glycerol and water. Miscellaneous applications Very low grade tragacanth gum can be used in textile print paste and sizes for high quality silks and crepes at a level 0.5–1%. It has good release properties and gives added body to these fabrics. It can be used as film forming and stabiliser in insecticide emulsions. In addition, it can be used in dressing leather and in the preparation of leather polishes, in furniture, floor and auto polishes. It can also be used as adhesive for reconstituted cigar wrapper leaves and as a binder and plasticiser for the graphite sticks in lead pencils, as well as in crayon pencils at about the 1–3% use level.24 Due to supply stability and price reasons, many applications traditionally using gum tragacanth have been replaced by xanthan gum or a combination of other hydrocolloids. But the properties of high viscosity, bifunctional emulsification, excellent acid stability and long shelf life with smooth and creamy mouthfeel are still attractive for many premium quality products.

13.1.6 Regulatory status Gum tragacanth is classified as ‘generally recognised as safe’ (GRAS) within the USA. It is also classified as ‘acceptable daily intake (ADI) not specified’ (the highest category of safety evaluation) by the Joint WHO/FAO Expert Committee on Food Additives (JECFA), and has the number E413 in the list of additives approved by the Scientific Committee for Food of the European Community. The toxicological studies that led to these regulatory decisions have been reviewed.8, 10, 25

13.2

Gum karaya

13.2.1 Introduction Gum karaya, known also as Sterculia gum, is defined by the Food Chemical Codex as ‘a dried gummy exudation from Sterculia urens Roxburgh and other species of Sterculia (Fam. Sterculiaceae), or from Cochlospermum gossypium A.P De Condolle or other species of Cochlospermum Kunth (Fam. Bixaceae)’.1 Commercial gum karaya are commonly obtained from tapping or blazing the mature Sterculia urens, a large bushy

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tree growing widely throughout the Indian sub-continent, and other related species of Sterculia such as S. villosa, which is also sometime tapped, S. setigera which is found in Sudan and other North African countries, or from Cochlospermum gossypium. The bulk of commercial gum karaya is obtained from S. urens trees from India. The trees grow to a height of about ten metres on the dry, rocky hills and plateaux of central and northern India. Their cultivation of the trees and the collection of the gum are closely controlled by the government. As with the other exudate gums, the trees are tapped. Exudation begins immediately and continues for several days in the form of large, irregular ‘tears’ or lumps which may weigh up to two kilos. The average tree can be tapped about five times during its lifetime, with a total yield of up to five kilos per season. The exudate is allowed to solidify on the tree. The native collectors pick this crude gum which is sold to dealers in Bombay. These gum ‘tears’ are broken into fragments less than 25mm in diameter, then cleaned, sorted and graded according to colour and purity before selling to importers and processors in Western counties. The best quality gum is picked in April, May and June, before the monsoon season. In September, the gum is again picked. This autumn crop has a greyish colour and is normally less viscous. Gum karaya was for many years a major and almost exclusively Indian export. In the past decade, however, world demand has fallen from around 6000 tons in the early 1980s to less than 3000 tons more recently.5 The demands slackened as other materials replaced gum karaya in some of its major applications. When the market dropped, the Indian government required export regulations in order to maintain high prices, which interfered with the supply chains. This exacerbated the problems, inducing Western importers to seek supplies from other sources. The result led to trade incentives for those African countries, such as Sudan and Senegal which have native stands of Sterculia setigera trees, becoming more important suppliers than previously. It appears that 50% of the tonnage lost by India has been acquired by African exporters. Gum karaya has been used as an emulsifier, stabiliser and thickening agent for many years. Like gum tragacanth, demand for gum karaya is decreasing.26 Many traditional applications have been replaced by more cost-effective gums, or by a combination of those hydrocolloids. Nevertheless, in particular applications, gum karaya continues to be the hydrocolloid of choice. Gum karaya, itself a strongly acidic polysaccharide, shows good stability in acidic preparations and is used as a food additive. Less than 10% of the total production is used in food applications, however. The principal use of gum karaya is in the pharmaceutical industry, as an emulsifier and binder in particular products, e.g., non-prescription laxative preparations and stoma surgical adhesive seals. A considerable tonnage was used in earlier years to prepare denture fixative powders, but this tonnage has decreased greatly since dental research showed an adverse effect on the remaining teeth consequent upon the habitual use of strongly acidic gum karaya.5

13.2.2 Manufacture Gum karaya is hand-picked, cleaned, sorted and graded by local labour, based solely on the criteria of pale colour and the absence of foreign matter. In earlier days, grading was based entirely on colour, but nowadays the gradings are allocated on the basis of Bark and Foreign Matter (BFM) content. The system of grading used for gum karaya has

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varied somewhat over time, but the current grades used in the international trade are Superior, No. 1, No. 2, No. 3 and Siftings. The highest grade of gum karaya is white in colour, translucent, and has minimum bark and foreign matter. In contrast, technical grades are brown to dark brown, and contain more impurities. The top quality is ‘Superior’ which should be white in colour with a foreign matter content of less than 0.5%. This grade gives a solution of light colour and normally has a high viscosity. For No. 1, No. 2 and No. 3, the colour gets darker from light grey to heavy tan and foreign matter content increases to around 0.5%, 1.5% and 3% respectively. Any product with a BFM content in excess of 3% is no longer in conformity with the standards permitted in the FCC.1 North American and European processors evaluate all incoming raw materials according to impurity content, solution viscosity and colour. After approval of a preshipment sample, the manufacturer utilises a processing system of size reduction, aspiration and density-table separation to remove bark, fibres, sand and other foreign matter from the gum. In order to meet customer specifications, the gum is further processed and blended for mesh size, colour and viscosity.

13.2.3 Structure Natural gum karaya is a complex, branched, partially acetylated polysaccharide with a reported molecular weight of 9 million and 16 million dalton.3, 27 Anderson and coworkers have chemically characterised commercial gum karaya and individual collected samples of gum karaya from S. urens, S. villosa and S. setigera trees and found them to be similar in terms of the chemical composition of gum karaya from the different species.28 On average, new gum karaya contains about 10–14% acetyl groups, from which free acetic acid is formed and is split off on ageing. Increased temperature, humidity and fine particle size increases the rate of acetic acid formation. After acid hydrolysis, gum karaya commonly produces D-galacturonic acid, Dgalactose, L-rhamnose and small proportions of D-glucuronic acid. The total uronic acid residue content in the gum can be up to 35–40%. The remaining sugar residues are neutral. About 1% of proteinaceous components are also bound on structure, but the amino acids compositions vary widely with the different species.29 Commercial gum karaya contains about 30–43% galacturonic acid, 13–26% galactose, and 15–30% rhamnose after acid hydrolysis. Calcium and magnesium are the major cations linked with uronic acid on the gum structure. Gum karaya has a much higher rhamnose content than other commercial exudate gums. Structurally, it is a heavily acetylated acidic polysaccharide containing -Dgalacturonic acid and -L-rhamnose residues as the main chains with O-4 of the acid and O-2 of rhamnose linkages, and the acid is linked by 1,2 linkage of -D-galactose or by 1,3 linkage of -D-glucuronic acid on side chains, where one half of the rhamnose units carry at O-4 by 1,4 linkage of -D-galactose units (Fig. 13.2).11

Fig. 13.2

Structure of gum karaya (Sterculia urens).

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13.2.4 Technical data Gum karaya has a slightly acetous odour and taste. Colour of the gum varies from white to tan depending on grade. Cost is based on purity and colour. Powdered karaya contains about 10–14% moisture, but a loss on drying figure is higher than this due to the volatile substance. A typical specification of commercial gum karaya is shown below. Appearance: Loss on drying: Ash: Acid insoluble ash: Insoluble matter: Viscosity 1% sol: Particle size: Heavy metals (as Pb): Pb: As: Microbiology: Salmonella: E.Coli: Shelf life:

White to buff fine powder 20% maximum1 7% maximum 1% maximum 3.0% maximum 900–1200 cps2 90% minimum pass 150 mesh BSS 20 ppm maximum 5 ppm maximum 3 ppm maximum Absent in 25g Absent in 10g Minimum 12 months when stored in unopened packaging in a cool, dry place.

Notes: 1 105ºC–60 minutes 2 Basis Brookfield Viscometer, Spindle No. 3 20 rpm @ 25ºC 24 hours. (Specification of Luxara 4408 gum karaya, courtesy of Arthur Branwell & Co Ltd.)

Solubility Gum karaya is the least soluble of commercial gums and forms true solutions only at very low concentrations (< 0.02% in cold water, 0.06% in hot),27 but highly viscous colloidal dispersions can be produced at concentrations up to 5%, depending on quality. Due to acetyl group on the gum structure, gum karaya does not fully dissolve in water to give a clear solution, instead absorbing water rapidly to form viscous colloidal dispersion at low concentration. The fine mesh gum hydrates much more rapidly than coarser gum, and gives a smooth, homogenous solution, whereas coarse granules yield a grainy dispersion. Up to 4% of gum may be hydrated in cold water to give a viscous gellike paste of uniform smoothness and texture. Karaya will form viscous solutions in 60% alcohol, but is insoluble at higher concentrations. Deacetylation by using alkali in solution can modify the gum’s characteristics from water-swellable to water-soluble.27 Generally, gum karaya of Indian origin (mainly S. urens) has a higher acid value and a more pronounced acetic odour than that of African origin (mainly S. setigera), resulting in African karaya having a better solubility than Indian gum karaya; a factor sought by some users. Viscosity The viscosity of karaya dispersions ranges from about 120–400 cps for 0.5% dispersions to about 10,000 cps for 3% dispersions depending on the grade. Gum karaya, in the dry state, loses viscosity on ageing and builds an acetic odour. The loss of viscosity is related to the loss of acetic acid. The fine powdered gum suffers

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greater viscosity loss than the granules or the whole exudate. This decrease is most noticeable in the first few weeks after the gum has been ground. High temperature or high humidity storage is harmful to its stability, therefore its recommended storage temperature should not exceed 25ºC.30 Climate and time of harvest also affects the viscosity. In solution, karaya is more viscous when hydrated in cold rather than in hot water. Boiling temperatures longer than two minutes particularly reduce the viscosity. The viscosity of karaya solutions may decrease with added electrolytes. The dispersion is not sensitive to weak electrolytes, but when certain strong electrolytes are added, even in small amounts, loss of viscosity occurs.3 Therefore, salts should be added only after the gum has been fully hydrated. There is no distinct correlation between viscosity and grade. Where viscosity is important, powdered karaya should be used within six months after processing. Rheological properties When gum karaya absorbs water, the particles do not dissolve but swell extensively. Gum karaya solutions are thixotropic. The hydrated swollen particles are not stable to mechanical shear, prolonged stirring causes viscosity decrease. It does not possess the ‘pourability’ characteristic of gum tragacanth. pH stability The pH of a 1% solution is about 4.5–4.7 for Indian origin and 4.7–5.2 for African origin. The viscosity of solutions decreases upon the addition of acid or alkali. Higher viscosity can be obtained if the gum is fully hydrated prior to pH adjustment.31 Above pH8, alkali irreversibly transforms the characteristic short-bodied solution into a ropey, stringy mucilage as the molecules lose their acetyl groups through rapid saponification. This has been ascribed to deacetylation of the karaya molecule. Due to high uronic acid content, karaya dispersions withstand acid conditions quite well and resist hydrolysis in 10% hydrochloric acid solution at room temperature for at least eight hours.3 Heat stability Heating karaya dispersions changes the polymer confirmation and increases the solubility, but results in a permanent viscosity loss. Concentrations of 4–5% can be prepared as the maximum by cold water hydration, but by heating, particularly under pressure, smooth, homogeneous, translucent and colloidal solutions at concentrations as high as 18–20% can be obtained.31 Water-binding properties Gum karaya has a strong water-binding ability. It can absorb water and swell to more than 60 times of its original volume. Film-form properties Gum karaya forms smooth films when plasticised with compounds such as glycols. Adhesive properties At high concentrations of 20–50%, gum karaya shows strong wet-adhesive properties. This enables karaya gels and pastes to resist loss of strength when diluted.31

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Compatibility Gum karaya is compatible with most gums as well as proteins and carbohydrates. Blending karaya with other gums, such as alginate, can modify the solution characteristics.32 However, karaya gels are incompatible with pyrilamine maleate, a strong hydrotrope and antihistaminic. Strong electrolytes or excessive acid cause a drop in viscosity, while alkalis make karaya solutions very ropey.33 Preservative The viscosity of karaya solution remains constant for several days and decreases gradually with ageing, unless preservatives are used to prevent bacterial attack. Preservatives such as benzoic or sorbic acid, methyl and propyl parahydroxybenzoate, glycerol, propylene glycol, chlorinated phenols, formaldehyde, and mercuric salts, are suitable.

13.2.5 Uses and applications Applications of gum karaya are mainly based on its stable viscosity on acidic conditions (although less so than gum tragacanth), excellent water-binding and adhesive properties. It can be used as thickener and emulsifier in low pH products due to its acid resistance; as a binder in meat products, paper and textile industries, as an adhesive when partially wet with water; and as a gelling agent when with alkali such as sodium borate to form soft gels; it can also be used as a bulking agent when it absorbs water and swells, and as a modified gum (deacetylated or partially hydrolysed) using mild alkali and its properties are pH-irreversible.33 Although gum karaya is one of the least soluble gums, precautions have to been taken as powdered karaya tends to form lumps if directly introduce to water. Suitable dispersion methods are mentioned in Section 13.1.5. Food applications In general, gum karaya is used as stabiliser in mayonnaise, dressings, ice cream and ice lollies, whipped cream products, cheese spreads, ground meat products, sauces and chutneys, tomato extender and brawn and tomato sauces. 1.

2.

3.

Dressings: gum karaya is used in sauces and chutney, where its high viscosity, suspension and acid stability properties are suitable. Use levels of 0.6–1.0% give smooth consistency and good suspension. High temperatures and high shear mixing should be avoided. In French dressings, gum karaya is used as a stabiliser increasing the viscosity of the oil-in-water emulsion, thereby preventing or slowing down the rate of separation. Sometimes gum arabic is combined effectively to enhance emulsion stability on such applications. Ice creams and frozen desserts: karaya, at 0.2–0.4% alone or at 0.15% with 0.15% locust bean gum, stabilises ice cream, ice lollies and sherbets by preventing the formation of large ice crystals and preventing the migration of free water or syneresis due to its excellent water-binding property. It effectively prevents the flavour and sugar to be sucked out of the ice lollies during consumption, leaving just a block of ice. It also helps to control overrun and minimise shrinkage. Dairy products: karaya has effective foam stabilisation properties and can be used as a stabiliser to keep whipped cream and other aerated dairy products from breaking. Due to water-absorbing properties, karaya is used in meringue powders to enable a greater volume of meringue to be prepared from a fixed amount of protein. In cheese

244

4.

5.

6.

Handbook of hydrocolloids spreads, at 0.8% or less, karaya is used as a binder to prevent water separation and to increase spreadability. Its acidic nature is not objectionable in these dairy products. Meat products: karaya, at about 0.3%, is used in sausages and comminuted meat products to improve adhesion between meat particles and to bind water during preparation and storage. It gives improved body, smooth texture and appearance, and serves to emulsify the protein, fat and moisture in the products. Bakery: karaya can be used in bakery products to improve tolerance to variations in water addition and mixing time. It is effective in retarding the staling of baked goods when combination with alginate or carrageenan at about level 0.01–0.02% when karaya is used at 0.1–0.9% level.31 Health foods: karaya may be used as a dietary supplement in health foods.34

Pharmaceuticals and cosmetics The major consumption of gum karaya is in the pharmaceutical industry. Karaya is utilised largely in medical colostomy bag fixings as an adhesive, such as for stoma seals after surgical operations. It was also largely used in dental fixatives, mainly for false teeth. As a denture adhesive, the gum, dusted on the dental plate, swells when it contacts with the moist surfaces of the mouth. This results in a more comfortable and tighter fit of the plate. The rapid swelling of the karaya particles, their relative insolubility, and their resistance to bacterial and enzymatic breakdown make the gum suitable for this purpose. A small amount of mild alkali added to the powder improves its adhesion. Coarse particles of karaya (8–30 mesh), with a protective coating, mainly in the form of chocolate-flavoured granules, are used as a bulk laxative as they absorb water and swell to 60–100 times their original volume on entering the gut, forming a discontinuous type of mucilage. The gum is not digested nor absorbed by the body. Gum karaya is also used in skin fixings for medical electrodes. Paper industry Gum karaya was used as a binder to make long-fibre, lightweight papers, such as condenser tissues and fruit wrap tissues. These long fibres form clusters or ‘flocks’ in the paper web. Karaya effectively deflocculates these fibres and maintains their uniform distribution in the paper web, resulting in improved formation and strength in the lightweight sheets. Before adding to the pulp suspension, the acetyl groups of the karaya must be removed by treating the gum with ammonia or other weak alkali. This exposes more active carboxyl and hydroxyl groups and increases the binding of the gum to the cellulose fibres. This deacetylated gum is added to the pulp suspension at about 1:200 ratio of pulp. This use of karaya in the paper industry is a very limited but important application in lightweight papers from long cellulose fibres. Textile applications Gum karaya was used as a thickener for the dye in the colour printing on cotton fabrics. For this use, the karaya must be cooked in water under pressure to make the karaya more soluble. Under these conditions, it forms a smooth, homogeneous, translucent, colloidal dispersion of 15–18% dry solids. Miscellaneous applications In blends with xanthan gum, gum karaya has been used as an ingredient in petroleum and gas recovery.33 Similar to gum tragacanth, more and more applications traditionally using

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gum karaya have been replaced by other hydrocolloids, such as guar gum, xanthan gum or gum combinations through product reformulation.

13.2.6 Regulatory status Gum karaya is classified as ‘generally recognised as safe’ (GRAS) within the USA. It is also classified as ‘ADI not specified’ by the Joint WHO/FAO Expert Committee on Food Additives (JECFA), and has the number E416 in the list of additives approved by the Scientific Committee for Food (SCF) of the EEC. However, it restricts the human intake of gum karaya to an upper limit of 12.5mg/kg body weight per day based on the toxicological studies of gum karaya.35 Recent registration in the European Union has limited gum karaya use in the following products:36 cereal and potato-based snacks; nut coatings; fillings, toppings and coatings for fine bakery wares; desserts; emulsified sauce; egg-based liqueurs; chewing gum; dietary food supplements.

13.3 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

References

Food Chemicals Codex, 4th edn, 1996. European Pharmacopoeia 3rd edn, 1998. WHISTLER, R. L. Exudate Gums in ‘Industrial Gums’ 3rd edn (Whistler, R. L. and BeMiller, J. N. eds) USA, Academic Press Inc, 1993, 318–37. DOGAN, M., EKIM, T. and ANDERSON, D. M. W. ‘The production of gum tragacanth from Astragalus microcephalus in Turkey’. Biological Agriculture and Horticulture, 1985, 2 (4), 329–34. ANDERSON, D. M. W. and WANG WEIPING, ‘The tree exudate gums permitted in foodstuffs as emulsifiers, stabilisers and thickeners’, Chemistry and Industry of Forest Products, 1994, 14 (2), 73–84. ROBBINS, S. R. J. ‘A review of recent trends in selected markets for water-soluble gums’, Overseas Development Natural Resources Institute Bulletin No. 2 London, 1988. ANDERSON, D. M. W. and BRIDGEMAN, M. M. E. ‘The composition of the proteinaceous polysaccharides exuded by Astragalus microcephalus, A. gummifer and A. kurdicus – The sources of Turkish gum tragacanth’. Phytochemistry, 1985, 24, 2301–4. ANDERSON, D. M. W. and BRIDGEMAN, M. M. E. ‘The chemical characterisation of the Test Article used in toxicological studies of gum tragacanth’. Food Hydrocolloids, 1988, 2 (1), 51–7. GRALEN, N. and KARRHOLM, M. ‘The physicochemical properties of solutions of gum tragacanth’, J. Colloid Sci., 1950, 5 (1), 21–36. ANDERSON, D. M. W. ‘Evidence for the safety of gum tragacanth (Asiatic Astragalus spp.) and modern criteria for evaluation of food additives’, Food Additives and Contaminants, 1989, 6 (1), 1–12. STEPHEN, A. M. and CHURMS, S. C. ‘Gums and Mucilages’ in Food Polysaccharides and Their Applications (Stephen, A. M. ed.), New York, Marcel Dekker Inc. 1995, 377–425. ANDERSON, D. M. W. and GRANT, D. A. A. ‘Gum exudates from four Astragalus species’, Food Hydrocolloids, 1989, 3 (3), 217–23. ASPINALL, G. O. and BAILLIES, J. ‘Gum tragacanth, Part I. Fraction of the gum and the structure of tragacanthic acid’, J. Chem. Soc., 1963, 1702–14. ASPINALL, G. O. and PUVANESARAJAH, V. ‘The hex-5-enose degradation: cleavage of 6-deoxy-6-iodo-(-Dgalactopyranosidic linkages in modified carboxyl-reduced methylated Tragacanthin acid’, Can. J. Chem. 1984, 62: 2736. ASPINALL, G. O. and BAILLIES, J. ‘Gum tragacanth, Part II. The arabinogalactan’, J. Chem. Soc., 1963, 1714–21. STAUFFER, K. R. ‘Gum tragacanth’ in Handbook of Water-soluble Gums and Resins (Davidson, R. L. ed.), New York, McGraw-Hill, 1980, Chap. 11. WAREING, M. V. ‘Exudate gum in Thickening and gelling agents for food 2nd edn (Imeson, A. ed.), London, Blackie 1997, 86–118. GLICKSMAN, M. ‘Gum tragacanth’ in Food Hydrocolloids Vol. II, USA, CRC Press, 1982, 49–59. STAUFFER, K. R. and ANDON, S. A. ‘Comparison of the functional properties of two grades of gum tragacanth’, Food Technology, 1975, 29 (4), 46. GRIFFIN, W. C. and LYNCH, M. J. Handbook of food additives 2nd edn (Furia, T. E. ed.), Cleveland, The Chemical Robber Co, 1972, p. 397. ANDERSON, D. M. W. and ANDON, S. A. ‘Water-soluble food gums and their role in product development’, J.

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Handbook of hydrocolloids American Association Cereal Chemists, 1988, 33 (10), 844–50. GLICKSMAN, M. ‘Gum Tragacanth’ in Food Hydrocolloids Vol. II, USA, CRC Press, 1982, 49–59. BEACH, D. C. ‘History, Production, and Uses of Tragacanth’ in Natural Plant Hydrocolloids, American Chemical Society, 1954, USA, 38–44. WANG WEIPING and ANDERSON, D. M. W. ‘Non-food applications of tree gum exudates’, Chemistry and Industry of Forest Products, 1994, 14 (3), 67–76. EASTWOOD, M. A., BRYDON, W. G. and ANDERSON, D. M. W. ‘The effects of dietary gum tragacanth in man’, Toxicology Letters, 1984, 21, 73–81. GORDON, I. R. ‘Food applications of hydrocolloids in Western Europe in the 90s’, Gums and Stabilisers for the Food Industry 6, (Phillips, G. O. et al. eds), 1991, Oxford, 29–42. LE CERF, D., IRINEI, F. and MULLER, G. ‘Solution properties of gum exudates from Sterculia urens (karaya gum)’ Carbohydr. Polym., 1990, 13 (4), 375–86. ANDERSON, D. M. W., MCNAB, C. G. A., ANDERSON, C. G., BRAOWN, P. M. and PRINGUER, M. A. ‘Gum exudates from the genus Sterculia (gum karaya)’. International Tree Crops J., 1982, 2, 147–54. ANDERSON, D. M. W., HOWLETT, J. F. and MCNAB, C. G. A. ‘Amino acid composition of proteinaceous component of gum karaya (Sterculia spp)’, Food Additives and Contaminants, 1985, 2, 159–64. British Pharmacopoeia, 1998. GLICKSMAN, M. ‘Gum karaya’ in Food Hydrocolloids Vol. II, USA, CRC Press, 1982, 39–48. LE CERF, D. and MULLER, G. ‘Mechanical spectroscopy of karaya-alginate mixed dispersions’, Carbohydr. Polym., 1994, 23 (4), 241–6. MEER, W. ‘Gum karaya’, in Handbook of water-soluble Gums and Resins (Davidson, R.L. ed.), New York, McGraw-Hill, 1980, Chap. 10. EDWARDS, C. A. and EASTWOOD, M. A. ‘Caecal and faecal short-chain fatty acids and stool output in rats fed on diets containing non-starch polysaccharides, Br. J. Nutr. 1995, 73 (5), 773–81. ANDERSON, D. M. W. ‘Evidence for the safety of gum karaya (Sterculia spp.) as a food additive’. Food Additives and Contaminants, 1989, 6 (2), 189–99. Official Journal of the European Communities, 1995, March 18th, L61, Vol. 38, 1–40.

14 Xyloglucan K. Nishinari (Osaka City University), K. Yamatoya and M. Shirakawa (Dainippon Pharmaceutical Co. Limited)

14.1

Introduction

Xyloglucan is a major structural polysaccharide in the primary cell walls of higher plants.1 Cell growth and enlargement are controlled by the looseness of a thin net of microfibrils made of cellulose. Xyloglucan cross-links these cellulose microfibrils and provides the flexibility necessary for the microfibrils to slide. Figure 14.1 shows potential linkage between xyloglucan and cellulose.1 It has been suggested that the cleavage of cross-linking xyloglucan by endolytic enzymes is necessary for cell enlargement during growth.1 Some of the xyloglucan oligosaccharides released by -glucanase have biological functions and are capable of promoting plant cell growth.2 Seeds of the tamarind tree (Tamarindus indica) contain a xyloglucan. Tamarind seed xyloglucan (TSX) is used as a food additive in Japan. The flow behaviour of the solution is very close to Newtonian, and very stable against heat, pH and shear. The various applications of tamarind seed xyloglucan include thick sauce, ice cream, dressing and processed vegetables. Tamarind xyloglucan is expected to find new food applications, serving as a thickener and stabiliser, gelling agent, ice crystal stabiliser and starch modifier, etc. It is called ‘ageing free starch’, because its property is similar to starch but is more stable. The enzymatic modification of xyloglucan to elicit new biological function or a unique property is also highlighted in this chapter.

14.2

Origin and manufacture

Xyloglucan is found in the primary cell walls of all higher plants. It has been characterised from Dicotyledons (e.g., hypocotyls of Phaseolus, fruits of Olea, Malus and Phaseolus, fibres of Lactuca, and cell cultures of Acer, Rosa, Glycine and Populus); Monocotyledons (e.g., bulbs of Allium, endosperm of Oryzae, seedlings of Oryza and Hordeum, shoots of Bamboos, and cell cultures of oryza), and Gymnosperms (e.g., cell cultures of Pseudotsuga).3 Xyloglucan may make up 20–30% of the dry weight of the primary wall; amounts are often lower (e.g. 5–10%) in the Gramineae. The seeds of a few

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Fig. 14.1

Potential linkages between xyloglucan and cellulose.

plants (e.g. Tamarindus, Impatients, Annona, Tropaeolum, Hymenaea, Detarium) have abundant deposits of xyloglucan surrounding their cotyledonary cells. Some xyloglucan is extractable from tissue with cold water. However, alkali is required for extraction of xyloglucan from cell walls in most cases. Tamarind seeds have long been consumed as food and feed for their sugars, protein and oils since before the birth of Christ.4 In India, Southeast Asia, West Indies, Brazil and elsewhere, numerous tamarind trees are grown for food (fruit) and ornamental use. Figure 14.2 shows areas where tamarind trees are grown. Tamarind trees grow fast; the mature evergreen tree can reach a height of 25–30m. The trees start to blossom and bear fruit 12– 13 years after planting. They continue to bear much fruit for more than 60 years, and are

Fig. 14.2

Areas where tamarind trees are grown.

Xyloglucan

249

said to survive over 120 years. Tamarind trees are popular for their splendid appearance and long life span, and the many benefits they provide; they are closely associated with many aspects of people’s everyday life. In the northern hemisphere, tamarind trees blossom April through May, and bear fruit from autumn through winter. A mature tamarind tree produces 200–250kg of fruit annually. The tamarind seed has a flat, irregular shape, being round, oval, or four-sided.5 The length of a side is about 0.6in. and the thickness is about 0.3in. The seed has an outer brown seed coating or hull or testa comprising about 30% of the weight and covering a whitish to tan coloured, cereal-like kernel which accounts for 70% by weight. The isolated tamarind seed kernels resemble cereals and have the following approximate composition: protein 15.4–22.7%; oil 3.9–7.4%; crude fibre 0.7–8.2%; dietary fibre 65.1– 72.2%; ash 2.45–3.3%. Tamarind gum (or tamarind kernel powder, unpurified xyloglucan) was discovered during a search for new sizing materials during shortages caused by the Second World War.5 It came into commercial production in 1943 as a replacement for starch in cotton sizing used in Indian textile mills. In the USA, its major industrial use has been as a wet end additive in the paper industry as a replacement for starches and galactomannans. Purified tamarind seed gum or TSX was successfully industrialised first in the world by Dainippon Pharmaceutical Co., Ltd., Japan in 1964. Regarding TSX preparation, basically the first process is washing the seeds with water and heating to make the testae or seed coating brittle and friable.5 The seeds are decorticated to leave the heavier crushed endosperm which is then ground to yield tamarind kernel powder (TKP). This TKP or tamarind flour usually contains at least 50% of the TSX. For more purification, TKP is boiled and agitated with about 30–40 times its weight of water for about 30–40 min. and allowed to sit overnight in a settling tank to allow the protein and fibre to precipitate and settle out. The supernatant liquid is concentrated to about half its volume, mixed with filter-aid and filtered through a filter press. The purified liquid is then dried and ground to yield a purified seed extract. Additional purification processes can be done using precipitation with salts and washing with alcohol. The only seed xyloglucan exploited commercially at present is obtained from the seed of the tamarind tree.4,5 This is manufactured by separating and purifying the major component of starting material tamarind seeds using water. It is available commercially as Glyloid from Dainippon Pharmaceutical Co., Ltd. in three types of TSX. Glyloid 3A is insoluble in cold water and requires heating to 75ºC for at least 15 min. for solubilisation. Glyloid 3S forms viscous solutions in cold water without heating. Glyloid 6C is also cold water soluble, but a highly transparent type. Non-commercial hydrolysed TSX was prepared as below.6 TSX (final concentration, 20%) was allowed to react with 2% endo-(1!4)- -glucanase from Trichoderma viride at pH4.0 (adjusted by citric acid) and 60ºC. After this reaction, which cleaved the main chain, the reaction mixture was heated at 100ºC for 30 min. to inactivate the enzyme. The mixture was then centrifuged at 8,000 rpm for 15 min. to remove insoluble materials, and then filtered. The filtrate was concentrated, neutralised, and dried to yield hydrolysed TSX. The hydrolytic reaction process was analysed by HPLC (column, Shodex Ionpak KS805 + Shodex Ionpak KS802; flow rate, 1 ml/min.) using water as the elute at 60ºC, with the aid of an RI detector. The constituent ratio of xyloglucan heptasaccharide, octasaccharide and nonasaccharide was determined by HPLC (column, TSK gel Amide 80; flow rate, 0.6 ml/min.; room temperature) using a 50% (w/w) aqueous solution of acetonitrile as the elute, with the aid of an RI detector.7

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14.3

Handbook of hydrocolloids

Structure

Xyloglucan polysaccharide has a -(1!4) linked D-glucan backbone that is partially substituted at the O-6 position of its glucopyranosyl residues with -D-xylopyranose.8 Some of the xylose residues are -D-galactosylated at O-2. The unit structures of tamarind xyloglucan, xyloglucan heptasaccharide (Glu4Xyl3), octasaccharide (Glu4Xyl3Gal) and nonasaccharide (Glu4Xyl3Gal2) are shown in Fig. 14.3. The ratio galactose:xylose:glucose was 1:2.25:2.89 and the constituent ratio of xyloglucan heptamer, octamer and nonamer was 13:39:486 in TSX. The architecture of tamarind seed xyloglucan has been investigated by light scattering, small angle X-ray scattering (SAXS) and synchrotron radiation.10 The data shows that TSX in aqueous solution consists of multi-stranded aggregates, with a high degree of particle stiffness. A statistical Kuhn length Lk is 150nm and the cross-sectional radius of ˚ . The conformation of xyloglucan oligosacchargyration is estimated as Rgcs = 6.00.5A ides chain was simulated by a Monte Carlo method. The radius of gyration Rg and the cross-sectional radius of gyration Rgc were evaluated for xyloglucan oligomers from SAXS.11 The SAXS profiles from xyloglucan heptamer, octamer and nonamer were fitted to those calculated from tri-axial bodies where an ellipsoid was found to represent scattering from each xyloglucan oligomer (Fig. 14.4). The results suggested that xyloglucan has a rather flat structure characterised by an elliptic cross-section of semi-

Fig. 14.3

The unit structures of tamarind xyloglucan.

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251

Fig. 14.4 Small-angle X-ray scattering from (a) xyloglucan heptamer, (b) octamer and (c) nonamer. The solid lines represent the scattering from respective ellipsoids with the radius of gyration and three semi-axes values shown in the figure.

axes 0.23nm and 0.88nm (for a nonamer). A larger radius of gyration was estimated for xyloglucan heptamer, indicating that the lack of galactose side chain promoted an aggregation in a solution (Rg; heptamer: 0.86nm, octamer: 0.62nm, nonamer: 0.77nm). The greater stiffness of the chain of TSX might be partially due to the high proportion of

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Fig. 14.5 Sterically refined molecular models of tamarind seed polysaccharide projected normal and parallel to their molecular axes for (a) ' ÿ60º, (b) ' 60º, and (c) ' 180º. The broken lines denote hydrogen bonds.

side chains.9 The stiffness of the chains or ‘hyperentanglements’ is not solely due to hindered rotation but also to specific aggregation in solution. Molecular weight of TSX was reported 115,0005 or 650,000 (GPC)4 or 880,000 (light scattering)9 or 2,500,000 (5 molecular aggregates?).10 Xyloglucan has been shown to occur fairly evenly over the area of the cell wall by using labelled antibodies and lectins.3 Differing from TSX, many of the galactose residues are -L-fucosylated at O-2 in cell wall xyloglucan. In grasses and in the other seed storage xyloglucans, fucose is rare or absent. The limited number of -glucanase- or cellulase-digestion products obtained from xyloglucan indicates a high degree of regularity in the arrangement of substituents along the -glucan backbone but their distribution or lining is random. Also the distribution is different in the xyloglucans from different species and has even been shown to differ slightly, but significantly, between two natural populations of the same species growing in different environments.12 Figure 14.5 shows models of TSX using X-ray fibre diffraction analysis and molecular modelling.13 The results show that, in the solid state, the backbone adopts a cellulose-like conformation, and that three types of conformation are sterically accessible to the sidechains. Xyloglucan isolated from the elongated regions of pea stems, which has fucosyl-galactosyl-xylosyl residues, was also suggested to have the backbone with an extended two-fold helix similar to cellulose.14

Xyloglucan

14.4

253

Technical data

Tamarind seed xyloglucan has unique physical properties in a water solution.4 • • • •

Newtonian flow (starch-like fluidity) synergism with sugars (thickening, gelling) outstanding stability (heat, acid, salt, mechanical) strong water retention.

14.4.1 Conformation and rheology Figure 14.6 shows the viscosities of aqueous solutions of TSX. Its 1% solution has a viscosity of about 150 cP (type B viscometer; 25ºC; shear rate 8.6sÿ1). TSX shows an intermediate position among polysaccharides, in terms of relation between solution concentration and viscosity. Figure 14.7 shows the temperature dependence of viscosity of TSX solutions with various concentrations. Aqueous solutions of TSX exhibit Newtonian fluidity, unlike most polysaccharides, such as xanthan gum (Fig. 14.8). Newtonian fluids offer the advantage of easy serving, owing to smooth flow from the

Figure 14.6

Viscosity of natural gums.

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Fig. 14.7

Viscosity of TSX (Tamarind Seed Xyloglucan) at various temperatures.

mouths of bottles and other containers. For TSX, an average intrinsic viscosity of [] = 60.5dL/g was obtained.9 From the radius of gyration and molecular weight from light scattering, an attempt can be made to predict the intrinsic viscosity from the FoxFlory theory. This approach yields a theoretical intrinsic viscosity = 50.4 dL/g, which is about eight times higher than a corresponding experimental value. This discrepancy may be due to polydispersity, excluded volume, and draining effects.10–12 Figure 14.9 shows the frequency dependence and temperature dependence of storage shear modulus G0 and loss shear modulus G00 of xyloglucan solutions of various concentrations.15 G00 was larger than G0 at all the frequencies from 10ÿ1 to 102 rad/s for a 1.05% TSX solution whilst G0 and G00 showed a crossover around the angular frequency 102 rad/s for TSX solutions with higher concentrations (Fig. 14.9(a)). Both G0 and G00 at a fixed frequency 1rad/s decreased monotonically with increasing temperature and G00 was always larger than G0 (Fig. 14.9(b)), which is consistent with a heating DSC curve showing no endothermic or exothermic peak.15 TSX offers natural texture or viscosity with starch-like body. The aqueous solution of TSX does not show a spinnability.

14.4.2 Synergism with sugars The viscosity of TSX solution increases by the addition of a sugar such as sucrose, glucose or starch syrup (Fig. 14.10). The degree of synergism varies to some extent, depending on the type of sugar.

Xyloglucan

Fig 14.8

255

Viscometer speed and viscosity.

14.4.3 Gelation TSX has the ability to form gels in the presence of sugar or alcohol. In this respect, it is similar to pectin. It forms a gel in the presence of 40–65% sugar over a wide pH range (Fig. 14.11). This gel shows high elasticity and low water release. If alcohol (up to 20%) is added, the amount of sugar needed to form a gel can be substantially reduced to get all of the gum into solution and even eliminated (Fig. 14.12). The correlation of the amounts of alcohol and sugar required for TSX gelation can be expressed by the following equation: 55 2A S where A is alcohol concentration, and S is sugar concentration. Gels with alcohol are harder and have lower melting point than gels with sugar.

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Fig. 14.9 (a) Frequency dependence of G0 and G00 for dispersions of xyloglucan. C.: Concentration of xyloglucan, Measurement temperature: 5ºC, Strain: 0.1 ● ; 1.05 wt% G0 , ❍ ; 1.05 wt% G00 , ■ ; 1.40 wt% G0 , ❒ ; 1.40 wt% G00 , ▼ ; 1.75 wt% G0 , 4; 1.75 wt% G00 . (b) Thermal scanning rheological measurements for dispersion of xyloglucan. Heating rate: 1ºC/min, Frequency: 1 rad/s, Strain: 0.05.

Fig. 14.10 Synergistic effect of TSX with sugar.

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Fig. 14.11 Sugar concentration and gel strength.

Fig. 14.12

TSX gelation with alcohol and sugar.

To form a gel, heating is required to dissolve polysaccharides, and upon cooling to room temperature, the gel will form. The freeze-thaw process makes TSK-sugar gel harder and more elastic. Preliminary X-ray fibre and powder diffraction or simulation of molecular conformations indicated xyloglucan backbone adopts a cellulose-like conformation.14,16 So in the condensed state, the conformation of the molecule is very similar to that of cellulose. Gelation in high sucrose concentration is, therefore, likely to involve chainchain association by aggregation of a regular, ribbon-like, two-fold, cellulose-like conformation.16,17 Gel formation rather than precipitation occurs because the sugar

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substituents along the chain limit the degree of aggregation. TSX might form random aggregates and a cross-linking domain was formed to make a gel.

14.4.4 Stability Figure 14.13 shows the heat stability of a TSX solution. TSX is very stable under a hightemperature heating process. Figure 14.14 shows the acid stability of a TSX aqueous solution. TSX is very stable to acid and salts over extended periods. TSX shows excellent stability in high salt concentrations of salt (20%). It is stable; salting-out or viscosity reduction does not occur even at high saline concentrations. TSX itself is very stable against mechanical shear. TSX also gives stability to starch. Corn starch dispersion alone decreased viscosity about 50% after treatment of colloid mill (clearance 0.3m/m, 5000rpm, recycle 3 times), but TSX itself or combining it with corn starch gave almost no change of viscosity when the same treatment was done.

Fig. 14.13 Heat resistance of TSX.

Fig. 14.14 Acid resistance of TSX and locust bean gum.

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14.4.5 Compatibility TSX exhibits no remarkable synergism with other hydrocolloids as found between xanthan gum and galactomannan. Nor is there any offsetting effect. With compatibility, TSX can be used in combination with other polysaccharides without interaction.

14.4.6 Iodine colour reaction When a solution of potassium iodide (0.1M) is added to xyloglucan solution to make the final concentration of potassium iodide to be 0.0002M, purple or light blue colour appeared. TSX or other xyloglucan, similar to starch, have been called ‘amyloids’ because of the characteristic blue stain that is produced with iodine/potassium iodide solution. The name of amyloid may have been derived from amylose or amylopectin.

14.5

Uses and applications

Tamarind seed xyloglucan (TSX) has been used widely as a food additive in Japan. The principal uses of TSX are in sauce, dressing and mayonnaise, ice cream and some flour products (Table 14.1).4,5 Usage of TSX depends on its purpose and foods, but generally speaking about 0.1% to 0.5% of TSX is used for food applications. Dissolution is the critical requirement in using TSX. Complete dissolution ensures full utilisation of the various features of TSX like other polysaccharides. The factors in successful dissolution are: • • • • • •

use as much water as possible (lower Brix recommended) use high mixing rate (rapid stirring) add little by little (to prevent formation of lumps). disperse not dissolve at first stage, if possible (to prevent formation of lumps). use low temperature solution use alcohol as dispersant (to prevent formation of lumps) (TSX does not dissolve in a high concentration alcohol) • use oil as dispersant. Basically, while water is stirred at high speed, TSX should be added little by little, followed by heating or TSX previously dispersed in a five-fold sugar (in dry blend) of alcohol or oil, is then added to water.

14.5.1 Thickener or stabiliser As a thickener, TSX provides good viscosity character without pastyness and spinnability. TSX also provides body or dense mouthfeel. Since the texture of TSX is similar to starch and more stable than starch, TSX can replace starch or in many cases TSX can be used in combination with starch. TSX has been used in the preparation of pork cutlet sauces and meat sauces, because these sauces are required to be highly viscous and stable at low pH for a long period. Very small levels (0.05%) of TSX provide body or improve texture in low-fat milk, fruit beverages or cocoa. As TSX provides suspension stability of small particles, it can be used in fruit-pulp beverages or shiruko (sweet soup with rice cake). In batter mix for fried products, it is important to hold viscosity stability during the battering process. Even if an enzyme in

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Table 14.1

Food applications

Action

Effects

(i)

❍

Thickener and stabiliser

❍ ❍ ❍ ❍

(ii) Gelling

❍ ❍ ❍ ❍

(iii) Ice crystal stabilisation

❍ ❍ ❍ ❍ ❍

(iv) Starch modification

❍ ❍ ❍ ❍

Provides good viscosity free of pastyness and trailing threads. Provides density and body. Provides stable emulsification. Suspends particles for stable dispersion. Retains water.

Subject foods Pork cutlet sauces, okonomiyaki sauces, meat sauces, fruit sauces, hamburger sauces, batters, pickles, yakiniku sauces, yakitori sauces, low-fat milk, coffee milk, dressings, mayonnaise-like seasonings, fruit juice beverages, shiruko, cocoa, seasoning solutions, seaweed tsukudani, enoki mushroom tsukudani.

Forms water release-free gel by Fruit jellies, cocktail jellies, low-sugar synergism with sugar. jams, youkan, kuzu mochi, frozen Forms gel by synergism with jellies, kelp tsukudani. alcohol. Forms gel resistant to freezethawing. Offers water retention by gel. Forms fine ice crystals. Offers excellent shape retention. Excellent acid resistance ensures stable overrun Thread-free viscosity offers good dissolution in the mouth. Good compatibility with other stabilisers offers versatile performance.

Ice creams, frozen dessert, glazing.

Suppresses starch ageing. Confers heat resistance upon starch, for protection. Improves wheat flour product texture. Confers mechanical strength upon starch, for protection.

Custard creams, flour pastes, gyoza shells, curry, stew, Chinese noodles, Japanese noodles, rice cake, dango, Japanese traditional confectionery.

wheat flour reduces the viscosity of a batter mix, TSX can provide viscosity stability during all battering processes.

14.5.2 Emulsion stabiliser As TSX ensures stable emulsification, it has been used in salad dressing and mayonnaise. TSX provides smaller oil particles in emulsion than xanthan gum or carrageenan, while the stabilisation effect of TSX is rather weaker than xanthan gum. The emulsion stabilising properties of TSX can be increased in combination with xanthan gum.

14.5.3 Ice crystal stabilisation TSX alone or in combination with other polysaccharides (e.g., guar gum, locust bean gum or carrageenan) is a very effective stabiliser in frozen dessert such as ice cream and

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261

sherbet. TSX gives a good overrun, no whey-off, excellent heat shock stability and good water-holding properties without separation of ice crystals and sugar after long storage.

14.5.4 Gelling agent TSX forms an elastic gel with concentrated sugar solutions over a wide pH range. The texture of gels of TSX is not affected by boiling in neutral aqueous solutions because of its heat stability. The gel is water-release free and resistant to freeze-thawing. It can be used as a substitute for pectin fruit jelly or jam. Some Japanese traditional desserts such as youkan or kuzu mochi can also be produced using TSX.

14.5.5 Water holding property TSX, as with other polysaccharides, has many OH groups in the molecules and an extended chain with branched side chains. Thus TSX chains are hydrophilic and bind to water strongly. This strong water holding capacity gives good appearance or burnishing in tsukudani (seaweed products). A small amount of TSX in carrageenan gel or agar gel prevents water release.

14.5.6 Starch modification TSX can suppress starch ageing in some conditions. Also it confers heat stability and mechanical strength upon starch for protection. TSX improves texture of starch, especially wheat flour. In combination with starch or as a replacement for starch, TSX has been used in custard cream, flour paste, stew, noodles and Japanese traditional confectionery (e.g. dango or rice cake). From the study of the effects of TSX on the gelatinisation and retrogradation of corn starch, it was suggested that xyloglucan molecules might entangle with corn starch and prevent the structure reordering, and hence retard the retrogradation during long storage.15 TSX was also effective in reducing the syneresis of starch dispersions.

14.5.7 Fat replacer With recent consumer demand for healthy foods and nutrition aspects it has been indicated that polysaccharides solution show the desired emulsion-like properties. As fat replacer or fat mimetic, TSX solution has excellent mouthfeel and fat/oil like properties (expect fried property and nutrition). Fat-reduced dressing and mayonnaise products are now an important application of TSX. In many cases xanthan gum or other polysaccharides and starch or dextrin are used in combination with TSX.

14.6

Regulatory status

As all higher plants contain xyloglucan polysaccharides, a large amount of xyloglucan may be eaten from many vegetables or fruits. As a food additive, purified TSX has been permitted in Japan and South East Asian countries (Taiwan and South Korea).4 TSX is not permitted in food in the USA and EU at the present time. TSX has undergone a battery of rigorous safety studies, including acute toxicity,

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subacute toxicity, mutagenecity study and three chronic toxicity studies. There were no TSX-related abnormal findings. It was reported that TSX incorporation at the levels of 4, 8, and 12% in a standard diet to rats for two years showed no significant changes.18 No significant changes were observed in body weight, food intake, biochemical analysis of urine and blood, hematological test, organ weight and histopathological findings of rats receiving TSX.

14.7

Modification of tamarind seed xyloglucan

14.7.1 Partial hydrolysis Xyloglucan is an indigestible polysaccharide that acts as a dietary fibre. Dietary fibre has been shown to control blood glucose and reduce cholesterol.19 These effects are important for the treatment and prevention of chronic diseases, including diabetes and cardiac disorders. Some water-soluble dietary fibres are so viscous that it is difficult to add them to food in amounts sufficient to have physiological effects without changing the texture of the food. To solve this problem, a strategy has been developed which involves the partial hydrolysis of these polysaccharides, which reduces their viscosity to allow for easy intake while retaining their physiological activities.20 TSX also was hydrolysed partially as described in a previous section. In rats receiving high-cholesterol diets, the addition of TSX and hydrolysed TSX significantly increased cecum weight and reduced adipose tissue.21 TSX significantly reduced plasma total cholesterol, plasma -lipoprotein, liver total lipid, liver cholesterol and liver triglyceride (Table 14.2). Hydrolysed TSX significantly reduced plasma triglyceride and liver triglyceride (Table 14.2). Intake of high-fat diets and hydrolysed TSX in rats also reduced total lipid, cholesterol, triglyceride and -lipoprotein in blood parameters (Table 14.3).6 Among the hepatic lipids, total lipid, cholesterol, triglyceride and phospholipid were significantly reduced by hydrolysed TSX (Table 14.4). These findings were the first to demonstrate the biological activity of hydrolysed TSX in animals. The mechanism by which water-soluble dietary fibres lower cholesterol levels remains to be established. This hypocholesterolemic effect of fibres may be due to two common Table 14.2

Plasma and liver lipids in rats fed a high-cholesterol diet

Lipids

Control

Xyloglucan

Hydrolysed xyloglucan

Plasma (mg/dl) Total cholesterol Triglyceride -lipoprotein

13127 19.14.8 277147

8611(**) 16.39.7 11331(**)

12322 10.03.0(**) 281110

1.100.22

0.950.15

0.990.11

24819 13321 49.56.1 9.31.4

16517(**) 7011(**) 41.55.84 7.71.18(*)

23517(*) 12214 40.75.7(*) 6.21.4(**)

Free fatty acid (mEq/l) Liver (mg/g) Total lipid Total cholesterol Triglyceride Adipose tissue weight (g)

Values are mean SD for 7 rats. * p < 0.05, ** p < 0.01: significantly different from the corresponding value in rats fed a control diet.

Xyloglucan Table 14.3

263

Plasma lipids (mg/dl)

Plasma lipids Total lipid Total cholesterol Triglyceride -lipoprotein

Control

Hydrolysed xyloglucan

1646105 16817 77465 1020111

1367258* 14425 656189 848219

Values are mean SEM for 6 rats. * p < 0:05: significantly different from corresponding value in rats fed a control diet.

Table 14.4

Liver lipids (mg/g)

Liver lipids Total lipid Total cholesterol Triglyceride Phospholipid

Control

Hydrolysed xyloglucan

1077.0 17.12.3 49.43.6 20.63.9

87.63.2** 11.62.4** 41.94.4* 15.31.3*

Values are mean SEM for 6 rats. ** p < 0:01, * p < 0:05: significantly different from corresponding value in rats fed a control diet.

attributes: they are viscous and/or fermentable by intestinal bacteria.19 Viscous intact TSX rather than hydrolysate has a greater effect in lowering cholesterol in a high cholesterol diet. The hypolipidemic effects of lower molecular weight dietary fibres may not be solely due to the inhibition of lipid absorption. Another possible mechanism related to low-viscosity dietary fibres is that they may affect the endogenous metabolism of lipids. The production of short-chain fatty acids upon the fermentation of fibres has been proposed to explain the link between fibre intake and cholesterol lowering. It has been postulated that propionate has an inhibitory effect on cholesterol synthesis.22

14.7.2 Tailoring of TSX properties using an enzyme As it is indicated that galactose side chains are the major structural features determining the water solubility of TSX, it is concluded that the removal of these residues from TSX increases the interaction among each polymer. It has been reported that gelation occurred when about 50% of galactose were released from TSX using -galactosidase from a plant.23 Otherwise, gelation occurred by removing about 35% of the galactose residues from TSX using fungal -galactosidase.24 This gel had the unique property of forming a gel on heating and reverting to a sol state on cooling. The gel strength at a higher temperature was larger than that at a lower temperature. The phase transition between sol and gel was completely reversible (Fig. 14.15). The gel strength became larger with increasing removal ratio of galactose from xyloglucan. Figure 14.16 shows the stressstrain curves of gels with a 56% galactose removal ratio at various temperatures. The gel was very weak at 10ºC. The gel strength and the elastic modulus both increased with heating temperature below 50ºC. However, those values decreased above 70ºC. There were two sol-gel transition points; one was at a low temperature (sol to gel) and the other was at a high temperature (gel to sol). Figure 14.17 shows the comparison of DSC and thermal scanning rheological measurements at low-temperature gel formation.25 The

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Fig. 14.15 The sol-gel transition temperature diagram of xyloglucan as a function of the removal ratio of galactose from xyloglucan. (❒ ; high-temperature transition point; ■ ; low-temperature transition point)

Fig. 14.16 The stress–strain curves of xyloglucan gels with 56% galactose removal ratio at various temperatures. (Sample diameter; 40 mm, sample height; 30 mm, plunger diameter; 8 mm for curves at 10ºC and 20ºC, 5 mm for 30ºC, 3 mm for 50ºC and 70ºC, compression speed; 1 mm/sec.)

temperature at which G0 drastically changes should correspond to the transition temperature. The gelation was believed to be induced by the association of main chains by hydrophobic interaction. Upon gelation, the cluster seems to become a flat plane of thickness about 3nm.11 Four to five glucan chains are supposed to stack laterally to form a flat plane constituting the cross-linking domain. No organised structure was found in

Xyloglucan

265

Fig. 14.17 Comparison of DSC (a) and rheological measurements (b) at low-temperature gel formation for a 1% xyloglucan solution (with a 43% galactose removal ratio). (Frequency: 1.0 rad/s, heating rate: 0.5ºC/min.)

the pre-gel state, and the clusters were formed by random association. This study described the biotechnological tailoring of polysaccharide properties using an enzyme, as a first step in modelling or designing a TSX molecule.26 This thermoreversible enzyme-modified TSX gel can be used as a sustained-release vehicle for the intraperitoneal administration of mitomycin C. Intraperitoneal administration of mitomycin C in a 1.5% TSX with 44% galactose removal ratio to rats resulted in a broad concentration-time profile for this drug in both the ascites and plasm over a three-hour period, compared with a narrow peak and rapid disappearance from both sites when this drug was given i.p. as a solution.27 Plasma levels of indomethacin after rectal administration of TSX gels (partially degraded by -galactosidase) to rabbits indicated a broader absorption peak following administration of the gels, and a longer residence time than the control system (no TSX gel).28 Enzymatic modification of TSX gel has many advantages in drug delivery systems.

14.7.3 Plant defence system Large quantities of agricultural chemicals have now been used to prevent microbial infections while crops are cultivated. However, there are various problems in respect of safety, such as influences on human bodies exerted by the chemicals remaining on the crops, and the environmental pollution by diffused chemicals. It has been known that a plant has various natural defence systems against infections, and that when a plant is infected with micro-organisms, an anti-microbial substance which is not present in normal tissue is induced for resisting the infection. This substance is referred to as a phytoalexin. A phytoalexin is induced by a cell component of a micro-organism, a

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fragment of a polysaccharides which construct the cell wall of the plant infected by a micro-organism, or the like. This substance, which induces a phytoalexin is called an elicitor. A -glucan fragment derived from the cell wall which had elicitor activity was found first.29 Xyloglucan oligosaccharide was also found to have an elicitor activity and is notably useful as an agent for inducing a phytoalexin.30 In the experiment using cotyledons of soybeans, hydrolysed TSX showed 7.8 times amounts of induction of glyceolin (a kind of phytoalexin). Both TSX oligosaccharides (hydrolysed TSX) and oligosaccharide fragments prepared from pea xyloglucan inhibited the development of necrotic lesions induced by tobacco necrosis virus on cotyledons of Cucumis L. cv. ‘Laura’. Inhibition of virus infection ranged from 44–84% when xyloglucan fragments were applied in amounts ranging from 10–100s per cotyledon 24 hours before virus inoculation. These results suggested the possible role of xyloglucan in the mechanism of induced resistance against viruses.31 The activity of chitinase and -1,3-glucanase increased more rapidly 24 hours after inoculation of tobacco necrosis virus on treated cotyledons.32 Stimulation of release and accumulation of these enzymes demonstrated the biological activity of xyloglucan oligosaccharides also in relation to the hypersensitive reaction to tobacco necrosis virus.

14.8 1. 2. 3. 4. 5.

References HAYASHI, T. (1989) Ann. Rev. Plant Physiol. Plant Mol. Biol., 40, 139. MCDOUGALL, G. J. and FRY, S. C. (1990) Plant Physiol., 93, 1042. FRY, S. C. (1992) Trends in Glycoscience and Glycotechnology, 4, 17, 279. DAINIPPON PHARMACEUTICAL CO., LTD. (1989) Technical Bulletin. GLICKSMAN, M. (1986) Food Hydrocolloids, Vol. III (Glicksman, M. ed.) p. 191,

CRC Press, Boca Raton,

Florida. 6.

YAMATOYA, K., SHIRAKAWA, M., KUWANO, K., SUZUKI, J.

and MITAMURA, T. (1996) Food Hydrocolloids,

10, 369. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

and KOSUGI, K. (1991) Abstract of the Annual Meeting of the Society for Fermentation and Bioengineering, Japan, p. 182. YORK, W. S., VAN HALBEEK, H., DARVILL, A. G. and ALBERSHEIM, P. (1990) Carbohydrate Research, 200, 9. MITSUISHI, Y.

GIDLEY, M. J., LILLFORD, P. J., ROWLANDS, D. W., LANG, P., DENTINI, M., CRESCENZI, V., EDWARDS, M., FANUTTI, C. and REID, J. S. G. (1992) Carbohydr. Res., 214, 299. LANG, P. and KAJIWARA, K. (1993) J. Biomater. Sci. Polymer 4, 517. YUGUCHI, Y., MIMURA, M., URAKAWA, H., KAJIWARA, K., SHIRAKAWA, M., YAMATOYA, K. and KITAMURA, S.

(1997) Proceedings of The International Workshop on Green Polymers – Reevaluation of Natural Polymers, Adisesha, H. T., Sudirjo, S. T., Panggabean, P. R., Arda, J. and Soetrono, C. W. eds, Indonesian Polymer Association, Indonesia, p. 306. REID, J. S. G. and EDWARDS, M. E. (1995) Food Polysaccharides and Their Applications, p. 155, Stephen, A. ed., Marcel Dekker, New York. MILLANE, R. P. and NARASAIAH, T. V. (1992) Gums and Stabilisers for the Food Industry 6 (Phillips, G. O., Williams, P. A. and Wedlock, D. J. eds) p. 391, IRL Press, Oxford. OGAWA, K., HAYASHI, T. and OKAMURA, K. (1990) Int. J. Biol. Macromol., 12, 218. YOSHIMURA, M., TAKAYA, T. and NISHINARI, K. (1999) Food Hydrocolloids, 13, 101. LEVY, S., YORK, W. S., STUIKE-PRILL, R., MEYER, B. and STAEHELIN, A. L. (1991) Plant J., 1, 195. DEA, I. C. M. (1993) Industrial Gums, 3rd edn (Whistler, R. L. and BeMiller, J. N. eds) p. 21, Academic Press, San Diego. DAINIPPON PHARMACEUTICAL CO., LTD. (1978) J. Toxicol. Sci, 3, 163. LAIRON, D. (1996) Eur. J. Clin. Nutr., 50, 125. YAMATOYA, K. (1994) International Food Ingredients, 4, 15. YAMATOYA, K., SHIRAKAWA, M. and BABA, O. (2000) Hydrocolloids 2: Fundamentals and Applications in Food, Biology, and Medicine, Nishinari, K. ed. Elsevier, p. 405. CHEN, W. J. L., ANDERSON, J. W. and JENNINGS, D. (1984) Proc. Soc. Exp. Biol. Med., 175, 215. REID, J. S. G., EDWARDS, M. E. and DEA, I. C. M. (1988) Gums and Stabilisers for the Food Industry 4 (Phillips, G. O., Wedlock, D. J. and Williams, P. A. eds) p. 91, IRL Press, Oxford. SHIRAKAWA, M., YAMATOYA, K. and NISHINARI, K. (1998) Food Hydrocolloids, 12, 25.

Xyloglucan 25. 26. 27. 28. 29. 30. 31. 32.

267

and NISHINARI, K. (1998) Gums and Stabilisers for the Food Industry 9 (Williams, P. A. and Phillips, G. O. eds), The Royal Society of Chemistry, UK, p. 94. YAMATOYA, K. (1997) Cellulose Communications, 4, 2, 61. SUISHA, F., KAWASAKI, N., MIYAZAKI, S., SHIRAKAWA, M., YAMATOYA, K., SASAKI, M. and ATTWOOD, D. (1998) International J. Pharmaceutics., 172, 27. MIYAZAKI, S., SUISHA, F., KAWASAKI, N., SHIRAKAWA, M., YAMATOYA, K. and ATTWOOD, D. (1998) J. Contorolled Release, 4, 56, 75. ALBERSHEIM, P. and DARVILL, A. G. (1985) Sci. Am., 253, 44. MISAKI, A., SEKIYA, K. and YAMATOYA, K. (1997) USP560211124. SUBIKOVA, V., SLOVAKOVA, L. and FARKAS, V. (1994) J. Plant Diseases and Protection, 101, 128. SLOVAKOVA, L., SUBIKOVA, V. and FARKAS, V. (1994) J. Plant Diseases and Protection, 101, 278. SHIRAKAWA, M., UNO, Y., YAMATOYA, K.

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15 Curdlan K. Nishinari and H. Zhang, Osaka City University

15.1

Introduction

Curdlan is an extracellular microbial polysaccharide and was first discovered and investigated by Harada et al. in 1964 who coined the name curdlan, derived from ‘curdle’ to describe its gelling behaviour at high temperatures.1, 2 Curdlan is composed entirely of 1,3- -D-glucosidic linkages, which occur widely in nature involved in cell structure and food storage in bacteria, fungi, algae and high plants.3 Cotton fibres are known to comprise ca. 10% 1,3- -D-glucosidic linkages twenty-five days after anthesis.4 1,3- -Dglucosidic linkages are present in pollen, root hair and stomata of plants. Mushrooms and yeasts are also known to contain 1,3- -D-glucosidic linkages.5 Curdlan has utility as a food additive in its ability to form an elastic gel. It forms a heat-set gel at both relatively high and low temperatures or on neutralisation or dialysis of alkaline solution of curdlan.6,7 The unique gelling characteristics of curdlan are not only opposite to cold-set gels such as agarose,8 gelatin,9 carrageenan10 and gellan,11 but also different from other heat-set gels, e.g., konjac glucomannan,12 methylcellulose13 and hydroxypropyl-methylcellulose.14 In addition, curdlan is believed to show strong bioactivities.15–18 These conspicuously unusual properties of curdlan have not been observed in other synthetic and natural compounds. For more than thirty years, quite a few works have been published since the discovery of curdlan. Because of its unique properties, curdlan has been the subject of remarkable investigation especially for use in the food industry. Recently rheological and thermal properties and conformational, morphological analysis as well as the physiological functions such as anti-tumour and anti-HIV activities have been studied. In the present work, physico-chemical properties of curdlan are reviewed and the structural characterisation of curdlan is also emphasised.

Dedicated to the memory of the late Professor Tokuya Harada, the discoverer of curdlan.

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Fig. 15.1

15.2

Typical process for the manufacture of curdlan.

Production

Curdlan is produced in a fermentation process (Fig. 15.1) from the mutant strain of the bacteria Alcaligenes faecalis var. myxogenes 10C3 which can be isolated from soil.1, 2 Commercial curdlan may contain cellular debris, proteins and nucleic acids19 and other organic acids.20 More than 100 tons a year of curdlan is now produced by Takeda Chemical Industries Co. Ltd., Osaka, Japan.

15.3

Chemical structure

The chemical structure of curdlan is shown in Fig. 15.2. Curdlan is one of the biopolymeric molecules known as 1,3- -D-glucans.21, 22 Such a polysaccharide is characterised by repeating glucose subunits joined by a linkage between the first and third carbons of the glucose ring, which differs only in the linkage manner of repeating units from cellulose, a well-known natural biopolymer consisting of cellulose residues glycosidically linked in the 1,4- -D-configuration. With respect to the 1,3- -D-linkage,

Curdlan

Fig. 15.2

271

Chemical structure of curdlan.

Fig. 15.3 Electron micrograph of curdlan granule.25

the structure of curdlan is similar to that of carrageenan, agarose and gellan gum. Unlike gellan and - and -carrageenan, curdlan is a neutral polysaccharide without acidic components. The number-average degree of polymerisation of curdlan determined using Manners’ method23 is about 450.6, 7

15.4

Native curdlan

In the solid state, curdlan may exist in a triple helical structure. Curdlan has been shown to have a triple helical structure by 13C NMR analysis. In its natural state, curdlan is poorly crystalline24 and is found as a granule in the form of doughnut shaped structure25 as shown in Fig. 15.3, much like that of starch. The granule is insoluble in distilled water but readily dissolves in an aqueous NaOH due to the ionisation of hydrogen bonds. There is extensive hydrogen bonding that is holding the granule together, most likely by strongly binding the helices to form microfibrils and then binding together the microfibrils. When these bonds are broken through swelling, the granule loses its structure, i.e., the microfibrils have dissociated from each other during hydrolysis.

15.5

Functional properties

15.5.1 Solution properties and conformations While the primary structure is a long linear chain, curdlan forms more complex tertiary structures due to intra- and intermolecular hydrogen bonding. Curdlan is not soluble in water at room temperature but dissolves in an alkaline aqueous solution, cadoxen [Tri(ethylene diamine) hydroxide] aqueous solution and DMSO (dimethyl sulfoxide).

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Table 15.1 25ºC30 Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Light scattering and viscosity data obtained for fractionated samples of curdlan at 10ÿ6MW

1011<s2>/cm2

104A2/gÿ2cm3mol

[]/cm3gÿ1

2.02 1.91 1.81 1.45 1.28 1.26 1.12 0.95 0.91 0.56 0.50 0.45 0.39 0.28 0.23 0.17 0.092 0.053

6.39 6.70 5.54 4.64 3.99 3.33 3.41 2.56 2.58 1.85 1.62 1.18 1.16 0.74 0.57 0.36 0.29 0.11

4.2 4.1 3.6 4.5 5.0 3.6 3.6 3.9 2.7 6.1 6.0 5.6 6.4 6.1 5.4 7.4 6.0 10.9

665 643 590 520 468 443 387 343 295 238 218 200 165 140 129 96 60 38

The water insolubility of curdlan may be attributed to the existence of extensive intraand intermolecular hydrogen-bonded crystalline domains like that of cellulose.26 It has been reported that 1,3- -D-glucans with a very low degree of polymerisation of below 25 DP is soluble in water27 and it was suggested that curdlan is soluble in water at elevated temperatures.25, 28 At present, however, there is no direct evidence to clarify the water solubility of curdlan though an aqueous suspension of curdlan becomes clear when heated at above 55ºC. Hirano et al.29 carried out light scattering and viscosity measurements on nine fractionated samples of curdlan in the 1:1 (v/v) water-diluted cadoxen in the range of molecular weight MW from 6.6104 to 6.8105 at 25ºC. The experimental results of z1=2 average mean-square radius of gyration < s2 >z and intrinsic viscosity [] of curdlan were concluded as ÿ2 M0:53 < s2 >1=2 z 3:2 10 W nm

1

3 ÿ1 2:5 10ÿ4 M0:65 W cm g

2

Recently Nakata et al.30 made light scattering and viscosity measurements on solutions of eighteen fractions of curdlan in 0.3M NaOH (Table 15.1). The relationship 1=2 between weight-average molecular weight MW and < s2 >z and [g] and second virial coefficient A2 were summarised as ÿ2 < s2 >1=2 M0:53 z 3:6 10 W nm

3

3 ÿ1 7:9 10ÿ3 M0:78 W cm g

4

ÿ2 cm3 mol A2 2:8 10ÿ2 Mÿ0:3 W g

5

They concluded that the behaviour of curdlan solution cannot be explained by the conventional two-parameter theory of flexible polymer chains by evaluating the Flory

Curdlan

Fig. 15.4

273

Molecular weight MW dependence of mean-square radius of gyration < s2 >1=2 for z curdlan and cellulose in 1:1 (v/v) water-diluted cadoxen at 25ºC.31

universal viscosity constant M=6 < s2 >3=2 , which is much smaller than the usual value about 2.51021 dl/molcm3. Compared with cellulose chain, the values of 1=2 < s2 >z for curdlan are only one-half of those for cellulose at fixed MW in 1:1 waterdiluted cadoxen, indicating that curdlan chain has a more contracted conformation and both curdlan and cellulose molecules are not so flexible as shown in Fig. 15.4.31 As for the conformation transformation of curdlan in aqueous NaOH solution, there are several changes that occur from a triple helix to random coil depending on the concentration of sodium hydroxide used. Ogawa et al.32 reported that the transition occurred at a NaOH concentration of 0.19–0.24M in studies on the optical rotatory dispersion, viscosity and flow birefringence. This transition was confirmed by Saito et al.33 by 13C NMR analysis. Stipanovic and Giammatteo34 measured the steady shear viscosity of curdlan solution at different NaOH concentrations. A significant increase in viscosity was observed in the range of 0.05–0.1M NaOH corresponding to the solvation of triple helices followed by a viscosity reduction above 0.25M NaOH as the triple molecules are dissociated into single chains of lower molecular weight, which were supported by their 13C NMR experiments. Figure 15.5 shows a plot of intrinsic viscosity of curdlan vs. NaOH concentration at 25ºC measured by Nakata et al.30 They believed that the sharp depression of [] at 0.22M NaOH in Fig. 15.5 might be due to the conformational transition of curdlan chain from helix to coil. Tada et al.35–37 studied the structure of molecular association of curdlan at diluted regime in alkaline aqueous systems by rheological, static light scattering and small-angle X-ray scattering measurements. They found that the degree of association of curdlan molecules increased with decreasing alkaline concentration and the viscoelastic properties depend strongly on the alkaline concentration, i.e., concentrated curdlan solutions show almost a Newtonian flow at high alkaline concentrations whereas a solidlike behaviour at low alkaline concentrations. Curdlan in DMSO at above 1% behaves like a concentrated polymer solution,38 similar to high concentrations of NaOH aqueous solutions of above 0.05M.35

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Fig. 15.5

NaOH concentration dependence of intrinsic viscosity [] for curdlan at 25ºC.30

15.5.2 Aqueous suspension properties Among extensive studies on curdlan, the majority in the literature having been concentrated on curdlan gels and curdlan solutions, there have been only a few investigations on curdlan suspensions. Curdlan can only be suspended in water due to insolubility. Using a homogeniser can make curdlan into well-dispersed suspension without separation when left standing. When curdlan is dispersed in water, it gives a paste-like suspension unlike konjac glucomannan which forms a suspension with very high viscosity.39 Tako and Hanashiro40 reported that curdlan preparations at 38ºC showed Newtonian behaviour at 0.1% but plastic behaviour at above 0.2%. According to Nishinari et al.,41, 42 plastic behaviours of curdlan suspensions were also observed. The values of yield stress were estimated to be 0.10, 0.12 and 1.2Pa for 2%, 3% and 4% suspensions, respectively. The frequency dependence of storage modulus G0 and loss modulus G00 of curdlan suspensions showed a solid-like behaviour at 40ºC (Fig. 15.6).

15.5.3 Gelation Gel formation Curdlan can form a gel through the heating process alone, rather than relying on accompanying conditions such as pH, sugar concentration, or the presence of cations. Curdlan aqueous gels can be formed by various methods, some of which are heating a curdlan aqueous suspension,43, 44 curdlan/DMSO/H2O suspension,38 neutralising or dialysing against water an alkaline solution of curdlan25, 44 or a solution of curdlan in DMSO25 in a stationary state at ambient temperature. Furthermore, by heat treatment a curdlan aqueous suspension is capable of forming two types of gels depending on heating temperature, one of which is a thermo-reversible gel termed as a low-set gel formed by heating up to about 55ºC then cooling, and the other thermo-irreversible gel termed as a high-set gel formed by heating at above 80ºC. This change is explained by the hypothesis that microfibrils dissociate at 60ºC as the hydrogen bonds are broken, but then reassociate at higher temperatures as hydrophobic interactions between the curdlan molecules occur. An additional change to an even more ordered form is suggested in some sources as the temperature is raised above 120ºC.45

Curdlan

275

Fig. 15.6 Frequency dependence of storage modulus G0 and loss modulus G00 of curdlan suspensions at various concentrations at 40ºC.41 (❒ G0 , ■ G00 for 2%; ❍ G0 , ● G00 for 3%; 4 G0 , ▲ G00 for 4%.)

Fig. 15.7

Concentration dependence of gel strength for curdlan at 30ºC.2 (Curdlan gel was obtained by heating at 90ºC for 10 min.)

Gel properties The physical properties of aqueous curdlan gels were studied extensively by Harada and co-workers. The gel strength increases with concentration of curdlan2 as do many other polysaccharide gels (Fig. 15.7). The relationship between the heating temperature and gel strength2 is shown in Fig. 15.8. Gel was formed at about 55ºC, the gel strength stays almost constant at 60–80ºC, and thereafter increases heating temperature until 100ºC. The gel strength is strongly dependent on heating temperature and it is found that the strength

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Fig. 15.8 Effect of heating temperature on gel strength of curdlan at a concentration of 3%.2

of a gel formed by heating for 3 min. at 90ºC is much greater than that obtained by heating for 4h at 70ºC. The gel strength does not change between pH3–10, and can be enhanced greatly by adding borate, but the presence of urea, a reagent which breaks hydrogen bonds, caused a decrease in the gelling temperature and a marked decrease in gel strength as the concentration of urea increased above 2M (Table 15.2)2 and elastic modulus of curdlan gel.46 However, the reason why the gel strength shows a maximum (Table 15.2) is unknown. The addition of inorganic salts does not exert significant influence on the gel strength2 and elastic modulus.46 The high-set gel7 has the properties of being much stronger and more resilient and syneresis (i.e. exude water because of the shrinkage of the gel) than the low-set gel (Table 15.3) and neutralised gel (Table 15.4) and is not broken when frozen and thawed.47 It was resistant to enzymatic and acidic hydrolyses, but the neutralised gel was not resistant at Table 15.2 Effect of urea on gel strength of curdlan (2%) obtained after heating at 90ºC for 10 min.2 Molar concentration of urea/M 0 1 2 3 4 5 6 7 8

Gel strength/gcmÿ2

Gel-forming temperature/ºC

1190 1660 1910 1380 1170 980 860 64 –

54 47 45 44 42 38 36 35 –

Curdlan

277

Table 15.3 Gel strength and syneresis at 30ºC of gels of curdlan (3%) obtained after heating at various temperatures in water for 20 min.7 Heating temperature/ºC

Gel strength/gcmÿ2

Syneresis/%

310 530 540 630 940 1100 1200 1800 2200

2 4 8 10 16 18 20 29 34

55 60 70 80 100 120 145 160 170

Table 15.4 Gel strength and syneresis at 32ºC of gels of curdlan formed by neutralising alkaline solutions of curdlan with different concentrations47 After freeze-thawing Concentration of curdlan/%

Gel strength /gcmÿ2

Syneresis /%

Gel strength /gcmÿ2

Syneresis /%

1 2 3 4 5

220 460 620 870 920

2 2 2 2 2

0 170 430 720 780

100 42 36 24 18

all.47 Gels set at above 90ºC are soluble only in concentrations of NaOH above 1M whereas neutralised and 60ºC-set gels are soluble in 0.01M NaOH.47 It has been reported that tannin, sugar and starch are capable of reducing the syneresis of curdlan gel.5, 42 Molecular conformations Marchessault et al.3, 24, 48, 49 determined the molecular and crystal structure of the anhydrous form of curdlan by X-ray diffraction analysis (Fig. 15.9). They proposed that the curdlan is composed of a triple stranded helix as shown in Fig. 15.10. The three strands of the glucan helix are parallel, right-handed and in phase along the helix axis, and the crystal structure is extensively hydrogen-bonded. Fulton and Atkins50 investigated the molecular structure and gelling mechanism of curdlan using X-ray diffraction and infra-red spectroscopy. They proposed that triple helices are dominant for most curdlan molecular chains. The triple stranded molecules are bound by hydrogen bonding to the interstitial water of crystallisation to form a micellar domain, in other words, interstitial water forms a hydrogen bonded network with the triple helices, binding them into a micellar structure (Fig. 15.11(a)). The gelling mechanism of curdlan involves the interactions between these micelles and not the untwining and retwining of single helices into triple stranded junction zones. It is the association of these micelles which forms the junction zones of the gel network (Fig. 15.11(b)). Stipanovic and Giammatteo34 demonstrated that three crystalline conformations, anhydrous, hydrate, and swollen, differing in the degree of lattice hydration and chain extension, are distinguishable by solid-state 13C NMR spectra. The anhydrous and

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Fig. 15.9

X-ray fibre diffraction pattern of the anhydrous form of curdlan. The fibre axis is vertical.3

Fig. 15.10 Projection of the triple helix in the xz plane (a) the crystallographic repeat of the helix is 1/3 of the molecular repeat and xy plane (b), showing the intrahelix hydrogen-bond scheme. Hydrogen atoms are omitted.3

hydrate forms adopt a six-fold triple-helical conformation, whereas the swollen polymorph is a seven-fold triplex. In the gel state, the conformation of curdlan is similar to that of the swollen seven-fold helical form for a low-set gel prepared at 60ºC, whereas a partial or dominant transformation from the swollen seven-fold form to the anhydrous or hydrate six-fold triplex was determined for a high-set gel formed at 95 or

Curdlan

Fig. 15.11

Fig. 15.12

279

Schematic gel network of curdlan.50

13

C NMR spectra of curdlan gels34 (a) 60ºC, (b) 95ºC, (c) 120ºC.

120ºC respectively (Fig. 15.12). The increase in gel strength is due to the additional degree of cross-linking resulting from such a transformation. However, Saito et al.51–53 suggested that the anhydrous form is ascribed to a single chain form and swollen curdlan adopted a conformation of single helix, whereas annealed (hydrate) curdlan is readily identified as the triple-helix form. They suggested that, in any kind of heat-set curdlan gels, the conformations of curdlan exhibit an identical single helix with a low proportion of a triple helix i.e., the single helix is present in any gel preparations. The constant gel strength between 60–80ºC (Fig. 15.8) corresponds to the

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Fig. 15.13 Schematic representation of structural change between three forms of curdlan (a) room temperature structure; (b) high temperature structure at high humidity; (c) high temperature structure at low humidity.31

formation of pseudo-crosslinks arising from hydrophobic association of the single helical chains while the increased gel strength in the high-set gel is due to increased proportion of triple helical conformation.53 Harada and co-workers7, 31, 54 also indicated that the helix structure was transformed from single strand to triple strands at higher temperatures. An elaborate interpretation of structural change was proposed as shown in Fig. 15.13. The gelation mechanism of the low-set gel is different from that of the high-set gel. For a low-set gel, curdlan micelle interior is packed mostly by 7/1 single helical molecules which are hydrogen-bonded to one another by water molecules and some parts of the micelle are occupied by triple helical molecules which are also hydrated, whereas for a high-set gel, curdlan molecules change their conformation to 6/1 triple helices and curdlan micelle is occupied by molecules of triple-stranded helix in which hydrophobic interactions between curdlan molecules take a predominant contribution to the formation of the gel.

15.5.4 Thermal and morphological analysis According to thermal analysis, the DSC curves of original curdlan in aqueous suspension show a sharp endothermic peak at 50–64ºC,6, 7, 38, 41, 42, 44 a shallow endothermic peak at 70– 100ºC,6, 7, 38 and another endothermic peak at ca. 150ºC (Fig. 15.14).38 The first endothermic peak is ascribed to swelling of curdlan due to the breakup of some hydrogen bonds and the second the occurrence of hydrophobic interaction between curdlan molecules. As for the

Curdlan

281

Fig. 15.14 DSC heating curves of curdlan aqueous dispersions at various concentrations. Figures beside each curve represent the curdlan concentration in wt%.38

Fig. 15.15 DSC heating curves of 5% aqueous dispersions of curdlan after heating at various temperatures for 60 min. Heating rate: 1ºC minÿ1. The numbers beside each curve represent the temperature in ºC at which the dispersion was kept.41

endothermic peak at ca. 150ºC, it might be caused by the structural change of curdlan gels by further heating, corresponding to some molecular conformation transformation mentioned in 15.5.3. It is noteworthy that no exothermic peaks were observed when heat-treated silver pans were used.38 One may find a marked difference in the shape of DSC curves obtained by using non-heated silver pans.6, 7, 44 It was suggested that such a large exothermic peak was attributed to an interaction between the non-heated silver pan and water.55 When curdlan dispersions were heated to different temperatures then cooled, double exothermic peaks appeared at about 38 and 31ºC, which were attributed to the structure ordering due to the formation of hydrogen bonds.41 When the cooled gel was heated again, an endothermic peak appeared at around 60ºC.7, 41 Figure 15.1541 shows the second run heating

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Fig. 15.16 The degree of thermo-irreversible gelation dG = 1ÿH2T/H1 for a 10% aqueous dispersion of curdlan as a function of heating temperature T, where H1 and H2 are the endothermic enthalpies determined from the endothermic peaks in the first and second run DSC heating curves, respectively.41

DSC curves for 5% dispersions of curdlan which had been heated at various specified temperatures, T, for 60 min. and then quenched to 10ºC. Dispersions heated at temperatures higher than 60ºC showed an endothermic peak much broader than those at lower temperatures below 60ºC. Figure 15.16 shows the degree of thermo-irreversible gelation dG which is defined as dG = 1ÿH2T/H1 for a 10% aqueous dispersion of curdlan, where H1 and H2T are the endothermic enthalpies determined from the endothermic peaks in the first and second run DSC heating curves, respectively. If gels formed by heating are completely thermo-irreversible, no endothermic peak should appear in the second run DSC heating curve. As is seen clearly from Fig. 15.16, the degree of thermo-irreversible gelation increases steeply in the temperature range 50–70ºC (a partially irreversible gel), and tend to become almost an irreversible gel when heated above 120ºC. How fast dG increases with increasing heating temperature T should depend not only on the concentration but also on the molecular weight of curdlan, and this should be explored in the future. Figure 15.17 shows the morphology7 of ultrastructure of curdlan obtained after heating a neutralised gel at various temperatures by transmission electron microscopy. Electron microscopy displayed microfibrils of endless length consisting of fibril units of about 100nm length and 10–25nm width. The fibril units may be formed by several connected subunits which are composed of several molecules of single stranded helix. Microfibrils in non-heating curdlan dispersions are similar to those in the dispesions heated at 55 and 60ºC. Above 70ºC hydrophobic interactions between microfibrils occur and microfibrils with released fine stubs were observed. On heating above 120ºC, triple-stranded helices were formed from single-stranded helices resulting in formation of pseudo-crystalline forms of 100nm length and 30nm width. Though curdlan may be considered as the best polymer to clarify the mechanism of gel formation due to its neutral characteristics, several conflicting gelation mechanisms of curdlan have been proposed. There is still a great deal of confusion concerning the exact structure of curdlan as well as the results of DSC analysis, because curdlan takes various different conformations. It has been suggested that curdlan can exist as a triple helix, single helix, single chain, or a random coil depending mainly on crystallinity of curdlan, heating temperature and type and concentration of solvents used. Obviously, Harada’s and Saito’s groups prefer that most molecular conformation of curdlan is a single helix, whereas Marchessault’s, Atkin’s and Stipanovic and Giammatteo’s groups hold that triple-stranded helices are predominant. In view of the heterogeneous nature of the

Curdlan

283

Fig. 15.17 Morphologies of neutralised curdlan gels heated at different temperatures7 (heating temperature: A, without heating; B, 55ºC; 2, 55ºC; C, 60ºC; D, 70ºC; E, 80ºC; F, 100ºC; G, 120ºC; H, 145ºC; I, 170ºC;) Bars represent 0.1m.

curdlan gel, the dissidence mentioned above might originate from the dissimilarity in the sample measured and its preparation and the techniques used to determine the conformation of curdlan chains.

15.6

Applications

15.6.1 Food applications Curdlan is tasteless, odourless and colourless. Curdlan produces a retortable, freezable food gel, making possible the development of heretofore impossible food products, such

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Table 15.5

Food applications of curdlan61

Application

Function

Noodle Kamaboko (boiled fish paste) Sausages, Hams Processed cooked foods

Texture modifier Texture modifier Texture modifier, water holding Binding agent, improvement in moisture retention and product yield Retention of shape Retention of moisture Retention of shape Gelling agent (stable against heating and freezing-thawing) Gelling agent (stable against heating and freezing-thawing)

Processed rice cake Cakes Ice cream Jellies Fabricated foods Noodle-shaped tofu Processed tofu (frozen, retorted, freeze-dried) Thin-layered gel food (frozen) Konjac-like gel food (frozen) Heat-resistant cheese food Edible films Dietetic foods

Film formation Low-energy ingredient

Use level (%) 0.2–1 0.2–1 0.2–1 0.2–2 4–6 0.1–0.3 0.1–0.3 1–5 1–5

1–10 30–100

as tofu noodles. It can form a gel even while incorporating large amounts of fats and oils. Curdlan used in noodle doughs reduces the leaching out of soluble ingredients and softening of the noodles, resulting in clearer soup broths. Frozen tofu containing curdlan keeps the smooth texture after thawing whilst when normal tofu is frozen, the texture becomes rough. This has been used as Koori tofu in traditional Japanese dishes. Adding to surimi products improves the elasticity. In frozen sweet products, it can improve the texture of cakes and the shape retention of ice creams. Funami et al.56–59 studied the effects of addition of curdlan to meat products and found curdlan can modify the texture and improve the water-holding capacity of the meat products. The applications of curdlan used as a food additive or an ingredient in new procedures for food production60, 61 are summarised in Table 15.5.

15.6.2 Other applications Hirata et al.62 reported that an addition of 3% curdlan into starch is sufficient to increase the water resistance of the extrudates which may have a potential use as an environmentally degradable material. Curdlan is effective as an agent in making superworkable concrete to prevent cement and small stones from segregation and is also used as an organic binding agent for ceramics and active carbon.5 Interestingly, curdlan and curdlan sulfates have anti-tumour and anti-HIV activities15–18 which may be induced by its specific chain conformation. It has been shown that there is no anti-tumour activity when curdlan assumes a random-coil conformation or is composed of shorter chains, but the anti-tumour activity is greatly enhanced when the curdlan takes a single helix conformation.52 As a relatively new object of study, curdlan has exhibited unique gelling properties and molecular conformations. The use of curdlan in foods as well as in other applications will increase, providing new opportunities for novel products.

Curdlan

15.7

285

Regulatory status

The available toxicological data on curdlan have indicated its safety by animal studies and in vitro tests.63 As an inert dietary fibre, curdlan has been approved for use in Korea, Taiwan and Japan. In 1997 Takeda received approval for curdlan from the Food and Drug Administration of the United States, which is the first direct food additive completely developed and petitioned by a Japanese company that the US government has approved.

15.8 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

References

HARADA, T., MASADA, M., FUJIMORI, K. and MEADA, I. (1966) Agric. Biol. Chem., 30, 196–8. MAEDA, I., SAITO, H., MASADA, M., MISAKI, A. and HARADA, T. (1967) Agric. Biol. Chem., 31, DESLANDES, Y., MARCHESSAULT, R. H. and SARKO, A. (1980) Macromolecules, 13, 1466–71. MEIER, H., BUCHS, L., BUCHALA, A. J. and HOMEWOOD, T. (1981) Nature, 289, 821–2. HARADA, T. (1992) Trends in Glycoscience and Glycotechnology, 4, 309–17. KONNO, A. and HARADA, T. (1991) Food Hydrocolloids, 5, 427–34. KONNO, A., OKUYAMA, K., KOREEDA, A., HARADA, A., KANZAWA, Y. and HARADA, T. (1994)

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‘Molecular association and disassociation in formation of curdlan gels’ in Food Hydrocolloids: Structures, Properties and Functions. Nishinari, K. and Doi, E. eds, Plenum Press, New York, 113–18. WATASE, M., NISHINARI, K., CLARK, A. H. and ROSS-MURPHY S. B. (1989) Macromolecules, 22, 1196–201. WATASE, M. and NISHINARI, K. (1980) Rheologica Acta, 19, 220–5. WATASE, M. and NISHINARI, K. (1987) Makromol. Chem., 188, 2213–21. MORITAKA, H., NISHINARI, K., NAKAHAMA, N. and FUKUBA, H. (1992) Biosci. Biotech. Biochem., 56, 595–9. NISHINARI, K., WILLIAMS, P. A. and PHILLIPS, G. O. (1992) Food Hydrocolloids, 6, 199–222. NISHINARI, K., HOFMANN, K. E., MORITAKA, H., KOHYAMA, K. and NISHINARI, N. (1997) Macromol. Chem. Phys., 198, 1217–26. SARKAR, N. (1995) Carbohydr. Polym., 26, 195–203. SASAKI, T., ABIKO, N., SUGINO, Y. and NITTA, K. (1978) Cancer Res. 38, 379–83. LEAO, A. M. A. C., BUCHI, D. F., LACOMINI, M., GORIN, P. A. J. and OLIVERIRA, M. B. M. (1997) J. Submicroscopic Cytology & Pathology, 29, 503–9. GAO, A., FUKUDA, A., KATSURAYA, K., KANEKO, Y., MIMURA, T., NAKASHIMA, H. and URYU, T. (1997) Macromolecules, 30, 3224–8. GAO, A., FUKUDA, A., KATSURAYA, K., KANEKO, Y., MIMURA, T., NAKASHIMA, H. and URYU, T. (1998) Polym. J., 30, 243–8. RENN, D. W. (1997) Carbohydr. Polym., 33, 219–25. KANZAWA, Y., KOREEDA, A., HARADA, A. and HARADA, T. (1989) Agric. Biol. Chem., 53, 979–86. HARADA, T., MISAKI, A. and SAITO, H. (1968) Arch. Biochem. Biophys., 124, 292–8. SAITO, H., MISAKI, A. and HARADA, T. (1968) Agric. Biol. Chem., 32, 1261–9. MANNERS, D. J., MASSON, A. J. and STURGEON, R. J. (1971) Carbohyd. Res., 17, 109–14. MARCHESSAULT, R. H., DESLANDES, Y., OGAWA, K. and SUNDARARAJAN, P. R. (1977) Can. J. Chem., 55, 300–3. KANZAWA, Y., HARADA, T., KOREEDA, A. and HARADA, A. (1987) Agric. Biol. Chem., 51, 1839–43. IMESON, A. (1997) Thickening and Gelling Agent for Food 2nd edn, Blackie Academic & Professional, UK. OGAWA, K. and TSURUGI, J. (1973) Carbohydr. Res., 29, 397–403. HARADA, T. (1983) Biochem. Soc. Symp., 48, 97–116. HIRANO, I., EINAGA, Y. and FUJITA, H. (1979) Polym. J., 11, 901–4. NAKATA, M., KAWAGUCHI, T., KODAMA, Y. and KONNO, A. (1998) Polymer, 39, 1475–81. KASAI, N. and HARADA, T. (1980) ‘ACS symposium Series, 141’ in Fiber Diffraction Methods. French, A. D., Gardner, K. H. eds, The American Chemical Society: Washington, DC, 363–83. OGAWA K., TSURUGI, J., WATANABE, T. and ONO, S. (1972) Carbohydr. Res., 23, 399–405. SAITO, H., OHKI, T. and SASAKI, T. (1977) Biochem., 16, 908–14. STIPANOVIC, A. J. and GIAMMATTEO, P. J. (1989) ‘Curdlan and scleroglucan: NMR characterization of solution and gel properties’ in Polymers in Aqueous Media, Edward Glass, J, ed, 73–87. TADA, T., MATSUMOTO, T. and MASUDA, T. (1997) Biopolymers, 42, 479–87. TADA, T., MATSUMOTO, T. and MASUDA, T. (1998) Chem. Phys., 228, 157–66. TADA, T., MATSUMOTO, T. and MASUDA, T. (1999) Carbohydr. Polym., 39, 53–9. WATASE, M. and NISHINARI, K. (1994) ‘Rheology and DSC of curdlan-DMSO-water system’ in Food Hydrocolloids: Structures, Properties and Functions. Nishinari, K., Doi, E. eds, Plenum Press, New York, 125–9. JACON, S. A., RAO, M. A., COOLEY, H. J. and WALTER, R. H. (1993) Carbohydrate Polym., 20, 35–41.

286 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63.

Handbook of hydrocolloids TAKO, M. and HANASHIRO, I. (1997) Polymer Gels & Networks, 43, 241–50. HIRASHIMA, M., TAKAYA, T. and NISHINARI, K. (1997) Thermochimica Acta, 306, 109–14. NISHINARI, K., HIRASHIMA, M., MIYOSHI, E. and TAKAYA, T. (1998) ‘Rheological and DSC

studies of aqueous dispersions and gels of curdlan’ in Gum and Stabilizers for the Food Industry, 9, Williams, P. A., Phillips, G. O. eds, 26–33. KONNO, A., KIMURA, H., NAKAGAWA, T. and HARADA, T. (1978) Nippon Nogei Kagaku Kaishi. 52, 247–50 (in Japanese). HARADA, T., OKUYAMA, K., KONNO, A., KOREEDA, A. and HARADA, A. (1994) Carbohydr. Polym., 24, 101–6. HARADA, T., KOREEDA, A., SATO, S. and KASAI, N. (1979) J. Electron Microscopy, 28, 147–53. KONNO, A., AZECHI, Y. and KIMURA, H. (1979) Biol. Chem., 43, 101–4. KANZAWA, Y. and HARADA, T. (1989) Carbohydr. Polym., 10, 299–313. MARCHESSAULT, R. H. and DESLANDES, Y. (1979) Carbohydr. Res. 75, 231–42. CHUAH, C. T., SARKO, A., DESLANDES, Y. and MARCHESSAULT, R. H. (1983) Macromolecules, 16, 1375–82. FULTON, W. S. and ATKINS, E. D. T. (1980) ‘The gelling mechanism and relationship to molecular structure of microbial polysaccharide curdlan’ in Fibre Diffraction Methods. Washington, DC, American Chemical Society, 385–410. SAITO, H., YOKOI, M. and YOSHIOKA, Y. (1989) Macromolecules, 22, 3892–8. SAITO, H., YOSHIOKA, Y., YOKOI, M. and YAMADA, J. (1990) Biopolym., 29, 1689–98. SAITO, H. (1992) ‘Conformation and dynamics of (1,3)- -D-glucans in the solid and gel state: highresolution solid-state 13C NMR spectroscopic study’ in Viscoelasticity of Biomaterials. Glasser, W. and Hatakeyama, H. eds, American Chemical Society, Washington, DC, USA, 296–310. OKUYAMA, K., OTSUBO, A., FUKUZAWA, Y., OZAWA, M., HARADA, T. and KASAI, N. (1991) J. Carbohyd. Chem., 38, 557–66. ZHANG, H., HUANG, L., NISHINARI, K., WATASE, M. and KONNO, A. (2000) Food Hydrocolloids, 14, 121–4. FUNAMI, T., YADA, H. and NAKAO, Y. (1998) Food Hydrocolloids, 12, 55–64. FUNAMI, T., YADA, H. and NAKAO, Y. (1998) J. Food Sci., 63, 283–7. FUNAMI, T., YOTSUZUKA, F., YADA, H. and NAKAO, Y. (1998) J. Food Sci., 63, 575–9. FUNAMI, T., YADA, H. and NAKAO, Y. (1999) Food Sci. Technol. Res., 5, 24–9. HARADA, T., TERASAKI, M. and HARADA, A. (1993) ‘Curdlan’ in Industrial Gums, 3rd edn, Whistler, R. L. and BeMiller, J. N., eds, Academic Press, New York, 427–45. MIWA, M., NAKAO, Y. and NARA, K. (1994) ‘Food application of curdlan’ in Food Hydrocolloids: Structures, Properties and Functions. Nishinari, K. and Doi, E. eds, Plenum Press, New York, 119–24. HIRATA, T., BHATNAGAR, S. and HANNA, M. A. (1997) Starch-Sta ¨ rke, 49, 283–8. SPICER, E. J. F., GOLDENTHAL, E. I. and IKEDA, T. (1999) Food Chem. Toxicol., 37, 455–79.

16 Cereal -glucans K. Morgan, Industrial Research Ltd, New Zealand

16.1

Introduction

Mixed linked (1!3), (1!4)- -glucans (referred to as -glucans within this chapter) are linear polysaccharides that are composed of cello-oligomers separated by single (1!3) -linkages. Generally those of high molecular weight form viscous solutions with the viscosity increasing with increasing molecular weight, while those of lower molecular weight show gel-like behaviour, with some types of -glucan able to form soft thermoreversible gels. Their major use in foods to date has been as texturising agents, especially as replacements for all or part of the fats in a range of dairy and bakery products, but there is also interest in including them in foods solely for their perceived health benefits. -glucan and grains containing high levels of -glucan have a useful physiological role as a dietary component and a significant part of research on -glucans has focused on this aspect. For coronary heart disease, a leading cause of death in industrialised countries, high serum cholesterol levels and high levels of LDL (low density lipoproteins) cholesterol appear to be risk factors. Consumption of foods rich in -glucan significantly reduces serum cholesterol levels and LDL cholesterol.1–5 The FDA, recognising the benefit of -glucans in diet, has allowed health claims for foods that contain more than 0.75 grams of oat-derived -glucan for each serving portion. Other health benefits of a diet rich in -glucans include moderation of the glycaemic response to the digestion of starchy foods6–9 and the lowering of serum lipids levels.10–12 -glucans are also a source of soluble fibre because humans produce no -glucan degrading enzymes and therefore -glucan is not hydrolysed in the small intestines. They are degraded by microbial fermentation in the large intestines. The fermentation produces beneficial short chain fatty acids, particularly butyric acid, which depending on where the degradation occurs may have a role in guarding against colo-rectal cancer.13 The health benefits of -glucan have stimulated interest in including -glucan in food products. Normally, cereal grains do not contain sufficient levels of -glucan to provide the most desirable intake of soluble fibre. So there has been interest in formulating foods enriched in -glucan by either supplementation with extracted -glucan or using cereal

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flours or brans enriched in -glucan. For this type of use any functionality that the glucan may impart to the food as a hydrocolloid is minor and the major reason for including the flour or bran is because of the perceived health benefits. -glucan has been extracted commercially, mainly from oats, but the extraction process is expensive so that the addition of the -glucan extract to foods has been uneconomic. However, since the early 1990s several key developments have occurred that should see the extensive use of significant amounts of -glucan in processed foods, not just because of their perceived health benefits, but also because the -glucan itself imparts significant functionality to the food, that is, it is a useful food hydrocolloid in its own right. In processed foods, the principal use to date for -glucan containing products has been as fat replacers. This in itself is a health benefit since the typical Western diet contains too much fat. The first key development was the invention of Oatrim by George Inglett at USDA in the early 1990s.14–18 Oatrim contains only moderate amounts of -glucan, but is inexpensive to produce and has shown new functionality in processed foods. Oatrim is currently being marketed by two companies. Mountain Lake Speciality Ingredients produces Oatrim under the name TrimChoice-5Õ, and Quaker Oats together with Rhodia produce a similar product called Beta-TrimTM. In 1998, Inglett invented Nu-trim19–21 which has similar functionality, is even less expensive to produce, but can contain higher levels of -glucan. (The ‘trim’ appendage is an acronym for Technical Research Involving Metabolism.) The second key development was a new, inexpensive process for extracting a novel form of -glucan, called Glucagel, from barley.22, 23 Glucagel is a new functional hydrocolloid containing up to 100% -glucan. It forms soft thermoreversible gels.

16.2

Manufacture

16.2.1 Origin -glucans occur in grasses of the Poaece family. They are constituents of the cell wall and appear during cell expansion.24–27 They are reabsorbed when the expansion has ceased, indicating that they may be acting as a scaffolding for the placement of other cell wall components. -glucans are also present in the cell wall of certain cereal grains, particularly those of oats and barley25, 26, 28 and are not readsorbed until germination. Their function here is uncertain but they may be convenient cell-wall materials that can be readily readsorbed during germination. In barley and oats, -glucans are the main non-starch polysaccharide, typically forming anywhere from 2 to 7% by weight of the grain.1, 6, 26, 28, 29 The variation in glucan content of the grain depends mainly on the cultivar type rather than environmental or agronomic factors during cropping. In oats, the bran contains from 5–10% -glucan30 and is enriched in -glucan compared to the groats which contain from 3–7% -glucan.6 In barley, the endosperm cell walls are composed of about 70% -glucan,26, 31 whereas the aleurone cell walls are about 20%.32 Barley was one of the first domesticated grains. As suggested by its scientific name, Hordeum vulgare (grain of the common people), it has had a long history of consumption by humanity. Over the past couple of centuries it has fallen from favour in the human diet, at least in the West, and in foods it appears only in a few speciality products such as soups. It is now used mainly as animal feed and for brewing. For both of these applications there has been considerable pressure to develop or select varieties low in -

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glucan. Low levels are preferred in brewing owing to the tendency of -glucans to produce hazing in beer and also to clog filters during clarification of beer.33 In animal feeds high levels of -glucan can act as an antinutrient, restricting the full metabolisation of the grain components.34 To avoid these shortcomings it is now common practice to add enzymes that specifically degrade -glucan to low molecular weight components. Oats are grown mainly for pasturage and as animal feed with a small amount used for human consumption. For oats there has not been the same selection pressure to produce varieties having low -glucan contents. To be able economically to extract and purify -glucan, cultivars having high glucan contents are desirable. Barley and oat flour fractions from varieties containing high levels of -glucan are being marketed today. High levels of -glucan in barley are associated with varieties that contain waxy or amylose-extended starch, mainly because in these varieties there is less starch synthesis occurring. An extreme example of high glucan content is the high-fibre barley flour called SustagrainTM marketed by Con-Agra. It is milled from a grain which has a 15% -glucan content. The flour is sold at a higher price than conventional flour because yields of the grain per hectare are low. -glucan was not used as a general food hydrocolloid until the 1990s, even though there has never been a shortage of suitable raw material for -glucan production. Current world production of barley, for example, is about 170 million tonnes per annum. This represents a considerable resource of -glucan. Even assuming that on average the barley contains about 2% -glucan then this is at least 3.0 million tonnes of -glucan that is synthesised and deposited in the cell walls of barley grain each year. -glucan production is, therefore, not limited by availability of raw material. Rather the reason that -glucan has not been promoted for use in foods has been due to the considerable cost of extracting and purifying the -glucan.

16.2.2 Extraction and purification Traditional methods for extracting -glucans from oats or barley flours have involved three key steps.35 Firstly enzymes present in the flour are deactivated to decrease hydrolysis of the -glucan to lower molecular weight products. Then warm or hot water is used to extract the -glucan from the flour. Lastly, the spent flour is removed, usually by centrifugation, and a -glucan containing gum is recovered by precipitation on addition of a water-miscible organic solvent. The gum contains about 40–60% -glucan and has an average molecular weight between 300,000 and 1,000,000. The properties of the gum are not conducive to inclusion in foods. For example, the high molecular weight of the -glucan leads to difficulties in redissolving the gum in water even for -glucan concentrations below 0.5%. Variations exist on this basic process and include extraction under alkali conditions,36, 37 and hydrolysis or precipitation of solubilised starch and protein after the extraction step, which increases the quantity of -glucan in the gum. The expense of extracting and purifying -glucan by the traditional methods and its undesirable functionality means that there are few applications for it in processed foods. The expense is incurred not from the extraction step as it simply uses water and elevated temperatures, but during the concentration and recovery of the gum. Innovation within the newer methods for producing economically a -glucan-containing hydrocolloid has, therefore, been in the recovery of the -glucan after the extraction step. For Oatrim14–18 the key development was reached on realising that it is not necessary for food use to have the -glucan particularly pure. A 5% -glucan containing solid could still have useful functionality in food systems if the other components were compatible

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with the end use as well. Thus, after the extraction process a very simple drying process could be used to form the -glucan containing solid, as long as the solids content of the extract was not too low. For the invention of Glucagel the key development was a simple method of concentrating -glucan which did not require the use of water-miscible organic solvents and produced a novel type of -glucan which forms soft thermoreversible gels.22

16.2.3 Oatrim and Nu-Trim Oatrim and Nu-trim are both isolated following a high temperature extraction of the cereal flour with water. Components of the flour that are not liquefied during the extraction are removed. A -glucan containing solid is then obtained from the liquid by conventional methods of evaporation, such as spray or roller drying. Oatrim is made by subjecting an aqueous dispersion of oat bran or flour to a high temperature hydrolysis with a thermostable -amylase.18 Reaction conditions are controlled so that the starch is liquefied to low molecular weight dextrins. Conditions that lead to protein solubilisation are avoided. Anything that is not solubilised is removed by centrifugation. The supernatant from centrifugation is then dried to give the Oatrim product. Although the dextrins are the major component of the final product after drying, the important functionality of Oatrim in foods appears to be due to a combination of both the -glucan and the dextrins. Dextrins by themselves are not known to have the texturising properties that Oatrim does. In Oatrim, the dextrins may also be acting as an adjunct that improves dispersion and solubilisation of the -glucan. As starch and starch hydrolysis products are components found naturally in the human diet, Oatrim can still be considered a natural food. A typical pilot plant production process involved slurrying six kilograms of oat flour in 18 litres of water containing 25ppm of calcium ion. The mixture was gelatinised by passage through a steam injection cooker and collected in a steam-heated kettle. Thermostable amylase such as ‘Enzeco Thermolase’ was added to provide 1 unit per gram of oat flour. After 5 min. the enzyme was inactivated by passing the slurry through the steam injection cooker. The warm slurry was centrifuged at 15,000rpm by a large Sharples centrifuge to separate the soluble component from the insoluble. The products were dried separately on hot rollers. The dried water soluble portion is Oatrim. Nu-trim is formed by high-temperature mechanical shearing of a suspension of oat or barley endosperm in water. Cellulosic fibres remaining are removed by filtration or gravity separation. The liquid remaining is dried to produce Nu-trim.21

16.2.4 Glucagel Traditional methods for extracting -glucan include an enzyme deactivation step. The process for forming Glucagel makes use of an enzyme,38–40 which appears to be of fungal origin but naturally present in barley flour, to achieve a small amount of hydrolysis of the -glucan to form products having average molecular weights between 15,000 and 150,000. The enzyme present in the flour preferentially cleaves cellooligomers within the -glucan that have a degree of polymerisation greater than about nine.40 Thus little in the way of oligosaccharides is produced. Enzyme hydrolysis appears to improve the extraction yield of -glucan, and also reduces solution viscosity so that less water is required during the extraction. Glucagel yields are the highest at extraction temperatures of 50–55ºC. Extraction above this temperature causes starch

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gelatinisation and this results in undesirable viscosity. Yields at extraction temperatures of 25ºC are approximately half those at 55ºC. After extraction the solids are removed and the supernatant is frozen for several hours. The supernatant is then thawed to produce a gelatinous or fibrous precipitate of Glucagel containing about 15% solids which can be dried. The -glucan content of the dried Glucagel can be as high as 90%, but it is typically 80% of dry weight. Glucagel can be purified to nearly a 100% glucan content by redissolving the Glucagel in water at temperatures above 70ºC and repeating the freezing and thawing steps. The Glucagel process requires less water than traditional methods and uses no organic solvents. Glucagel has different functionality compared to -glucans extracted by traditional methods. Low molecular weight Glucagel dissolves readily in water at temperatures above about 75ºC and at concentrations as high as 20%. High molecular weight Glucagel dissolves less readily in water to solution concentrations of about 5%. Both high and low molecular weight Glucagel solutions set at temperatures below 55ºC to form soft gels. A typical pilot plant production of Glucagel started with the extraction of -glucan from a slurry of 80 kg of barley pollard flour in 400 litres of water at 50ºC. After 30 minutes the slurry was passed through an APV Scroll Decanter centrifuge and then a Westfalia SAOH Disc Stack centrifuge to remove solids. The supernatant was frozen for 24 hours at ÿ18ºC, and then allowed to thaw. The precipitate from the thawed supernatant was separated on a Sharples basket centrifuge and dried in a freeze/drier.

16.3

Structure

16.3.1 Molecular structure -glucans are unbranched polysaccharides formed of glucopyranosyl units joined by groups of contiguous (1!4)- -linkages and isolated (1!3)- -linkages (Fig. 16.1). Isolated (1!4)- -linkages never occur, instead most of the (1!4)- -linkages are in groups of two or three. This forms the main structural motif: chains of cellotriosyl and cellotetraosyl residues, joined by single (1!3)- -linkages. The -glucan thus has a cellulose-like backbone but contains kinks at the position of the (1!3)- -linkages. The kinks disrupt the strong hydrogen bonding network that is normally found in cellulose, thus unlike cellulose the cereal -glucans can be dissolved in water. The structural sequence of -glucan has been probed in some detail.40–45 The enzyme, lichenase, solely cleaves the (1!4)- -linkage immediately following a (1!3)- -linkage (moving towards the reducing end of the polymer). Lichenase treatment of the -glucan generates a series of oligomers that have the same number of glucopyranosyl residues as

Fig. 16.1

Molecular structure of a mixed linked (1!4), (1!3)- -glucan.

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Fig. 16.2 Lichenase treatment of a -glucan (horizontal lines are (1!4)- -linkages, angled lines (1!3)- -linkages and vertical dashed lines are the site of lichenase hydrolysis).

the cello-oligomer residues in the original -glucan polymer (Fig. 16.2), although they contain a (1!3)- -linkage at the end of the chain instead of a (1!4)- -linkage. The oligomers can be characterised by chromatography to obtain the distribution of the cellooligomers in the original -glucan (Fig. 16.3). About 90% of the -glucan consists of cellotriosyl and cellotetraosyl oligomers joined by (1!3)- -linkages. The other 10% contains cello-oligomeric residues having a higher degree of polymerisation (DP).43, 44 Cello-oligomers having DP in the range of five to nine are the most common. On lichenase treatment of some types of -glucan, especially those extracted at high temperatures, a precipitate is formed. This precipitate contains oligomers of a DP from eight to as high as 19.43, 44 Thus -glucan in the native state, that is within the cell wall, consists mainly of cellotriosyl and cellotetraosyl residues joined by single (1!3)- linkage, but incorporates longer cello-oligomers up to at least DP 19. Since adjacent (1!3)- -linkages are not found in -glucans and neither are single (1!4)- -linkage flanked by two (1!3)- -linkage, it is apparent that the glycosidic linkages are not completely random. Analysis of fragments from enzyme hydrolysis shows that the repeat sequence follows a second-order Markov chain rather than a random polymer model.41, 46 The linkage sequence only depends on preceding linkages that are no further then two glucose residues away, although there is significant autocorrelation between glycosidic linkages 15 to 20 glucose units away. Ratios of cellotriosyl to cellotetraosyl residues in -glucan are in the ranges from 2.10.1 for oats, 3.20.3 for barley and 3.50.4 for wheat and vary according to the

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Fig. 16.3 High-perfomance ion-chromatography of oligomers released from lichenase treatment of barley -glucan.

temperature and conditions under which the -glucan is extracted.45 Barley -glucan extracted at temperatures of 65ºC have higher molecular weights, higher content of (1!4) -linkages and greater viscosity than those extracted at 45ºC.42

16.3.2 Tertiary structure The tertiary structure of -glucan in solution can be deduced in part from viscoelasticity measurements. Complications arise in interpretation of these measurements because the solution behaviour of -glucan depends not just on the molecular weight of the -glucan, but also the history of the -glucan solution and conditions used for preparing the glucan. The behaviour of -glucan in solution appears to be quite complex. -glucan of high molecular weight forms viscous solutions.28, 47–50 Usually, the higher the molecular weight of the -glucan the more viscous will be the solution at a given concentration. Low molecular weight -glucans can associate and aggregate51–54 which alters the solution behaviour of the -glucan. In the extreme case of Glucagel there appears to be a very strong association and soft gels are formed.22 The intrinsic viscosity of -glucan in solution is consistent with an extended conformation that can be modelled by a partially stiff worm-like cylinder.51,52 Gomez et al.53 found that the viscoelastic properties of -glucan solutions are consistent with a labile cooperative association of -glucan chains which can be disrupted by mechanical shearing. They found that the viscosity of solutions of -glucan increase with increasing temperature, an observation that was difficult to explain. Samples of -glucan that had different mechanical and thermal histories had different viscoelastic properties.53 Doublier and Wood54 found gel-like behaviour for oat -glucan that is of low molecular weight, but not for that of high molecular weight. For unhydrolysed oat gum, the rheology was similar to that of other nongelling polysaccharides such as guar gum. With hydrolysed gum, aggregation was observed. Va˚rum et al.49 found that ~10% of the -glucan extracted from oat aleurone undergoes reversible association forming large

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Fig. 16.4

G0 and G00 of a gel containing 5% Glucagel.

cooperatively stabilised aggregates. The difference in behaviour between high and low molecular weight -glucan may be because once an association has occurred it is easier for the rest of a shorter chain to rearrange to extend the region of association than it is for a longer chain.53 For Glucagel the gel melting temperature, 65ºC, is reasonably high22 suggesting that there can also be a strong stable association of -glucan chains. Structural studies of Glucagel, see below, have confirmed this. This suggests that the labile association observed by Gomez et al.53 can be extended into a stable network structure as observed in gels formed from Glucagel. Glucagel forms soft gels at concentrations above about 2%. For 5% gels, viscoelastic measurements55 (Fig. 16.4) show that the storage modulus, G0 , is unaffected by oscillatory frequencies extending from 1.0 to 200 rad/s, and G00 , the loss modulus, is much smaller than G0 . The large difference between G0 and G00 and the independence of G0 on oscillatory frequency is a characteristic of a strong gel.56 However, the marked increase of G00 with increasing oscillatory frequency, indicating breakdown of the gel into smaller clusters, is more characteristic of a weak gel. This behaviour is similar to that of galactose-removed xyloglucan at the transition point from a weak to a strong gel.57 By definition, strong gels rupture above a critical deformation, whereas Glucagel flows under increasing deformation, thus Glucagel must be classified as a weak gel at least at concentrations below 5%. Below a concentration of 2%, solutions of Glucagel still gel (at low concentration gelation times can be several hours), but the gel mixture readily flows. This behaviour is characteristic of a fluid gel where there is a particulate gel suspended in an aqueous phase. For Glucagel, the particles in the fluid phase are irregularly shaped and vary in

Cereal -glucans

Fig. 16.5

13

295

C CP/MAS NMR spectrum of moistened Glucagel. Peaks assigned to -glucan chains in the A-conformation and amorphous conformation are marked.

size from 2–20m. This is similar to the size range of oil emulsions and may in part explain why certain -glucan products can impart creaminess to food, partly replacing oil and fats in processed foods. For Oatrim and Nu-Trim the tertiary structure of the -glucan has not been reported. However, Inglett has reported that heating and cooling a 24% Oatrim dispersion58 produces a shortening-like gel. The structure of the gel and of the dried Glucagel itself has been examined using solidstate NMR59 (Fig. 16.5). In all the samples, portions of the -glucan chains were found to be in one of two conformations. One of the conformations is similar to that adopted by glucan chains in solutions which suggest that the chains are in an amorphous conformation. The other conformation is due to an association of two -glucan chains. It has been named the A-conformation, after similar nomenclature for starch structures. It is possible to observe the association of -glucan chains directly using atomic force microscopy (AFM).59 For very dilute solutions of Glucagel the -glucan chains require several days to associate. AFM images of a freshly prepared, very dilute Glucagel solution cast onto a freshly cleaved mica surface show no fine detail, since the -glucan chains are mobile within the surface hydration layer on the mica surface. For solutions that have been aged for several days before being imaged, fibres, networks and microgels can be observed, which is good evidence for chain association. Although the fibres are generally straight, details of the fibres show irregular deviations away from the axis of the fibre. This may reflect the random nature of the -glucan chains. The evidence then is that the chains that form Glucagel are arranged in a mixture of an amorphous conformation where there is no chain association and the A-conformation where association between two chains occurs. The melting temperature of a gel formed from Glucagel (100% -glucan) is about 60ºC. Differential scanning calorimetry of a 5% gel (Fig. 16.6) shows that the melting transition is not a simple single endotherm.22 A large endotherm occurs at 58ºC and a smaller endotherm at 68ºC. The large endotherm appears to be associated with melting of the gel. On the molecular level this is probably due to melting of -glucan chains in the A-conformation. The cause for the smaller endotherm is uncertain.

16.3.3 Health benefits The structure of -glucan in solution is directly related to a number of its health benefits as a food ingredient. The hypocholesterolemic, that is cholesterol-lowering, ability of glucan is ascribed to the increase in viscosity of gut gastrointestinal contents as -glucan dissolves, and that this reduces the reabsorption of bile acids.

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Fig. 16.6

DSC of a gel containing 5% Glucagel.

There have been a number of studies examining the hypocholesterolemic properties of -glucan and soluble fibres in general. For milled, oat-bran fractions containing -glucans of high molecular weight, serum cholesterol reduction was found by Ma¨lkki et al. to be as much as 30% in rats fed diets containing 3.3% -glucan.2 Oat bran preparations that yielded -glucan of low viscosity were less effective in reducing serum cholesterol levels. Studies on human subjects have shown that -glucan lowers total cholesterol as well as LDL cholesterol (‘bad’ cholesterol), whereas HDL cholesterol levels remain the same.1,4,5,60 It was also noted in a number of these studies that low molecular weight glucans were ineffective. Similar results have been obtained for other viscous polysaccharides that are effective hypocholesterolemic agents. For instance, a study on guar gum noted that without high viscosity the gum was ineffective in lowering serum cholesterol levels.61 However, not all studies have shown that high viscosity is required for efficacy (see below). There is good evidence that the -glucan may not be the only component affecting cholesterol levels. Peterson and Qureshi11 found that the addition of tocotrienols to an oat bran diet was more effective in lowering cholesterol in chickens than oat bran by itself. In contrast to results of other researchers, they found that removing -glucan from barley diets did not change the efficacy of the diets. They assumed that perhaps another component of the barley was lowering serum cholesterol. This could be due to barley arabinoxylans which also form viscous solutions or tocotrienols naturally present in the grain. There have been several studies examining the postprandial glycaemic response, especially in diabetic subjects, after digesting foods rich in -glucan.7–9 Wood6 found that the -glucan was effective in reducing the glycaemic response to an oral dose of glucose and that the effectiveness depended on the logarithm of the viscosity. For high glycaemic foods such as cereals, a 50% reduction in peak glucose levels can be obtained with glucan levels of 10%. Similar decreases in serum glucose levels have been observed in human diabetic subjects consuming meals containing high levels of -glucan.7, 9

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Fibre is considered to be beneficial for lessening the chance of developing colo-rectal cancer13 although a recent study by Fuchs et al.62 suggests that there is little or no benefit of a high fibre diet. In a study examining 88,000 nurses over 16 years, women who ate a high fibre diet developed the same rate of colo-rectal cancer as those who ate little fibre. The meaning of these results, however, is the subject of continuing debate and there are other health benefits that remain attributable to ingestion of soluble fibres such as -glucan. To date, only Oatrim has been investigated for health benefits. Both Nu-trim and Glucagel are recent inventions and significant studies of health benefits are yet to be undertaken. Yokohama et al. found that Oatrim was particularly effective in lowering serum cholesterol levels in hamsters.63 Chickens fed a diet containing Oatrim showed a significant drop in blood cholesterol levels.64 In a clinical trial with human volunteers Oatrim was found to significantly reduce blood cholesterol and LDL cholesterol levels.65 In addition there was a 7–12% drop in blood glucose levels. The volunteers also lost an average of 2kg in weight without feeling hungry although they were consuming slightly more calories. In another study, Behall et al.12 found significant reduction in total and LDL cholesterol in twenty-three volunteers with above average cholesterol levels for their age and sex, when fed a diet containing 50–75g per day of Oatrim with a 10% -glucan content. Levels for use of -glucan as a functional agent in foods will probably be between 0.2– 2%. Since both the Oatrim and Glucagel products contain -glucan of lower molecular weight than those extracted by traditional methods it is unlikely that at this usage level they will contribute significantly to product viscosity. Therefore any claim that these products can moderate glycaemic response or reduce blood cholesterol levels will have to be carefully substantiated, especially when used at low levels, since these health properties have frequently been correlated with high viscosity of the gut contents. Also, only a small part of the daily diet is likely to include foods containing -glucan. The major health benefit is therefore likely to be from replacing some or all of the fat in the diet with a lowcalorie alternative without compromising taste or texture. For hydrocolloid use these glucans products should be recognised primarily for the functionality they impart to foods, and secondly to the reduction in fat they engender in processed foods, rather than to any other perceived health benefit. It is interesting to note in this regard that neither manufacturer of the Oatrim products Beta-Trim or TrimChoice-5 make any specific health claim for their products, except as a reduced-calorific additive to foods.

16.4

Technical data

16.4.1 GlucagelÕ At the time of writing, Glucagel was still undergoing commercialisation thus the following data is only indicative of potential applications and properties. Product data sheet (GlucagelÕ) Description Glucagel is a high-purity -glucan product produced by an aqueous extraction and purification process. It has neutral flavour and forms soft thermoreversible gels. Characteristics • White solid • Forms soft gels at concentration above 2% • Forms fluid gels below concentration of 2%

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• Pleasant mouthfeel • Neutral flavour • Dissolves readily in water above 75ºC forming high-clarity solutions and sets below temperatures of 60ºC forming soft translucent gels • Very good solubility (>25% w/w in water) • High purity, typically 85–100% • Fat mimetic • Stable gels formed from pH10.0–pH2.0, unaffected by salt and sugar • Forms strong transparent films Composition • Moisture 5–10% • -glucan 85–95% • pH7.0 • Different grades are available with the average molecular weight able to be tailored from 5,000–150,000 Daltons to suit the end use. Applications Bakery, dairy, dressings, edible films. Disclaimer While the above suggestions and data contained herein are based on information believed to be reliable, Industrial Research or its supplier make no representation or warranties of any kind for the product described here. The formulation, examples and use recommendations as may be described herein should not be construed as permission to violate any patent or as a warranty of non-infringement of any patent.

16.4.2 Oatrim Two ventures are licensed to produce Oatrim. Mountain Lake Specialty Ingredients, a joint venture between ConAgra and A. E. Staley, a Tate and Lyle company, hold licences for the USA, France, Germany, Belgium, United Kingdom and Canada. They market two products, TrimChoice-5Õ and TrimChoice-OCÕ. Quaker Oats and Rhodia have a USA licence and market their product as Beta-Trim.

Product data sheet (Beta-TrimTM/QuakerÕ Oatrim) Introduction One of the outstanding qualities of the Beta-Trim Brand of Quaker Oatrim is its heritage. Born of a partnership between two food industry leaders, Quaker Oatrim combines the strengths of The Quaker Oats Company – representing more than a century of experience with oat technology, raw material, and milling – and Rhodia Food Ingredients a world leader in food applications research and development. Partnership is the cornerstone of our business. It is the way we developed this product and it is the way we approach our customers. As our motto says, we are ‘Your Food Tech Partner’. And we prove it every day. We are committed to help you get the most of your food process. Our technical support staff will work with you to reduce your costs, increase efficiency, and further enhance product taste, texture, and nutritional composition – either on-site at your location or in one of our Rhodia food applications laboratories.

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Description Beta-Trim Brand of Quaker Oatrim is enzymatically hydrolysed oat flour containing approximately 5% -glucan. Beta-Trim is produced by treating oat flour with a natural food-grade enzyme, resulting in two products, (water soluble and insoluble portions) which are physically separated and dried. The dried water-soluble portion is Beta-Trim with a very neutral flavour. Benefits and features Tastes like the real thing – Beta-Trim gives reduced fat foods the mouthfeel, texture, and taste consumers expect from full-fat products. Beta-Trim provides: • Lubricity and creaminess of foods that starches and maltodextrin cannot provide. • Desirable flavour properties – Beta-Trim has a neutral flavour that does not interfere with the natural flavour of foods. • Easy to use – Beta-Trim is available as a free-flowing powder, and may be used as is or prepared as a gel. • All-natural Derived naturally from grain, Beta-Trim enhances your product’s appeal to healthconscious consumers. Typical properties • Moisture 4–8% • pH6.0–7.7 • Fat 1.5% • Dextrose equivalence less than 5 • -glucan 4.5–5.5% Label statement Oatrim or Hydrolyzed Oat Flour Applications Bakery Products Cakes Cookies Muffins Frosting Dairy Frozen Desserts Yogurts Sour Cream Cheese spreads

Meat Products Wieners Kielbasa Patties Pork Sausage Convenience Soups Sauces Dressings Beverages

Product data sheet (TrimChoice-5Õ) Converting oat starches to maltodextrins • TrimChoiceÕ is a line of functional fat replacers derived from oats and other cereal grains. The line is manufactured from a process invented and patented by the USDA at

300

•

•

• •

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the National Center for Agriculture Utilization Research (Inglett, 1991). The TrimChoiceÕ process involves the conversion of cereal starches in flour to maltodextrins using -amylase enzymes for starch liquification. Maltodextrins are created by cleaving the starch amylose and amylopectin chains. The degree of polymerisation of the maltodextrins produced is important in determining functionality. Maltodextrins with low dextrose equivalent give the most suitable fat substitute. The conditions of enzyme treatment are selected to achieve conversion of the substrate so that the soluble fibres bound in the cellular matrix are liberated. glucan is the major soluble fibre found in oats. After the enzyme is inactivated, the soluble materials containing the maltodextrin and the soluble fibre -glucan are separated from the insoluble components by centrifugation and dried. TrimChoiceÕ is the only carbohydrate-type fat substitute that contains -glucan soluble fibre which has been shown to have positive health benefits when included in most diets. It is the unique combination of -glucan and low dextrose equivalent maltodextrin that gives TrimChoiceÕ its superior functional properties and its fat-like mouthfeel. (Other maltodextrin gels from the starches of corn, tapioca or potatoes do not possess equivalent fat-like qualities.) TrimChoiceÕ forms a fat-like gel on heating and cooling at 25% dispersion. This gel has slightly less than one calorie per gram compared to fat having nine calories per gram. All of the ingredients in TrimChoice are natural and GRAS (Generally Recognised As Safe) by the FDA.

Specifications for TrimChoiceÕ Proximate Moisture Mineral (ash) Fat (ether extract) Protein (nitrogen6.25) pH 10% solution Dextrose equivalent, DE -glucan* Functionality: Gel strength** (Thermo-reversible gel)

TrimChoice-5 (% on dry basis)

TrimChoice-OC (% on dry basis)

Method

4.0–8.0 3.5 max 0.5 max 5.0 max 5.5–6.5 3.0–5.0 4.0–5.5

4.0–8.0 3.5 max 0.4 max 4.0 max 5.5–6.5 3.0–5.0 1.5–3.0

AACC 44–16 AACC 08–01 AACC 30–20 AACC 46–10 AACC 02–52 Copper number AACC 32–22

100–140

80–120

ML 100–1

* McCleary, B. V. and Glennie-Holmes, M. J. Inst. Brewing 91285 (1985); other than betaglucan, the remaining dietary fibre components are pentosans based on rye, oat and wheat studies. ** 25% solids, blended (Waring commercial blender) 2 minutes in 100ºC water, held at 4ºC for 24 hours and gel strength determined using a Universal Penetrometer fitted with a 35g aluminum cone. Gel strength is measured in mm of penetration. Extraneous matter: To comply with federal tolerances. Ingredient statement: Each container shall be clearly and properly labelled with the following information: ● ● ● ● ●

Ingredient name and product number Processing date or code Country of origin Manufacturer’s name & address Net weight

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Colour and flavour: Lightly creamy white with typical flavour and odour. Label declaration: TrimChoiceÕ-5 TrimChoiceÕ-OC Flour

Hydrolysed oat flour Hydrolysed oat and corn

Packaging: Moisture-resistant multi-wall bags Staples and metal ties are unacceptable Storage Clean, dry area at ambient temperature.

16.5

Uses and applications

The major uses for the -glucan containing hydrocolloids Glucagel, Oatrim and Nu-trim appear to be as texturising agents in foods, where they are able to replace all or part of the fats in processed foods. Oatrim already has found use in meat products, dairy products, bakery products, beverages and convenience foods. It seems likely that both Glucagel and Nu-trim will have similar uses. In addition Glucagel can form strong transparent edible films which could have a wide variety of applications in food coating products. Inglett has reported on the current applications of Oatrim: Currently, Oatrim is found in a nationally distributed extra lean ground beef which is 96% fat-free meat which has the natural taste and texture of a 80% fatfree hamburger. A 112 gram portion has 130 calories compared with 300 calories from an equal weight of the 80% beef. It is also used to replace fat in 97% fatfree franks, fat-free cheeses, and various deli meats. New products containing Oatrim are appearing frequently and many major reduced-fat or fat-free products are under active development which include reduced-fat meats, frozen desserts, salad dressings, sauces, gravies, soups, mayonnaise, margarine, breads, waffles, granola bars, muffins, cookies, brownies, beverages, and cakes. Nu-trim was evaluated for replacing coconut cream in six Thai desserts: coconut jelly, taro conserve, crispy pancake, steamed banana cake, coconut-cantaloupe ice cream. Almost 100% substitution could be made although many panellists favoured 80% substitution to allow for some coconut flavour. Co-processed Oatrim and soy flour was found to replace some or all of the coconut milk in the Thai dishes: chicken green curry and fermented soybean dip, and two Thai desserts: cassava paste and mungbean conserve. Glucagel has been evaluated in bread, cakes, dressings and ice-cream, by an ad hoc panel. Results indicate acceptable flavour and texture when replacing part of the fats in the foods. Individual formulations are given below.

16.5.1 Reduced fat pie crust Product description TrimChoiceÕ-OC produces a tender, flaky crust. KRYSTARÕ crystalline fructose provides a gold brown crust colour.

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Ingredients Untreated pastry flour, White Spray1 Cold water All purpose shortening, Creamtex2 Baker’s margarine, chilled3 TrimChoiceÕ-OC (Mountain Lake Specialty Ingredients Co.) KRYSTAR 3003 Salt Emulsifier, Panodan 1504 Total

Composition (%) 52.01 24.83 10.60 5.49 4.35 0.99 0.99 0.74 100.00

Note: In the development of this formula, the indicated ingredients were obtained from the following suppliers. Their use does not necessarily constitute exclusive endorsement. 1ConAgra, 2Van den Bergh, 3A.E. Staley Co., 4 Danisco.

Preparation Mixer used: three-speed Hobart C-100 with paddle. 1. 2.

3.

4. 5. 6. 7. 8. 9.

Dry blend flour, emulsifier, KRYSTAR and TrimChoiceÕ-OC for 5 minutes at Speed 1 with a Hobart mixer using a flat paddle. Add shortening and margarine. Jog mixer 5 times to break up large lumps of shortening. This should take a total of 5 seconds. Scrape down sides of mixing bowl. Then mix at Speed 1 for an additional 10–15 seconds, scraping bowl as needed. Shortening mixture should be in small lumps. Do not overmix. Preblend the water and salt. Add the water/salt mixture to the ingredients in the Hobart mixer. Jog mixer 5 times. Scrape down sides of bowl. Jog mixer for an additional 10–15 seconds. Using a reversible sheeter and a lightly floured surface, sheet dough to 2mm thick. Avoid using excess flour during sheeting. Place dough into pan and gently press bottom and sides of dough into pan. Avoid stretching dough. Add pie filling. Place rolled dough for top crust on top of filled pie and gently crimp edges of bottom and top dough together to seal pie. Cut ventilation slits on top dough. Bake in preheated 420ºF (216ºC) oven for approximately 38 minutes or until done.

Note: Recipe supplied by Mountain Lake Specialty Ingredients Company, Box 3100, Omaha, NE 68103-0100, USA. The information contained in this bulletin should not be construed as recommending the use of our product in violation of any patent, or as warranties (express or implied) of non-infringement or its fitness for any particular purpose. Prospective purchasers are invited to conduct their own tests and studies to determine the fitness of Mountain Lake’s products for their particular purposes and specific applications. 16.5.2 No-fat blueberry muffin Product description Our fat replacement, TrimChoiceÕ-OC, is used in this system to retain moisture in addition to mimicking the fat characteristics. MIRA-GEL Õ463 and MIRA-SPERSEÕ help provide the desired texture and crumb structure.

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Composition (%)

A

B

Sucrose STALEYDEXÕ3331 Hydrated monoglycerides, GMS 902 Sodium stearoyl lactylate (SSL)

12.17 1.77 1.06 0.24

Cake flour, Velvet3 TrimChoiceÕ-OC (Mountain Lake Specialty Ingredients Co.) STA-SLIMTM-1501 Defatted soy flour, Soyafluff 200w4 Non-fat dry milk, Super Heat6 MIRA-GELÕ4731 MIRA-SPERSEÕ Baking soda Pan-O-Lite6 Sodium propionate Salt Blueberry flavour no. 2935367 Dicalcium phosphate dihydrate (DCPD)7 Xanthan gum, Keltrol TF8

24.74 1.67 1.67 1.42 1.06 0.92 0.92 0.43 0.39 0.35 0.33 0.22 0.05 0.04

Water Liquid egg white

28.48 13.24

Frozen blueberries

8.83

C

D Total

100.0

Note: In the development of this formula, the indicated ingredients were obtained from the following suppliers. Their use does not constitute endorsement. 1A.E. Staley Co, 2American Ingredients, 3ConAgra, 4Central Soya Co, 5Land O’Lakes, 6Monsanto, 7Tastemaker, 8Kelco.

Preparation Mixer used: Hobart C-100 with 3.5 quart mixing bowl and paddle. 1. 2. 3. 4. 5.

Place part A in bowl and blend on speed 3 for 15 seconds. Add part B and blend on speed 1 for 2 minutes. Add part C and mix on speed 1 for 15 seconds. Scrape bowl. Mix an additional 15 seconds on speed 1. Handmix in blueberries. Line muffin pan with paper cups. Weigh 63 grams per muffin and bake at 375ºF (191ºC) for 25 minutes or until done.

Note: Recipe supplied by Mountain Lake Speciality Ingredients Company, Box 3100, Omaha, NE 68103-0100, USA. The information contained in this bulletin should not be construed as recommending the use of our product in violation of any patent, or as warranties (express or implied) of non-infringement or its fitness for any particular purpose. Prospective purchasers are invited to conduct their own tests and studies to determine the fitness of Mountain Lake’s products for their particular purposes and specific applications.

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16.5.3

Extra lean (5% fat) beef patty

Ingredients

Composition (%)

95% Lean Beef Ice/water TRIMCHOICEÕOC (Mountain Lake Specialty Ingredients) TALOTM TF55 flavouring (Staley) Salt Natural beef flavour 1016581 Total 1

89.00 8.52 1.50 0.33 0.40 0.25 100.00

F & C International

Preparation 1. 2. 3. 4.

00

Grind lean beef (28–32ºF (ÿ2–0ºC)) to 12 . Into meat mixer load all ingredients. Mix only long enough to blend, mix for approximately 30 seconds. Remove meat from mixer and grind to 3/1600 . Form patties and freeze.

Note: Recipe supplied by Mountain Lake Speciality Ingredients Company, Box 3100, Omaha, NE 68103-0100, USA. The information contained in this bulletin should not be construed as recommending the use of our product in violation of any patent, or as warranties (express or implied) of non-infringement or its fitness for any particular purpose. Prospective purchasers are invited to conduct their own tests and studies to determine the fitness of Mountain Lake’s products for their particular purposes and specific applications. 16.5.4 Dry mix no-oil creamy Italian dressing Product description TrimChoiceÕ-OC and Instant STELLAR give this cold water dispersible dry mix dressing its smooth and creamy texture. Ingredients Powdered vinegar1 KRYSTARÕ crystalline fructose2 Salt Sweet dairy whey3 Sugar Spice blend, F303784 Instant STELLARÕ2 TrimChoiceÕ-OC (Mountain Lake Specialty Ingredients Co.) MIRA-THIKÕ 4692 Citric acid Xanthan, Keltrol5 Guar, Supercol U6 Parsley, dehydrated4 Titanium dioxide7 Total

Composition (%) 24.00 12.20 12.00 12.00 11.00 8.50 6.00 5.00 4.00 3.00 1.00 0.70 0.30 0.30 100.00

Note: In the development of this formula, the indicated ingredients were obtained from the following suppliers. Their use does not constitute endorsement. 1Kerry Ingredients, 2A.E. Staley Co., 3 Land O Lakes, 4McCormick, 5 Kelco, 6Aqualon, 7Warner-Jenkinson Co.

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Preparation (dry mix) 1. 2.

Mix all the ingredients together thoroughly. Package 50 grams per unit for consumer use.

Preparation (dressing) 1. 2.

In a bowl, combine content of the packet with 12 cup of water. Mix well with wire whip or electric mixer. Add 12 cup of skim milk and mix well. Refrigerate at least 1 hour before serving.

Note: Recipe supplied by Mountain Lake Speciality Ingredients Company, Box 3100, Omaha, NE 68103-0100, USA. The information contained in this bulletin should not be construed as recommending the use of our product in violation of any patent, or as warranties (express or implied) of non-infringement or its fitness for any particular purpose. Prospective purchasers are invited to conduct their own tests and studies to determine the fitness of Mountain Lake’s products for their particular purposes and specific applications.

16.6

Regulatory status

Oatrim has FDA GRAS (generally regarded as safe) status. Nu-Trim would require reaffirmation of FDA GRAS status which is to be sought by the licensee for manufacture. Glucagel, at the time of writing, has not been evaluated for regulatory status. An informal approach to ANZFA (Australian and New Zealand Food Association) indicates that it will be classified as a food since it is produced by an extraction process from barley that uses only water, heating and cooling.

16.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

References

and GRAHAM, H. ‘The hypocholesterolemic function of barley -glucans’, Cereal Foods World, 1989 34 (10) 883–6. ¨ LKKI, Y., AUTIO, K., HA ¨ NNINEN, O., MYLLYMAKI, O., PELKONEN, K., SUORTTI, T. and TO ¨ RRO ¨ NEN, R. ‘Oat MA bran concentrates: physical properties of -glucan and hypocholesterolemic effects in rats’, Cereal Chem, 1992 69 (6) 647–53. ˚ MAN, P., GRAHAM, H., NEWMAN, C. W. and NEWMAN, R. K. ‘Chemical studies on mixedBENGTSSON, S., A linked -glucans in hull-less barley cultivars giving different hypocholesterolaemic responses in chickens’, J Sci Food Agric, 1990 52 435–45. ˚ ., HALLMANS, G., SANDBERG, A., SUNDBERG, B., A ˚ MAN, P. and ANDERSSON, H. ‘Oat -glucan increases LIA, A bile acid excretion and a fiber-rich barley fraction increases cholesterol excretion in ileostomy subjects’, Am J Clin Nutr, 1995 62 1245–51. HECKER, K. D., MEIER, M. L., NEWMAN, R. K. and NEWMAN, C. W. ‘Barley -glucan is effective as a hypocholesterolaemic ingredient in foods’, J Sci Food Agric, 1998 77 179–83. WOOD, P. J. ‘Evaluation of oat bran as a soluble fibre source. Characterization of oat -glucan and its effects on glycaemic response’, Carbohydr Polym, 1994 25 331–6. BRAATEN, J. T., SCOTT, F. W., WOOD, P. J., RIEDEL, K. D., WOLYNETZ, M. S., BRULE, D. and COLLINS, M. W. ‘High -glucan oat bran and oat gum reduce postprandial blood glucose and insulin in subjects with and without type 2 diabetes’, Diabetic Medicine, 1994 11 312–18. WURSCH, P. and PI-SUNYER, F. X. ‘The role of viscous soluble fiber in the metabolic control of diabetes’, Diabetes Care, 1997 20 (11) 1774–80. ¨ GOLZ, E. and WU ¨ RSCH, P. ‘Effects of breakfast cereals containing various amounts of TAPPY, L., GU glucan fibers on plasma glucose and insulin responses in NIDDM subjects’, Diabetes Care, 1999 19 (8) 831–4. DANIELSON, A. D., NEWMAN, R. K., NEWMAN, C. W. and BERARDINELLI, J. G. ‘Lipid levels and digesta NEWMAN, R. K., NEWMAN, C. W.

306 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Handbook of hydrocolloids viscosity of rats fed a high-fiber barley milling fraction’, Nutr Res, 1997 17 (3) 515–22. PETERSON, D. M. and QURESHI, A. A. ‘Effects of tocols and -glucan on serum lipid parameters in chickens’, J Sci Food Agric, 1997 73 417–24. BEHALL, K. M., SCHOLFIELD, D. J. and HALLFRISCH, J. ‘Effect of beta-glucan level in oat fiber extracts on blood lipids in men and women’, J Am Coll Nutr, 1997 16 (1) 46–51. CHAPLIN, M. F. ‘Bile acids, fibre and colon cancer: the story unfolds.’, J Roy Soc Health, 1998 118 (1) 53– 61. INGLETT, G. E. ‘Oatrim – a functional ingredient in foods from licensed USDA/ARS technology’, Am. Assoc. Cereal Chem. AACC 27. San Antonio, Texas, 1995. KNUCKLES, B. E., CHIU, M. C. and INGLETT, G. E. ‘Physical characteristics of -glucan in oat and barley after treatment by the Oatrim and Nu-Trim processes’, Am. Chem. Soc. 216th ACS National Meeting. Boston, Massachusetts, 1998. CARRIERE, C. J. and INGLETT, G. E. ‘Nonlinear solution viscoelastic properties of amylolytic hydrolyzed oat-based -glucan-containing materials’, Am. Chem. Soc. 216th ACS National Meeting. Boston, Massachusetts, 1998. CARRIERE, C. J. and INGLETT, G. E. ‘Solution viscoelastic properties of OATRIM-10 and cooked oat bran’, Cereal Chem, 1998 75 (3) 354–9. INGLETT, G. E. ‘Method for making a soluble dietary fiber composition from oats’ US Patent 4996063, 1991. INGLETT, G. E. ‘Nu-trim, a new -glucan-rich hydrocolloid as a phytonutrient for increasing health benefit of functional foods’, Am. Chem. Soc. 216th ACS National Meeting. Boston, Massachusetts, 1998. CARRIERE, C. J. and INGLETT, G. E. ‘Solution viscosity properties of an oat-based -glucan-rich hydrocolloidal extractive: Nu-Trim.’, Am. Chem. Soc. 216th ACS National Meeting. Boston, Massachusetts, 1998. INGLETT, G. E. ‘Soluble hydrocolloid food additives and method of making’ US Patent 5766662, 1998. MORGAN, K. R. and OFMAN, D. J. ‘Glucagel, a gelling -glucan from barley’, Cereal Chem, 1998 75 (6) 879–81. MORGAN, K. R. ‘Beta-glucan products and extraction processes from cereals’ No. 98/13056. CARPITA, N. C. and GIBEAUT, D. M. ‘Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth’, Plant J, 1993 3 (1) 1–30. FINCHER, G. B. in Barley: genetics, biochemistry, molecular biology and biotechnology. ed. Shewry P R, Wallingford, C.A.B. International, 1992. FINCHER, G. B. and STONE, B. A. in Advances in Cereal Science and Technology, ed. Pomeranz Y, St Paul MN, Am. Assoc. Cereal Chem. 1986. CARPITA, N. C. ‘Structure and biogenesis of the cell walls of grasses’, Annu Rev Plant Physiol Plant Mol Biol, 1996 47 445–76. WOOD, P. J. in Oats: Chemistry and Technology, ed. Webster, F. H., St Paul MN, Am. Assoc. Cereal Chem., 1986. MCLEARY, B. V. and GLENNIE-HOLMES, M. ‘Enzymatic quantification of (1!3)(1!4)- -glucan in barley and malt’, J Inst Brew, 1985 91 285–95. ` , R. ‘Extraction of oat gum from oat bran: effects of process on yield, BEER, M. U., ARRIGONI, E. and AMADO molecular weight distribution, viscosity and (1!3)(1!4)- -D-glucan content of the gum’, Cereal Chem, 1996 73 (1) 58–62. BALLANCE, G. M. and MANNERS, D. J. ‘Structural analysis and enzymic solubilization of barley endosperm cell-walls’, Carbohydr Res, 1978 61 107–18. BACIC, A. and STONE, B. A. ‘Isolation and ultrastructure of aleurone cell walls from wheat and barley’, Aust J Plant Physiol, 1981 8 453–74. ¨ GER, E. and BURCHARD, W. ‘Solution properties of -D-(1, 3)(1, 4)-glucan isolated from GRIMM, A., KRU beer’, Carbohydr Polym, 1995 27 205–14. BHATTY, R. S. in Barley: Chemistry and Technology. eds MacGregor A W and Bhatty R S, St. Paul MN, Am. Assoc. Cereal Chem. 1993. WOOD, P. J., WEISZ, J., FEDEC, P. and BURROWS, V. D. ‘Large-scale preparation and properties of oat fractions enriched in (1!3)(1!4)- -D-glucan’, Cereal Chem, 1989 66 (2) 97–103. BHATTY, R. S. ‘Extraction and enrichment of (1!3), (1!4)- -D-glucan from barley and oat brans’, Cereal Chem, 1993 70 (1) 73–7. BHATTY, R. S. ‘Laboratory and pilot plant extraction and purification of -glucans from hull-less barley and oat brans’, J Cereal Sci, 1995 22 163–70. YIN, X. S., MACGREGOR, A. W. and CLEAR, R. M. ‘Field fungi and -glucan solubilase in barley kernels’, J Inst Brew, 1989 95 195–8. YIN, X. S. and MACGREGOR, A. W. ‘An approach to the identification of a -glucan solubilase from barley’, J Inst Brew, 1988 95 327–30. YIN, X. S. and MACGREGOR, A. W. ‘Substrate specificity and nature of action of barley -glucan solubilase’, J Inst Brew, 1989 95 105–9. STAUDTE, R. G., WOODWARD, J. R., FINCHER, G. B. and STONE, B. A. ‘Water-soluble (1!3), (1!4)- -D-

Cereal -glucans

42. 43. 44. 45.

46. 47.

48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

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glucans from barley (Hordeum vulgare) endosperm. III. Distribution of cellotriosyl and cellotetraosyl residues’, Carbohydr Polym, 1983 3 299–312. WOODWARD, J. R., PHILLIPS, D. R. and FINCHER, G. B. ‘Water-soluble (1!3, 1!4)- -D-glucans from barley (Hordeum vulgare) endosperm. IV. Comparison of 40ºC and 65ºC soluble fractions’, Carbohydr Polym, 1988 8 85–97. IZYDORCZYK, M. S., MACRI, L. J. and MACGREGOR, A. W. ‘Structure and physicochemical properties of barley non-starch polysaccharides. I. Water-extractable -glucans and arabinoxylans’, Carbohydr Polym, 1998 35 249–58. IZYDORCZYK, M. S., MACRI, L. J. and MACGREGOR, A. W. ‘Structure and physicochemical properties of barley non-starch polysaccharides. II. Alkali-extractable -glucans and arabinoxylans’, Carbohydr Polym, 1998 35 259–69. WOOD, P. J., WEISZ, J. and BLACKWELL, B. A. ‘Molecular characterization of cereal -D-glucans. Structural analysis of oat -D-glucan and rapid structural evaluation of -D-glucans from different sources by highperformance liquid chromatography of oligosaccharides released by lichenase’, Cereal Chem, 1991 68 (1) 31–9. HENRIKSSON, K., TELEMAN, A., SUORTTI, T., REINIKAINEN, T., JASKARI, J., TELEMAN, O. and POUTANEN, K. ‘Hydrolysis of barley (1–3), (1–4)- -D-glucan by a cellobiohydrolase II preparation from Trichoderma reesei’, Carbohydr Polym, 1995 26 109–19. ˚ RUM, K. M., MARTINSEN, A. and SMIDSROD, O. ‘Fractionation and viscometric characterization of a (1– VA 3), (1–4)- -D-glucan from oat, and universal calibration of a high-performance size-exclusion chromatographic system by the use of fractionated -glucans, alginates and pullulans’, Food Hydrocolloids, 1991 5 (4) 363–74. DAWKINS, N. L. and NNANNA, I. A. ‘Studies on oat gum [(1!3, 1!4)- -D-glucan]: composition, molecular weight estimation and rheological properties’, Food Hydrocolloids, 1995 9 (1) 1–7. ˚ RUM, K. M., SMIDSROD, O. and BRANT, D. A. ‘Light scattering reveals micelle-like aggregation in the VA (1!3), (1!4)- -D-glucans from oat aleurone’, Food Hydrocolloids, 1992 5 (6) 297–511. BEER, M. U., WOOD, P. J. and WEISZ, J. ‘Molecular weight distribution and (1!3)(1!4)- -D-glucan content of consecutive extracts of various oat and barley cultivars’, Cereal Chem, 1997 74 (4) 476–80. ´ MEZ, C., NAVARRO, A., MANZANARES, P., HORTA, A. and CARBONELL, J. V. ‘Physical and structural GO properties of barley (1!3), (1!4)- -D-glucan. Part I. Determination of molecular weight and macromolecular radius by light scattering’, Carbohydr Polym, 1997 32 7–15. ´ MEZ, C., NAVARRO, A., MANZANARES, P., HORTA, A. and CARBONELL, J. V. ‘Physical and structural GO properties of barley (1!3), (1!4)- -D-glucan. Part II. Viscosity, chain stiffness and macromolecular dimensions’, Carbohydr Polym, 1997 32 17–22. ´ MEZ, C., NAVARRO, A., MANZANARES, P., HORTA, A. and CARBONELL, J. V. ‘Physical and structural GO properties of barley (1!3), (1!4)- -D-glucan. III. Formation of aggregates analysed through its viscoelastic and flow behaviour’, Carbohydr Polym, 1997 34 141–8. DOUBLIER, J-L. and WOOD, P. J. ‘Rheological Properties of Aqueous Solutions of (1!3), (1!4)- -DGlucan from Oats (Avena sativa L.)’, Cereal Chem, 1995 72 (4) 335–40. SIMS I. (unpublished) 1997. LAPASIN, R. and PRICL, S. in Rheology of Industrial Polysaccharides: Theory and Application, London, Chapman and Hall, 1995. SHIRAKAWA, M., UNO, Y., YAMATOYA, K. and NISHINARI, K. in Gums and Stabilizers for the Food Industry 9, eds Williams P A and Phillips G O, Wrexham, Roy. Soc. Chem. 1997. INGLETT, G. E. (Personal communication) 1998. 13 MORGAN, K. R., ROBERTS, C. J., DAVIES, M. C., WILLIAMS, P. M. and TENDLER, S. J. B. ‘A C CP/MAS NMR spectroscopy and AFM study of the structure of Glucagel, a gelling -glucan from barley’, Carbohydr Res, 1999 315 169–79. BRAATEN, J. T., WOOD, P. J. and COLLINS, M. W. ‘Oat -glucan reduces blood cholesterol in hypercholesterolemic subjects.’, Eur J Clin Nutr, 1994 48 465–74.

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DAVIDSON, M. H., DUGAN, L. D., STOCKI, J., DICKLIN, M. R., MAKI, K. C., COLETTA, F., COTTER, R., MCLEOD, M. and HOERSTEN, K. ‘A low-viscosity soluble-fiber fruit juice supplement fails to lower cholesterol in

62.

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63. 64. 65.

hypercholesterolemic men and women’, J. Nutr., 1998 128 (11) 1927–32. ‘Dietary fiber and the risk of colorectal cancer and adenoma in women’, New Engl J Med, 1999 340 (3) 169. YOKOHAMA, W. H., KNUCKLES, B. E. and INGLETT, G. E. ‘Raw and processed oat ingredients lower plasma cholesterol in the hamster’, J Food Sci, 1998 63 (4) 713–6. INGLETT, G. E. and NEWMAN, R. K. ‘Oat -glucan-amylodextrins: preliminary preparation and biological properties’, Plant Foods Hum Nutr, 1994 45 53–61. MCBRIDE, J. ‘Two thumbs up for Oatrim’, Agriculture Res, 1993 41 (12) 4–7.

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17 Soluble soybean polysaccharide H. Maeda, Fuji Oil Co., Ltd., Osaka

17.1

Introduction

Soluble soybean polysaccharide, ‘SSPS’, is a water soluble polysaccharide extracted and refined from soybean. Fuji Oil has been marketing SSPS under the brand name ‘SOYAFIBE-S’ since 1993. SOYAFIBE-S is mainly composed of the dietary fibre of soybean and has relatively low viscosity and high stability in aqueous solution. During the manufacture of soy protein isolate, soymilk and tofu (soybean curd), ‘okara’, which is the insoluble residue from protein extraction, is produced.1, 2 Okara is a rich source of dietary fibre and has bowel-conditioning effects.3, 4 Therefore, okara can be made into various types of food, including salads, soups, sauces, baked goods, desserts, sausages and okara burgers.5, 6 However, most okara is used ineffectively and is treated as an industrial waste, because it contains about 80% moisture and spoils quickly. The author and his colleagues started to study the high utilisation of okara about 11 years ago and developed the SSPS, SOYAFIBE-S. SSPS has various functions such as dispersion, stabilisation, emulsification, and adhesion.7, 8 Therefore, SSPS can be used not only as a dietary raw material for fibrefortified foods, but also for pharmaceutical and industrial applications as well as for many other food applications. In this chapter, the material structure, properties and applications of SSPS are introduced.

17.2

Manufacture

The water soluble soybean polysaccharide, SSPS, is made from a cotyledon in the seed of Glicine max MERRILL. SSPS is produced by heating in weak acidic conditions from okara, which is the insoluble substance simultaneously produced in the manufacture of soy protein isolate. After a rather efficient and patented extraction technique followed by refining, pasteurising, and spray-drying process, SOYAFIBE-S comes out in powder The author wishes to thank Professor G. O. Phillips for his critical reading of the manuscript and valuable suggestions.

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Fig. 17.1 The manufacturing process of SOYAFIBE-S.

form as shown in Fig. 17.1. Some reports about the extraction of SSPS from okara have already been published.9–11 No hazardous chemical material whose residue may cause any safety problems is used in the production of SOYAFIBE-S.

17.3

Structure

17.3.1 General analytical values Table 17.1 shows typical analytical values of SOYAFIBE-S-DN of the most common type. The component sugars are mainly galactose, arabinose, galacturonic acid, but also include many others such as rhamnose, fucose, xylose and glucose. SOYAFIBE-S is supplied in different types, each modified to have specific functionality. For example, a type containing less than 5% of crude protein is produced.

17.3.2 Structure of major components Soybean okara, which is the residue after oil and protein extraction from soybean, contains soluble and non-soluble dietary fibre from soybean cotyledon. In soluble dietary fibre from soybean cotyledon, the sugar compositions and sequences have been reported,14–17 but the full aspects of their structure are poorly understood. The gel filtration chromatographic analysis of SSPS by HPLC shows roughly three components having approximate molecular weight of 550,000, 25,000, and 5,000 respectively, and the mean value is several hundred thousand (Fig. 17.2). Recently, Nakamura et al. identified most of the chemical structure of soluble soybean polysaccharides having molecular weight of 550,000, as shown in Fig. 17.3.18 This major component of SSPS has three types of galacturonic acid main back-bone consisting of rhamnogalacturonan and homogalacturonan. It consists of long-chain rhamnogalacturTable 17.1

Chemical composition of SOYAFIBE-S-DN

(%)

Crude protein (%)

Crude ash (%)

Dietary fibre content1 (%)

Rha

Fuc

Ara

Xyl

Gal

Glc

GalA

5.8

9.2

8.6

66.2

5.0

3.2

22.6

3.7

46.1

1.2

18.2

Moisture

Sugar composition2 (%)

1. The analysis was conducted by AOAC official method (Prosky method).12 2. Neutral saccharides were analysed by GLC after being converted to alditol acetate, and galacturonic acid were by Blumenklanz method.13

Soluble soybean polysaccharide

Fig. 17.2

311

Molecular weight distribution of SOYAFIBE-S-DN.

Fig. 17.3

Structure of main fraction from SSPS.

Fig. 17.4 Distribution of galacturonan regions in SSPS and citrus pectin. Straight lines, notched lines and arrows indicate homogalacturonan regions, rhamnogalacturonate regions and the reducing end, respectively. Figures express the percentage of galacturonate distribution. In SSPS, neutral sugar side chains consisted of arabinan and galactan mainly linked to rhamnogalacturonan main back-bone. This result indicates that SSPS has a spherical structure compared with linear structure of pectin.

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onan and short-chain homogalacturonan, while citrus pectin consists of short-chain rhamnogalacturonan and long-chain homogalacturonan (Fig. 17.4). Homogeneous galactosyl and arabinosyl neutral sugar side chains combine with the rhamnogalacturonan region through rhamnose and are longer than the galacturonosyl main back-bone.

17.4

Basic material properties and characteristics

17.4.1 High dietary fibre content As shown in Table 17.1, the content of dietary fibres measured by the AOAC official method is more than 60%. Therefore, SSPS has the same physiological functions as those of other dietary fibres. We have found that SSPS is partially metabolised and changed into organic acid by enteric bacteria and effectively shortens the gastrointestinal transit time in rats.19

Fig. 17.5

Fig. 17.6

Viscosity comparison of various polysaccharide solutions at 25ºC.

Viscosity change by heating at various pH ranges (10% SOYAFIBE-S aqueous solution).

Soluble soybean polysaccharide

Fig. 17.7

313

Effect of various salts on viscosity of a 10% SSPS solution at 20ºC.

17.4.2 High solubility and stable viscosity against heat, acid and salts SSPS is soluble in both cold and hot water without gelation, and shows a relatively low viscosity compared to the viscosity of other gums/stabilisers such as guar gum, allowing a highly concentrated (more than 30%) solution to be produced (Fig. 17.5).20 Furthermore, the viscosity of the solution is not significantly affected by acid, heat or salts (Figs 17.6 and 17.7).

17.4.3 Excellent adhesive and film forming property SSPS has a strong adhesive property. The adhesive strength was tested according to the JIS standards, K6848-1987 and K6851-1976. As shown in Table 17.2, the adhesive strength was as strong as or better than that of Pullulan, an adherent generally regarded as having very high adhesive strength.21 With this property, SSPS functions as a binder for not only dried foods like snacks and cereals, but also papers, woods or glass (Table 17.2). In tension tests, the film processed without using any additives showed a resistance to tension approximately as high as that of Pullulan as shown in Table 17.2.21 The film forming property allows us to make a colourless, transparent, water soluble, and edible film easily. Also, this filmability can be used to coat the surface of foods such as tablets and other materials.

Table 17.2

Adhesive strength and material property of film

Material SOYAFIBE-S Pullulan Gum arabic

Adhesive strength (kgf/cm2)

Tensile strength (kgf/cm2)

Young ratio (kgf/cm2)

46.6 40.5 30.7

540 509 N/D*

9,730 12,800 N/D*

* The film was easily cracked not allowing the tension to be measured.

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Fig. 17.8

Antioxidative effect of SOYAFIBE-S on soybean oil (stored at 60ºC).

17.4.4 Anti-oxidative property It has been confirmed that SSPS prevents oxidation of oils and flavour oils. This is presumed to be due to the stabilisation of free radicals by the pectic polysaccharides which are the main component of SSPS.22 Figure 17.8 shows the anti-oxidative effects on soybean oil of SOYAFIBE-S-LA200 developed for use in powdered flavour. On the other hand, gum arabic did not show an anti-oxidative effect such as SOYAFIBE-S.

17.5

Functions and applications of SSPS

Table 17.3 shows the functions and applications of SOYAFIBE-S, among which dispersion stabilising, emulsifying, and anti-sticking effects are outstanding. SOYAFIBE-S is supplied in a variety of types specialised for each purpose (Table 17.4). Table 17.3

Functions and applications of SOYAFIBE-S

Functions

Applications

Type of SOYAFIBE-S

Soluble Dietary Fibre Stabilising effects under acidic conditions Emulsifying Emulsion stabilising Adhesion Film forming property Dispersing Foam stability

General dietary fibre, fortified foods Drinkable yoghurt, ice cream, acidic dessert, sour cream Flavour emulsion, powdered flavour, coffee cream, dressing, cleaner Edible film, coating agent, particle forming agent, snack, bakery, coating for printing rolls Various types of paint, agricultural chemicals, ceramics, cement Meringue, surfactant

All types DN DA100 EN100 LN LA200 DN FA100 DN LA200 LN1 DN DA100 LA200

Anti-sticking effect

Various types of cooked rice and noodles

DN DA100 LA200

Softening effect

Bread, cake, ham, sausage, Kamaboko (boiled fish paste), cream sauce

All types

Soluble soybean polysaccharide Table 17.4

315

Varieties of SOYAFIBE-S Crude protein (%)

Crude ash (%)

Viscosity (mPa/ sec)

SOYAFIBE-S-DN

9.2

8.6

56

SOYAFIBE-S-DA100

6.2

8.4

62

SOYAFIBE-S-LN SOYAFIBE-S-LN1 SOYAFIBE-S-LA200

10.4 13.9 7.5

6.6 6.8 6.5

24 13 16

SOYAFIBE-S-EN100

8.7

7.2

71

SOYAFIBE-S-FA100

5.8

6.0

140

Type

Characteristics Developed as a stabiliser of protein particles under acidic conditions Improved flavour over DN For a stabiliser of protein particle under acidic conditions Developed as an emulsifier With the lowest viscosity Improved flavour over LN/LN1 For powdering bases Improved stability over LN in suspensions Developed for flavour emulsions Excellent colour and high viscosity For edible films and coatings

17.5.1 Dispersion stabilisation effects on protein particles under acidic conditions Under acidic conditions, SOYAFIBE-S prevents protein particles from coagulating and precipitating. Unlike HM-pectin (high methoxyl pectin), which is the most commonly used stabiliser under acidic conditions as in drinkable yoghurt,23, 24 the point of interest with SOYAFIBE-S is its ability to stabilise protein particles at low pH conditions without raising viscosity. CMC (sodium carboxymethylcellulose)25 and PGA (propylene glycol alginate)26 may also be used as such a stabiliser. These hydrocolloids, on the basis of their characteristics, tend to give a high viscosity to final products, which means acidic milk drinks with low viscosity, light taste and no sticky mouthfeel can be produced.27 Figure 17.9 shows the viscosity, rate of precipitation and particle size of protein of drinkable yoghurts made with SOYAFIBE-S or HM-pectin as a stabiliser. Acidic milk drinks with the stabiliser SOYAFIBE-S show a lower viscosity than drink stabilised with HM-pectin. In lower pH products (pH < 4.0), SOYAFIBE-S shows excellent stabilising effect. At higher pH (pH = 4.4), however, SOYAFIBE-S is less effective than HM-pectin. In other words, SOYAFIBE-S is suitable for acidic milk drink with lower pH and less non-fat milk solids. This function of SOYAFIBE-S can be applied especially to many other products, such as beverages, ice creams, desserts, etc., and provides an excellent stability and refreshing taste under acidic conditions. Further, SSPS has the merit of having low reactivity with calcium, thus ensuring its full performance even if applied at an early stage of processing before fermentation. This beneficial feature allows manufacturing processes to be improved. From the result of particle size shown in Fig. 17.10, the mechanism of dispersion with SSPS is rather different from that with HM-pectin. As shown in Fig. 17.10, SSPS originally contains about 20% of galacturonic acid, which is located in the back-bone of a major molecule. The anion groups of the acid probably bind to the surface of cationic protein particles, so polysaccharides coat the particles. The anionic polysaccharides are prevented from coagulation with each other by the electric charge. It is assumed that the coating layer of the polysaccharides is thick, because each has many side chains of galactose or arabinose.

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Fig. 17.9 Effects of SSPS and HM-pectin on dispersion of acidic milk protein.1 1. Formulation of acidic milk drink: non-fat milk 8.0%, stabiliser 0.4%, sugar 7.0%. 2. Measured with a B type Viscometer Model BM at 10ºC (Rotor no. 1, 60rpm). 3. After each 50 gram solution has been centrifuged at 3,000G for 20 min., the supernatant was decanted and the residue was allowed to stand for 20 min. in order to remove the remaining supernatant before measurement of the extent of precipitation. Rate of precipitation = {Precipitate (g)/50 (g)} 100 (%) 4. Measured with Laser Diffraction Particle Size Distribution Analyzer (SALD-2000A, Shimadzu Corp.)

Fig. 17.10 Stabilising mechanism of protein particles under acidic conditions.

17.5.2 Emulsifying and suspending stability Flavour emulsions are often used to suspend the whole liquid of a drink 0.1% or so to a colourless transparent liquid to give it a juicy feel as well as scent and colour. Gum arabic is used widely in the field of flavour emulsion by virtue of its excellent emulsifying property.28 SOYAFIBE-S has an emulsifying function compatible with gum arabic, and

Soluble soybean polysaccharide Table 17.5

Formulation of flavour emulsions SOYAFIBE-S-EN100

Test no.

1

Oil phase1 Gum arabic SOYAFIBE-S-EN100 Glycerol Water Citric acid2 Total

317

2

Gum arabic

3

4

20 20 20 – – – 20 15 10 20 20 20 60 65 70 sufficient sufficient sufficient 120

120

120

5

6

20 20 20 30 20 15 – – – 20 20 20 50 60 65 sufficient sufficient sufficient 120

120

120

1. Lemon oil:MCT:SAIB = 5:55:40 (Specific Gravity 1.010) 2. To adjust pH at 4.0

Table 17.6

Results of emulsification test SOYAFIBE-S-EN100

Test no. Water phase viscosity (mPa/sec)1 Particle size (m) Heat stability2 Clouding stability3

Gum arabic

1

2

3

4

5

6

1600 0.62 0.0% A

413 0.73 2.7% B

95 0.83 14.5% C

485 0.50 6.0% B

105 0.58 10.3% C

45 0.77 10.4% D

1. Measured by BM Type Viscometer at 20ºC. 2. Shown in the increased rate of the oil particle size in the flavour emulsions after four-week storage at 35ºC. 3. Dispersed 0.1% of flavour emulsion in water containing 8.7% sugar and 0.3% citric acid, and observed clouding stability after four-week storage at 50ºC. A; Superior, B; Good, C; Acceptable, D; Not acceptable.

1. 2. 3. 4.

Fig. 17.11 Preparation of flavour emulsions. Dissolve polysaccharide and glycerol in water and mix completely. Adjust the solution’s pH at 4.0 with citric acid. Pour oil phase into the solution, and mix it by homomixer at 8000rpm for 30 min. at 35ºC. Homogenise twice at 150 kg/cm2, 35ºC.

can be used as an emulsifier and stabiliser for any emulsified foods including flavour emulsions and powdered flavours. Table 17.5 and Fig. 17.11 show the recipe and processing method in a comparison with gum arabic using SOYAFIBE-S as an emulsifying agent for the flavour, including changing the ratios of the oil phase and the emulsifying agent. From the results shown in Table 17.6, it can be seen that compared with gum arabic, a smaller volume can formulate a flavour emulsion with good suspension stability.

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Table 17.7

Results of foam stability test1

Sample solution

SOYAFIBE-S -Carrageenan No additive

Foaming ratio2

Quantity added

Viscosity

(%)

(mPa/sec)

After 10 min.

After 3 hrs

0.2 0.2 –

4.9 11.3 4.4

2.0 2.6 1.6

1.8 1.4 0.1

1. 50ml of the hydrolysed soybean protein solution was placed in a 100ml measuring cylinder and a change was observed on standing after shaking strongly for one minute and allowing to stand. 2. Foaming ratio = volume of foam (ml)/volume of liquid layer (ml).

SSPS contains glycoproteins, whose structure we presume is similar to that of the Wattle Blossom Model suggested for gum arabic.29, 30 Unlike low molecular surfactants, a high molecular surfactant of this type can stabilise one oil particle with fewer molecules and remain adhering to many parts of the surface. With SPSS, even if one part of SSPS falls apart from the surface, another part can come back to the surface. Therefore, this will maintain a stable state of suspension. Additionally, SSPS suppresses deterioration of flavours with its antioxidative effects on oils, as shown in Fig. 17.8. 17.5.3 Foam stabilising function SOYAFIBE-S has a foam stabilising function. The stability of foams when 2% of SOYAFIBE-S was added to 2% water solution of hydrolysed soy protein, a common foaming agent, is shown in Table 17.7, from which we can see that SOYAFIBE-S is more effective than -Carrageenan. This function can be used for stabilising meringue foam. 17.5.4 Anti-sticking effect of cooked rice and noodles SOYAFIBE-S keeps cooked rice, such as plain rice or rice with other ingredients like pilaf, not sticky for many hours after being boiled. This means the addition of SOYAFIBE-S can be a great help when mixing other ingredients with cooked rice. The same effect is obtained when SOYAFIBE-S is used in retort or frozen rice. Furthermore, as rice is boiled hard by the addition of SOYAFIBE-S, more water can be added before being boiled and so the yield of boiled rice increases. The rice obtained in this manner has a benefit that it does not easily get hard during cold storage, as shown in Fig. 17.12. On the other hand, SOYAFIBE-S keeps cooked noodles without sticking for many hours. Just dipping the boiled noodles in SSPS water solution or spraying the solution on the noodles prevents the noodles from sticking to each other for many hours. The same effect is obtained when the noodles are boiled in the SSPS solution. SOYAFIBE-S also keeps spaghetti and chow mein not sticky for a long time without oil. When SOYAFIBE-S is added in sauce for spaghetti or chow mein, the noodles remain unsticky for many hours. SSPS is absorbed on the surface of cooked rice or noodles and coats the surface. It is assumed that the driving force of absorption is the galacturonan of the main back-bone in SSPS and the coating layer of SSPS is thick. 17.5.5 Other applications SOYAFIBE-S is a dietary fibre and can be used for a fibre enrichment in various foods. Many other applications are possible, such as film coating agent for tablets and softener

Soluble soybean polysaccharide

319

Fig. 17.12 Change in the taste value of boiled rice (stored at 10ºC). Rice was boiled with 1.2 times of water in no additive case. On the other hand, where SOYAFIBE-S was added, rice was boiled with 1.44 times of water containing 1.0% of SOYAFIBE-S for the rice. The boiled rice was stored at 10ºC for 48 hours and the change in the taste value was analysed by the Rice Taste Analyzer STA-1A (Satake Corporation).31

of baked foods. Thus, it may be useful not only in various foods, but also in various industrial applications. SSPS has dispersing effects for inorganic particles like its stabilising effect with protein particles. Therefore, applications as a dispersion agent for water colour, ceramics and cement are possible.

17.6

Regulatory status

In Japan, SSPS is classified, both as a food ingredient and food additive with no limitation of application. It must be labelled as ‘soybean polysaccharide’ or ‘soybean hemicellulose’ as a food additive, according to supplement 3 of Food Sanitation Law 1996. On the other hand, it can be labelled as ‘soybean fibre’ for use as a food ingredient. In the USA, SSPS has the status of self-affirmed GRAS according to a law firm’s written opinion.

17.7 1. 2. 3. 4. 5. 6. 7. 8. 9.

References

and MINO, M. ‘Studies on the carbohydrate of soybeans’, Bull Agr Chem Soc, 1955 19 69–76. LIU, K. SOYBEANS, New York, Chapman & Hall, 1997. TSAI, A. C., MOTT, E. L., OWEN, G. M., BENNICK, M. R., LO, G. S. and STEINKE, F. H. ‘Effects of soy polysaccharide on gastrointestinal functions, nutrient balance, steroid excretions, glucose tolerance, serum lipids, and other parameters in humans’, Am J Clin Nutr, 1983 38 504–11. TAKAHASHI, T., EGASHIRA, Y., SANADA, H., AYANO, Y., MAEDA, H. and TERASHIMA, M. ‘Effects of soybean dietary fiber on growth rate, absorption and gastrointestinal transit time in rats’, J Jpn Soc Nutr Food Sci (in Japanese), 1992 45 277–84. SHURTLEFF, W. and AOYAGI, A. The Book of Tofu, Berkeley CA, Ten Speed Press, 1975. SHURTLEFF, W. and AOYAGI, A. Tofu and Soymilk Production, Lafayette CA, The Soyfoods Center, 1984. MAEDA, H. ‘Development and application of soybean polysaccharides’, Shokuhin to Kaihatsu (in Japanese), 1992 27 47–9. MAEDA, H. ‘Soluble soybean polysaccharide: Properties and applications of SOYAFIBE-S’, The Food Industry (in Japanese), 1994 37 no. 12 71–4. YOSHII, H., FURUTA, T., MAEDA, H. and MORI, H. ‘Hydrolysis kinetics of okara and characterization of its water-soluble polysaccharides’, Biosci. Biothec Biochem, 1996 60 (9) 1406–9. KAWAMURA, S., KOBAYASHI, T., OSIMA, M.

320 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Handbook of hydrocolloids and MAEDA, H. ‘Extraction of water-soluble soybean polysaccharides under acidic conditions’, Biosci. Biothec Biochem, 1998 62 (12) 2300–5. MORITA, M. ‘Polysaccharides of soybean seeds: Polysaccharide constiuents of hot-water-extract fraction of soybean seed and an arabinogalactan as its major component’, Agr Biol Chem, 1965 29 564–73. PROSKY, L., ASP, N.G., FURDA, I., DEVRIES, J. W., SCHWEIZER, T. F. and HARLAND, B. F. ‘Determination of total dietary fiber on foods and food products: Collaborative study’, J Assoc Off Anal Chem, 1985 68 677–9. BLUMENKRANTZ, N. and HANSEN, G. A. ‘New method for quantitative determination of uronic acids’, Annal Biochem, 1973 54 484–9. ASPINALL, G. O., COTTRELL, I. W., EGAN, S. V., MORRISON, I. M. and WHYTE, J. N. C. ‘Polysaccharides of soybean. part IV, J Chem Soc (C), 1967 1071–80. KAWAMURA, S. ‘A review on the chemistry of soybean Polysaccharides’, Nippon Shokuhin Kougyo Gakkaishi (in Japanese), 1967 14 514–23, 553–62. KIKUCHI, T. and SUGIMOTO, H. ‘Detailed structure of acidic polysaccharide in soy sauce, confirmed by use of two kinds of purified pectinase’, Agric Biol Chem, 1976 40 87–92. LABAVITCH, J. M., FREEMAN, L. E. and ALBERSHEIM, P. ‘Structure of plant cell walls’, J Biol Chem, 1976 25 5904–10. NAKAMURA, A., FURUTA, H., MAEDA, H., NAGAMATSU, Y. and YOSHIMOTO, A. ‘The structure of soluble soybean polysaccharide’, Hydrocolloids: Part 1 (Ed. K. Nishinari) Elsevier Science, 2000 235–41. TAKAHASHI, T., MAEDA, H., AOYAMA, T., YAMAMOTO, T. and TAKAMATSU, K. ‘Physiological effects of water-soluble soybean fiber in rats’, Biosci. Biothec Biochem, 1999 63 (8) 1340–5. FURUTA, H. and MAEDA, H. ‘Rheological properties of water-soluble soybean polysaccharides extracted under weak acidic condition, Food Hydrocolloids, 1999 13 267–74. NAKAMURA, S. ‘Function and application of pullulans’, Fragrance Journal (in Japanese), 1986 78 69–74. YOSHIKI, Y. and OKUBO, K. ‘Chemiluminescence of DDMP saponin and Chemiluminescence substance in soy sauce’, Syushi Seiriseikagaku Kenkyukai Youshisyu (in Japanese), 1994 15 26. GLAHN, P. E. ‘Hydrocolloid stabilization of protein suspensions at low pH’, in: Prog Fd Nutr Sci 6, Phillips G O, Wedlock D J and Williams P A, eds, Oxford, Pergamon Press, 1982 171–7. PEDERSEN, H. C. A. and JORGENSEN, B. B. ‘Influence of pectin on the stability of casein solutions studied in dependence of varying pH and salt concentration, Food Hydrocolloids, 1991 5 323–8. ANON. ‘Develops milk-orange juice’, Food Eng, 1971 Apr 97–101. LUCK, H. and GROTHE, J. ‘Fruit juice-flavored milk’, S Afr J Dairy Technol, 1973 5 47–52. ASAI, I., WATARI, Y., IIDA, H., MASUTAKE, K., OCHI, T., OHASHI, S., FURUTA, H. and MAEDA, H. ‘Effect of soluble soybean polysaccharide on dispersion stability of acidified milk protein’, in Food Hydrocolloids Structure, Properties, and Functions, Nishinari K and Doi E, eds, New York, Plenum Press, 1993. TAN, C. T. and HOLMES, J. W. ‘Stability of beverage flavor emulsions’, Perfumer and Flavorist, 1988 13 Feb/Mar 23–41. CONNOLLY, S., FENYO, J. C. and VANDEWELDE, M. C. ‘Heterogeneity and homogeneity of an arabinogalactan-protein: Acacia senegal gum’, Food Hydrocolloids, 1987 1 477–80. RANDALL, R. C., PHILLIPS, G. O. and WILLIAMS, P. A. ‘The role of proteinaceous component on the emulsifying properties of gum arabic’, Food Hydrocolloids, 1988 2 131–40. MIKAMI, T. ‘Machine design and development for rice other grains in chugoku area (Development of rice taste analyzer)’, Sekkei Kougaku (in Japanese), 1997 32 113–19. FURUTA, H., TAKAHASHI, T., TOBE, J., KIWATA, R.

18 Bacterial cellulose T. Omoto, Y. Uno and I. Asai, San-Ei Gen, FFI Inc., Japan

18.1

Introduction

It is well known that cellulose is produced by plants and that cellulose is a substance making skeletons of plants. Fine crystal cellulose or fibrillated cellulose, which is obtained by pulverising cellulose through chemical and physical treatments, is widely used as a food additive. Not only is cellulose produced by plants but also it is produced by micro-organisms. A micro-organism which can produce cellulose is Acetobacter. Acetobacter is a bacterium used for manufacturing vinegar. In vinegar manufacture, gel-like membrane has often been found on the surfaces of culture liquids. About a century ago this material was determined to be cellulose. This cellulose of bacterial origin is called bacterial cellulose, distinguished from plant cellulose. Bacterial cellulose has been eaten for many years as a dessert food called ‘nata de coco’.

18.2

Manufacture

Cellulose by fermentation is a kind of microbial polysaccharide consisting of cellulose fibre produced by strains of xylinum, a subspecies of Acetobacter aceti, a nonpathogenic bacterium, and is named bacterial cellulose or fermentation-derived cellulose, distinguished from fine crystal cellulose or fibrillated cellulose prepared from pulp, etc.

18.2.1 Fermentation process The genenal fermentation goes through five stages, the final stage being the production sized fermentation. In the first stage, vials of Acetobacter xylinum (stored at ÿ140ºC) are added to 2.8l Fernbach flasks containing sterile growth media. The operating volume is 500ml at pH5 and the temperature is maintained at 30ºC. The second stage is again in a 2.8l Fernbach flask containing 1000ml media. The inoculation is 50ml from the stage one flask. The bacteria are grown until an optical density (OD) of >1 is reached. Four preseed Fernbach flasks are used for the primary seed fermentor.

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The primary seed fermentor has a 36 litre volume and contains the same growth media as the previous flask cultures. It is maintained at 30ºC with sufficient airflow to avoid oxygen limiting conditions. Transfer criterion is an OD > 1.5, with a final negative Gram stain. This fermentation is used to inoculate the secondary seed fermentor. The secondary seed fermentor has a ca. 3800 litre volume. The temperature, pH and dissolved oxygen are all monitored. At this point cellulose strands will be visible. The secondary seed is used to inoculate the final fermentor, in this case ca. 19,000 litre. The parameters monitored include pH, dissolved oxygen, generated carbon dioxide, temperature, glucose, cellulose and cell growth. Fermentation is run for approximately 60 hours. Glucose feed is turned off one hour before harvest.

18.2.2 Bacterial cellulose purification and recovery The fermentor broth is initially dewatered on a twin wire belt press to yield a cake of approximately 20% solids. This cake is reslurried to between 1.5–3% solids. Solid sodium hydroxide is added to bring the pH up to 13.1, and the system is heated to 65ºC for two hours with agitation to dissolve the micro-organisms. After two hours the pH is adjusted with sulfuric acid to 6–8. The slurry is then dewatered as above and reslurried and dewatered two additional times to purify the bacterial cellulose.

18.3

Structure

Bacterial cellulose has the same chemical structure as cellulose from plants, and it is a straight chain polysaccharide having D-glucose molecules connected by -1,4 bonding (Fig. 18.1). Although bacterial cellulose has the same chemical structure as cellulose from plants, it consists of much finer cellulose fibres being produced by bacteria. Its single fibres have a diameter of about 50nm, and bacterial cellulose exists in the form of a single fibres’ bundle having a diameter of 0.1–0.2m. The length of the fibres is not determinable because the bundles and fibres are entangled with each other to form a network structure (Fig. 18.2). For comparison, the diameter of typical fine crystal cellulose is 10–30m (Fig. 18.3).

Fig. 18.1 Chemical structure of bacterial cellulose.

Bacterial cellulose

Fig. 18.2

Network structure of bacterial cellulose in aqueous solution.

Fig. 18.3

Typical fine cellulose fibre.

323

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Fig. 18.4 The effects of NaCl and retorting in acidic solution.

18.4

Technical data

18.4.1 The structure in aqueous solutions Bacterial cellulose is not soluble in water, but it can be dispersed in water forming a network, resulting in good functions. When bacterial cellulose is allowed to swell in water, a continuous three-dimensional network structure is formed with cellulose fibres connected partly to each other. The three-dimensional network structure has a good ability to hold water and develops viscosity based on such a structure itself. On the other hand, cellulose from plants, which has been well studied until now, is also insoluble in water, but, unlike bacterial cellulose, it exists in the form of fine particles in water; it is poor at forming a three-dimensional structure and holding water; and thus it develops almost no viscosity and precipitates unless polysaccharide is used in combination with it. Bacterial cellulose dispersed in water which has formed a three-dimensional network structure shows pseudoplasticity in its viscosity. It has only weak reactivity with various additives because of its insolubility in water. Its three-dimensional network structure is little affected by acids, salts and heat (Fig. 18.4), because the structure is formed by cellulose strongly connected partly to each other. Besides, by virtue of this network structure, bacterial cellulose gives good dispersion-stability or emulsion-stability to insoluble matters, oils, etc. even in low-viscosity systems.

18.4.2 Viscosity As shown in Fig. 18.5, liquids containing swelled bacterial cellulose have pseudoplastic viscosity like that of xanthan gum solutions, and has a good dispersion- and suspensionstability. Pseudoplastic viscosity is a viscosity which decreases when the shear applied to the liquid increases and restores to the initial level when the shear decreases, and xanthan gum is the most famous of the polysaccharides that show such a viscosity. Dilatant viscosity is the opposite of pseudoplastic viscosity. In a dilatant liquid, the viscosity increases when the shear increases, and the viscosity decreases when the shear decreases.

Bacterial cellulose

Fig. 18.5

325

The viscosity of bacterial cellulose and xanthan gum (B-type viscometer, 20ºC).

An example of the dilatant system is starch or water in which sand is dispersed. Besides there is Newtonian viscosity, which remains constant regardless of change in the shear applied to the liquid. Water, -carrageenan, and locust bean gum are typical substances which give Newtonian viscosity. Of these three types of viscosity, only pseudoplastic viscosity gives a good dispersion- and suspension-stability. Most of the solutions having pseudoplastic viscosity have the yield value; this is considered to be the reason why they have a good dispersion-stability. The yield value of a solution is said to be a force which resists, until the solution starts to flow, a force acting to make the solution flow. Take, for example, an insoluble solid matter going to precipitate (flow downward) by gravity. If the yield stress is greater than the force acting to make the solid matter precipitate, the solid matter cannot precipitate and remains dispersed in the solution; i.e., the greater the yield value is, the more dispersion- and suspension-stable the solid matter is. Liquids containing swelled bacterial cellulose have very great yield values, and have a good suspension- and dispersion-stability. This can be explained by the fact that bacterial cellulose, which consists of very fine cellulose fibres, forms in liquid a continuous and strong three-dimensional network structure by cellulose connecting partly to each other. The network structure thus formed is very strong and remains undissolved in water, so the structure is not changed by heating and the suspension- and dispersion-stability is kept good even at high temperatures. As shown in Fig. 18.6, the viscosity of liquids containing swelled bacterial cellulose depends only weakly upon temperature, and the viscosity at higher temperatures has almost no difference compared to that at lower temperatures. However, in xanthan gum solutions, which have a suspension- and dispersion-stability as good as that of liquids containing bacterial cellulose, the viscosity at a low shear rate (6 rpm) decreases when the temperature becomes higher. This decrease in viscosity is considered to be a result of xanthan gum not being able to retain the network structure at higher temperatures and changing its configuration into the random coil.

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Fig. 18.6

The effect of temperature on the viscosity of bacterial cellulose and xanthan gum.

18.4.3 The method for swelling A strong shear force is needed to swell bacterial cellulose powder in water, but no heating is needed. Although swelling bacterial cellulose powder in water can be achieved by agitation using a common propeller stirrer or mixer, treatment with a strong homogeniser is needed to allow bacterial cellulose to function fully. Besides, the procedure sequence for pH adjustment and salt addition has to be arranged carefully. Change in pH or addition of salts has no effect on the liquid properties after the bacterial cellulose has fully swelled in water and the network structure has formed. In a solution in which salts have been dissolved beforehand, however, bacterial cellulose has difficulty in swelling to some extent, so sometimes it does not function well. Therefore, in order for bacterial cellulose to work fully with its excellent function, pH adjustment and salt addition have to be conducted after the three-dimensional network structure has formed (Fig. 18.7).

18.5

Uses and applications

The addition of bacterial cellulose in a very small amount will give foods good dispersion- and emulsion-stability, and also will give foods short mouthfeel based on a good shape-retainability. These functions are largely due to the three-dimensional network structure of cellulose fibres and are stable to physical and chemical treatments; they are also resistant to heat, acids and salts. These characteristics enable bacterial cellulose to be applied to foods for a wide range of purposes such as stabilisation of thickening, dispersion, suspension, and emulsion, replacement of fat, prevention of protein aggregation, etc. (Table 18.1).

Bacterial cellulose

Fig. 18.7

327

Procedures for swelling bacterial cellulose.

18.5.1 Application to drinks Bacterial cellulose has an excellent function of stabilising the dispersion of insoluble solid matters in cocoa drinks, powdered green tea drinks, Ca-fortified drinks, etc. For example, cocoa powder used in cocoa drinks is insoluble in water, so it precipitates without being dispersed. Various polysaccharides were used to prevent the precipitation; stabilisers consisting mainly of fine crystal cellulose have been used until now, but their effects have not been good enough. Fine crystal cellulose improves the dispersionstability primarily by preventing the insoluble solid matters from sticking to each other, which effect is brought by the loose network structure (a three-dimensional structure formation by the Brownian motion of fine crystal cellulose itself, and a network structure formation by the interaction of fine crystal cellulose and polysaccharides) and the connection of the insoluble solid matters to fine crystal cellulose. This method neither increases the viscosity nor has influence on the mouthfeel, but the effect is temporary and the precipitate increases over time. The use of bacterial cellulose can solve the problems mentioned above. This is because bacterial cellulose forms in water an insoluble network structure of very fine cellulose fibres which takes in the insoluble solid matters to prevent them from precipitating completely. In addition, the network structure, which has been formed by insoluble cellulose connected partly to each other, has a high yield value and hence a very good ability to disperse. Therefore, the addition of bacterial cellulose in a small amount can have a sufficient effect without large increase in viscosity. Although the viscosity of drinks obtained becomes a little higher, the mouthfeel is not thick and is clear-cut, raising no problem; this is because bacterial cellulose remains undissolved in water. Another

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Table 18.1

Examples of the applications of bacterial cellulose

Food

Effect

Cocoa drinks Powdered green tea drinks Ca-fortified drinks

Stabilising dispersion of insoluble solid matters Improving heat stability Achieving good mouthfeel without raising viscosity much

Non-oil dressings

Stabilising dispersion of insoluble solid matters Achieving clear-cut mouthfeel without raising viscosity much Achieving appropriate flowability Improving stability at low pH Achieving fatty mouthfeel

Sauce for roast meat

Stabilising dispersion of insoluble solid matters Decreasing threading Achieving non-gluey good mouthfeel Improving stability at high salt concentration Improving stickiness

Low-fat mayonnaise

Improving creamy mouthfeel Replacing fat Achieving appropriate flowability Achieving body

Jellies with fruit flesh

Stabilising dispersion of insoluble solid matters Achieving heat stability Achieving good meltability in the mouth

Retorted pudding

Preventing milk protein from aggregation Achieving heat stability Achieving good meltability in the mouth

Lacto-ice

Improving creamy mouthfeel Improving shape retainability

Soft-mix

Preventing whey from separating Improving mouthfeel

feature of bacterial cellulose is that its ability to disperse is not lowered extremely, even at higher temperatures, because its network structure remains undissolved in water.

18.5.2 Application to frozen desserts Bacterial cellulose can improve the stability of soft-mix and give it a creamy and rich mouthfeel. Soft-mix contains polysaccharides added for the purpose of stability increase, mouthfeel improvement, etc. It has been found that the stability of soft-mix decreases and the whey separates when the amount of polysaccharides added is increased for improvement of mouthfeel, etc. When the amount of polysaccharides added is not so much, a portion of the polysaccharides reacts with milk protein and the remaining portion of the polysaccharides disperses in the system or connects partly with other milk protein. This behaviour of the polysaccharides keeps a milk protein molecule electrically or physically away from other milk protein molecules and thus prevents the aggregation of milk protein. However, when more polysaccharides are added to improve mouthfeel, etc., the polysaccharides connect excessively with milk protein leading to the separation of whey.

Bacterial cellulose

329

In contrast, when bacterial cellulose is used, the three-dimensional network structure of cellulose fibres is built in the soft-mix and the milk protein is taken in to the network structure to be stabilised; hence, more polysaccharides can be added with whey separation suppressed. Thus soft-mix having a creamy and rich mouthfeel can be obtained without causing whey separation. Bacterial cellulose can also be expected to have the effect of improving shape-retainability and its application to lacto-ice, etc., will be effective.

18.5.3 Application to retorted pudding Bacterial cellulose enables chilled pudding to be retorted without causing protein aggregation. When common chilled pudding is retorted, protein aggregation occurs, so in the manufacture of retorted pudding, protein aggregation has had to be prevented by increasing the solid matter content, raising the viscosity, etc. This has resulted in a heavy mouthfeel and lack of meltability in the mouth. The use of bacterial cellulose in combination with a gelling agent for chilled pudding, however, enables chilled pudding to have resistance to retorting; so a new type of retorted pudding which has the same light and melting mouthfeel as chilled pudding can be prepared. This resistance to retorting is considered to be due to the formation of the threedimensional network structure in the chilled pudding which stabilises the milk protein by taking it in the network and suppresses the milk protein aggregation physically by keeping milk protein molecules away from each other beyond the distance allowing them to aggregate.

18.5.4 Application to jellies with fruit flesh Until now, to disperse pieces of fruit flesh in jellies, some levels of viscosity and sugar content and adjustment of gelling temperature have been needed. Even at high temperatures in germ-killing processes, etc., bacterial cellulose retains the threedimensional network structure and can keep fruit flesh pieces in the preparation at a constant level of dispersion. Thus the use of bacterial cellulose enables solid matters to be uniformly dispersed without complicated adjustments.

18.5.5 Application to dressings The application of bacterial cellulose to dressings can improve and stabilise the dispersion of seasonings, etc. This dispersing ability is retained even during long storage at high temperatures. The mouthfeel of dressings using bacterial cellulose is plainer and more clear-cut than those using xanthan gum. As for dressings of the emulsion type, a long-term emulsion-stability can be given.

18.5.6 Application to sauces Sauces often have a very high salt content and so the use of thickeners has been limited, but there has been no means of dispersing insoluble solid matters, other than viscosity raising. Much thickener has therefore been added, often resulting in high cost and a heavy mouthfeel. Bacterial cellulose can achieve dispersion-stability of insoluble solid matters by adding in a small amount, and give less threading products having a plain mouthfeel. It

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can also improve shape-retainability (stickiness) and give salt-resistance, so it enables application of sauces to foods to which sauces have not been applicable until now.

18.5.7 Replacement of fat in mayonnaise and dressings In dressings and mayonnaise, a substance used for replacing fat is mainly starch. In using starch for that purpose, products have been given an odour unique to starch or been influenced by the retrogradation of starch. In addition, starch does not have a good meltability in the mouth, so it is poor at releasing flavours and often has a thick mouthfeel. Starch also changes its physical properties to a great extent depending upon heating conditions, so its mouthfeel is hard to control. Bacterial cellulose can give to products good body and at the same time a very creamy and short mouthfeel. It also has a good meltability in the mouth and can improve flavour release. Further, it changes its physical properties only little with heat, so can always give stability in mouthfeel to products.

18.6

Regulatory status

Bacterial cellulose has been established as a generally recognised as safe (GRAS) food ingredient through the self-determination process under 21 CFR 182.1. This was accomplished using scientific procedures in accordance with 201 (s) (21 USC Section 321 (s)) of the Federal Food, Drug and Cosmetic Act. This GRAS determination was filed as a GRAS affirmation petition on December 11, 1991, and accepted by the FDA for filing on April 13, 1992. A request to amend this GRAS affirmation petition to GRAS notification has been filed under the Interim Policy provision of the FDA’s April 17, 1997, GRAS notification proposal (Sec. 21 CFR 170.36 (g) 2).

19 Microcrystalline cellulose: an overview H. Iijima and K. Takeo, Asahi Chemical Industry Co. Limited

19.1

Introduction

Microcrystalline cellulose (MCC) is a unique ingredient used mainly in the pharmaceutical and food industries to solve products or processing problems. It can be called a multifunctional ingredient because it can work as a viscosity controller, gelling agent, texture modifier, suspension stabiliser, fat mimetic, ice crystal suppressant, form stabiliser, water absorber, non-adhesive binder, emulsifier, etc. MCC is prepared from naturally occurring cellulose that has been purified. The hydrolysis under controlled conditions brings out the stable cellulose microcrystals, which are composed of tight bundles of cellulose chains in a rigid linear arrangement. Dr O. A. Battista of FMC Corporation, USA, discovered and established its preparation method in 1955. Since then, FMC Corporation (USA) has commercialised microcrystalline cellulose under the brand name of AvicelÕ and Asahi Chemical Industry Co., Ltd. (Japan) has acquired the licence to use the AvicelÕ trademark. In 1996, Asahi Chemical started to commercialise new types of MCC under its own brand name of CeolusÕ. For more than 30 years, MCC has been used widely as an additive for pharmaceuticals, foods, cosmetics, general industrial use, etc. Recently its application for dietary fibre has been drawing keen attention in the food industry field. MCC has a number of unique properties as a water insoluble, rod-like shaped particle, so that it has wide applicability, especially in the field of foods.

19.2

Manufacture

There are three types of commercialised MCC products used in foodstuffs, (a) powder type MCC: AvicelÕ FD and CeolusÕ ST; (b) colloidal type MCC: AvicelÕ RC and CeolusÕ SC and (c) cream paste type MCC: CeolusÕ Cream FP. Figure 19.1 shows the process for manufacturing these three types of MCC.

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Fig. 19.1

Process for manufacturing three types of microcrystalline cellulose.

19.2.1 Acid hydrolysis MCC is prepared by acid hydrolysis of highly purified wood pulp under controlled conditions. In the first step, the pulp is treated with aqueous dilute mineral acid solution. During the hydrolysis, the acid molecules penetrate the amorphous regions and cleave the -1,4 linkages between glucopyranose units of cellulose. Resultant water-soluble cellooligosaccharides and glucose are removed by subsequent rinsing and filtration. The remaining wet cake contains only pure crystalline regions of natural cellulose itself.

19.2.2 Powder type MCC The refined MCC wet cake is mixed with water and the resultant slurry is neutralised and dried to produce powder type MCC grades. The dry particle has a free-flowing property. Its porosity is rather high, because the dry particle is an aggregate of many bound materials composed of the primary MCC particle. The primary particles are bound together through tight hydrogen bonding and never released individually.

19.2.3 Colloidal type MCC and cream type MCC The hydrolysed cellulose is broken into small fragments and subjected to shear to liberate the cellulose microfibrils and crystallite aggregates (the primary MCC particles). The functionality desired is based on the distribution of microfibrils and crystallite particles. This fracturing process of hydrocellulose fragments, which results in the liberation of microfibrils and crystallite aggregates, is called attrition.1 MCC concentration is adjusted to produce the cream paste type MCC. When the attrited cellulose is dried, intermolecular hydrogen bonds are formed between surfaces of adjacent primary MCC particles and they are not rehydrated easily. In order to give a dispersible ability to the dry particulate MCC, a surface of the primary MCC particle is coated with hydrocolloids. Colloidal type MCC is a co-processed mixture of MCC and hydrocolloids (watersoluble cellulose derivative or water-soluble polysaccharide such as sodium carboxymethylcellulose, xanthan gum and karaya gum). The hydrocolloids act as the barrier dispersant for the adjacent MCC particles to keep them from reaggregating during the drying process. Accordingly, the primary MCC particles can be redispersed uniformly in water when the colloidal type MCCs are placed in water with adequate shear.

Microcrystalline cellulose: an overview

333

Aqueous dispersions of AvicelÕ RC and that of CeolusÕ SC are white opaque dispersions without supernatant liquid and any sedimentation. The hydrophilic barrier dispersants are hydrated and dissolved in water and it allows the cellulose microcrystals to disperse independently. These dispersed primary MCC particles form a threedimensional network structure which gives various functional properties to AvicelÕ RC and CeolusÕ SC. The grades and properties of AvicelÕ and CeolusÕ are listed in Table 19.1.

19.3

Structure

Figure 19.2 shows the molecular structure of cellulose. A cellulose molecule has a linear arrangement of D-glucose units connected by -1,4 linkage: [C6H1005]n. It has many hydroxyl groups available to take part in hydrogen bonding between adjacent cellulose molecular chains. The strong hydrogen bonding brings about a bundle of cellulose molecular chains called microfibrils. In the secondary wall of wood, cellulose chains form microfibrils with 5–10nm width. It may be that this width corresponds to that of a bundle of a few tens to a hundred cellulose chains. Within the bundles, there exist crystalline and amorphous regions in the microfibrils. Native cellulose in the higher plants shows the X-ray diffraction pattern of cellulose I: three distinct peaks corresponding to (110), (110) and (200). Planes are observed at diffraction angle 2 = 14.8º, 16.3º and 22.6º, respectively.2 Most plants synthesise biologically cellulose molecules and construct their cell wall. Soft woods contains about 50% wt of cellulose. Degree of polymerisation (DP) of the native cellulose depends on its origin; in woods, ca. 8000. DP of cellulose in wood pulp is smaller than that of original woods; ca. 1000–1500. When cellulose is hydrolysed with mineral acid, the amorphous region is removed as water-soluble cello-oligosaccharides and glucose. In an initial stage of the hydrolysis, DP of cellulose decreases dramatically, but it approaches to a constant DP value, which is known as the levelling-off DP. In the case of using wood pulp as a precursor of microcrystalline cellulose, the levelling-off DP ranges from 200–300. In this sense, an element of microcrystalline cellulose should be a fragment of cellulose microfibril (width is approximately 5nm) of which length is equal to that of the levelling-off molecular weight (approximately, 100–150nm). Actually, all the elements bind together with strong hydrogen bonding, constructing much larger crystallite aggregates (the primary MCC particles). Sugiyama et al.3 studied MCC aqueous dispersions using small-angle neutron scattering, demonstrating that the scattering profiles showed a fractal structure with a dimension of 2.2. The minimum size of the fractal structure was approximately 6nm.4 It is worth pointing out that the length of the element mentioned above is much larger than the minimum size of the fractal structure (6nm) and also that the width of the element

Fig. 19.2

Molecular structure of cellulose.

Table 19.1

Grades of microcrystalline cellulose AvicelÕ and CeolusÕ and their properties Powder type Avicel

Grade

(Hydrocolloids)

Water soluble

Disperse

Õ

Ceolus

Colloidal type Õ

Avicel

Cream paste type

Õ

Ceolus

Õ

CeolusÕ

FD-101

ST-01

RC-N81

RC-N30

RC-591

SC-N43

FP-03

Standard

Highly compressible (non)

Standard

Easily dispersed

Easily dispersible

Easily dispersible

Dispersed

(karaya, dextrin)

(dextrin, xanthan)

(CMC-Na)

(dextrin, xanthan)

(non)

(non)

Water/oil

Insoluble

Partially soluble to water

Dil-alkali

Partially soluble Swell Insoluble

Partially soluble Swell Partially soluble

Organic solvent

Insoluble

Insoluble

Insoluble

Insoluble

Cold/hot water

–

Easily dispersed

Easily dispersed

Easily dispersed

Dil-acid

Partially soluble to water Partially soluble Swell Insoluble/swell Partially soluble

Insoluble Partially soluble Swell Insoluble

Apparent specific gravity

0.3

0.13 ~ 0.23

0.6

0.6

–

Water absorption

2~3

1.7 ~ 2.7

–

–

–

Oil absorption

1.2 ~ 1.4

1.7 ~ 2.0

–

–

–

ca. 40m

ca. 60m

–

–

3–4m

White ~ slightly yellowish white

White ~ slightly yellowish white

White

Max. absorb. value

Ave. particle diameter Colour

White

3

Notes: Specific gravity of microcrystalline cellulose is approximately 1.55g/cm . Max. absorption value – according to JIS K-5101. All grades are produced by Asahi Chemical Industry Co., Ltd., Japan.

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agrees fairly well with the minimum size of the fractal structure in microcrystalline cellulose. In order to understand MCC particles, it is helpful to have an image of the fractal structure. During hydrolysis, the crystalline region of cellulose I increases, so that the crystallinity of MCC becomes higher that that of cellulose in the wood pulp. It is noteworthy that the degree of crystallinity of MCC is not 100%, but 70–80% using Xray diffraction.5–8 This fact is consistently explained by considering the size of a microfibril, the number of cellulose molecules constructing the microfibril and the surface effect on crystallinity. If the width of a microfibril is 5nm, the width of the cellulose molecular chain is 0.5nm and a cross-section of the microfibril is a square: the microfibril consists of 100 cellulose molecules. Even if these 100 cellulose chains form strong hydrogen bonds between them, creating a complete cellulose crystal in the microfibril, 36 cellulose molecules existing on the surface layer of the microfibril cannot make strong hydrogen bonds with cellulose molecules in a surface layer of an adjacent microfibril. Accordingly, 36% of cellulose molecules in the microfibrils in the crystalline region do not entirely participate in making the cellulose crystal. Therefore, if the width of the microfibril is 5nm, the degree of crystallinity would not be 100% and also would be larger than 64%. If the width of the microfibril in 10nm, the degree of crystallinity would be larger than 81%.

19.4

Technical data

19.4.1 Powder type MCCs Compressibility Powder type MCCs for food use are AvicelÕ FD and CeolusÕ ST. They have excellent properties as a binding agent for direct compression of tablets. As shown in Fig. 19.3(a) and (b), powder type MCCs are composed of aggregated MCC particles with unique, irregular rod-like shape. When compressed using a punch and die, it gives a hard tablet with a flat glassy surface under low pressure due to entanglement of particles. Aggregated MCC particles of CeolusÕ ST-01 have unique morphology of long and narrow rod-shape, giving significantly higher compressibility than AvicelÕ FD grade.9 Adsorption As the aggregated MCC particle has rather high porosity, its apparent specific gravity is smaller than the real specific gravity of microcrystalline cellulose itself. It has excellent properties of absorbing and retaining water, oil and other useful materials. Therefore, AvicelÕ FD can be used to make powder-like oil and flavouring agent, etc. In the case of powdered cheese, oil in cheese diffuses, coming out to a surface of powder gradually and this causes adhesion of cheese powder. AvicelÕ FD can retain the oil and prevent adhesion. Granulation AvicelÕ has excellent water-holding capacity which enables us to make pasty products in a wide range of water content. It can improve fluidity of dilatancy fluid. Furthermore, AvicelÕ has a function in the binder, it can prevent adhesion between adjacent drug powders, so that one can obtain hard granules or spheres with uniform size distribution efficiently. AvicelÕ has been used as a granulation aid for fluidised bed granulation and tumbling granulation, etc.

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Fig. 19.3 Scanning electron micrographs of microcrystalline cellulose particles: a, AvicelÕ FD-101; b, CeolusÕ ST-01; c, AvicelÕ RC-591; scale bar stands for 50m.

19.4.2 Colloidal type MCCs The primary MCC particle composing colloidal type MCCs (AvicelÕ RC and CeolusÕ SC) are aggregated microcrystalline cellulose particles (the secondary MCC particles) whose surface is coated with water-soluble polymers. When colloidal type MCCs are placed in water with adequate agitation, the water soluble polymers swell and dissolve easily, the aggregated secondary MCC particles are disintegrated into the primary particles. Those countless primary MCC particles play an important role in achieving many excellent functions in their aqueous dispersion. Figure 19.3(c) shows a scanning electron micrograph of AvicelÕ RC-591 powder.

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Suspension stability The primary MCC particles move randomly by Brownian motion, repelling each other due to their electric charge in aqueous dispersion. Above a critical concentration of AvicelÕ RC, they gradually form a three-dimensional network structure, which can suspend fine solid particles, forming stable suspensions without sedimentation or syneresis. Rheological property Aqueous suspensions of MCC particles exhibit one of the most unusual and useful characteristics as a viscosity stabiliser over a wide range of temperatures. Figure 19.4 shows the effect of temperature on viscosity of aqueous suspension of CeolusÕ Cream FP-03. The viscosity increases with an increase in temperature from 20–60ºC and keeps almost the same value as 60ºC in a temperature range above 60ºC. Nishinari et al.10 studied the dynamic viscoelastic properties of aqueous suspensions of MCC particles, clarifying that the entropic contribution of the storage modulus was found to be (a) positive for the temperature range from 5–70ºC and (b) to increase with increasing temperature. They concluded that these facts suggest that a tenuous network is formed in the dispersion, which becomes more solid-like at higher temperature. According to the same principle, the three-dimensional network of MCC particles brings about heat stability in a MCC/water-soluble hydrocolloids system. Therefore, viscosity of the suspension is almost constant or slightly decreased over a high temperature range though viscosity would be dramatically decreased under high temperature conditions if MCC were omitted. Figure 19.5 shows shear thinning behaviour. Gel containing AvicelÕ RC readily breaks down under shear, but when the shear is removed, the gel reforms quickly with minimal loss in viscosity. When MCC particles in a dispersion solution undergo shear stress larger than a yield value, a three-dimensional network structure becomes loose. A rod-shaped MCC particle is oriented to the direction of shearing, because this position of the rod particle makes the stress smallest in the shear field. The decrease in the stress leads to a remarkable drop in apparent viscosity of the dispersion. When the stress is removed, MCC particles take a random arrangement again and form a three-dimensional structure in a short time. Suspensions of AvicelÕ RCs exhibit a thixotropy. Figure 19.6 shows the effects of MCC concentration on the thixotropy loop of AvicelÕ RC-591 suspensions. No hysteresis

Fig. 19.4 Effect of temperature on viscosity of aqueous suspension of CeolusÕ Cream FP-03: cellulose concentration, 2.0 %wt; shear rate, 16.4 sÿ1; a Brookfield model BL viscometer.

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Fig. 19.5

Fig. 19.6

Fluidity of AvicelÕ RC dispersion: (a) Before shaking, (b) After shaking; AvicelÕ RC-591 3.0 %wt aqueous dispersion.

Effect of MCC concentration on the thixotropy of AvicelÕ RC-591; cellulose concentration, 0.5–2.0 %wt; temperature, 25ºC.

is observed at 0.5% wt. At 1.0% wt, the MCC network is rather coarse, but the whole suspension is covered with the network and thixotropic behaviour begins to appear. With an increase in MCC concentration, the network grows rapidly to become thicker and the suspension shows an extremely large stress and a remarkable thixotropy. Yield values of AvicelÕ RC-591 suspension were observed above 1.0% wt.11 Texture Microcrystalline cellulose is a water insoluble rigid particle. Therefore, compared with other water-soluble gums, AvicelÕ RC’s network structure exhibits lower viscosity (for example, viscosity of 1.0% wt CMC-Na aqueous solution is of the order of 102–103 mPas; that of 1.0% wt AvicelÕ RC-N30 aqueous dispersion is approximately 10 mPas). This gives a refreshing texture, i.e., non-gummy taste. AvicelÕ RC can be used to shorten the texture of various foods and drinks. It replaces soluble gums without imparting a gummy or pasty texture and provides an excellent opacity to foods and a glossy and deep milkwhite appearance to gel-type foods. Water-holding capacity A three-dimensional network structure of MCC particles can hold a large amount of water, it prevents syneresis induced by heat-shock and pressure change. MCC particles

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339

provide a unique body and texture to creamy, pastry foods, improving the form retention of the foods. Health aspects • Fat replacement. The water-holding capacity and thixotropic property of MCC dispersions give similar rheological and textural properties together with similar appearance to foods containing oil or fat such as salad dressings and frozen desserts. • Low calorie. Microcrystalline cellulose is undigested by humans. It is useful to reduce the effective calorie content of foods. • Dietary fibre. Microcrystalline cellulose can be used as a source of insoluble dietary fibre.

19.4.3 How to disperse colloidal type MCCs In order to obtain the optimum functions of colloidal type MCCs, three points should be noted: 1. 2. 3.

Use high-shear equipment, e.g., homogeniser. Disperse in hot water. Provide sufficient dispersing time.

Dispersion pattern of AvicelÕ RC AvicelÕ RC shows the same pattern of time-related change of viscosity compared with water-soluble polymers (e.g. gum) when it is swollen, disintegrated and dispersed in water. When AvicelÕ RC-591 is dispersed in water using a Waring blender, the apparent viscosity changes as shown in Fig. 19.7. When dispersed at 10ºC, it reaches the maximum

Fig. 19.7

Dispersing time and apparent viscosity: AvicelÕ RC-591 1.0 %wt aqueous dispersion; using a Waring blender at 15,000 rpm, dispersing temperature, 10ºC and 50ºC.

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swollen point in 10 sec. After this point, the apparent viscosity decreases with time. At 300 sec. it can be regarded as fully dispersed. At 50ºC, it attains the maximum swollen point within 10 sec. Dispersing energy and viscosity change Figure 19.8 shows an effect of dispersion rate on the relation between apparent viscosity measured after 24 hours from preparation and dispersion time in the case of 2.0% wt aqueous dispersions of AvicelÕ RC-591. When a mixture of colloidal type microcrystalline cellulose and water was dispersed at 6000rpm by using a Waring blender, apparent viscosity increased rapidly, but it reached a plateau at 30 sec. keeping constant even after 300 sec. without showing a maximum. When dispersed at 8000 rpm, it showed the maximum viscosity around 30 sec. then decreased gradually. In the case of a dispersing rate of 14,700 rpm, the apparent viscosity reached the maximum value, which was larger than that obtained at 8000 rpm around 30 sec. After reaching the maximum, the viscosity decreased gradually. At the point of maximum viscosity, attained at 14,700 rpm, CMCNa was completely swollen by water, showing the largest viscosity. At 6000 rpm, the swollen point did not appear. These facts mean that RC-591 is still in the process of swelling at 6000rpm. As the maximum viscosity of 2%wt RC-591 dispersion is approximately 900–1000 mPa.s at 20ºC, we can fully disperse AvicelÕ RC-591 under conditions of 15,000 rpm for 300 sec. by using a power blender at room temperature. When the apparent viscosity does not reach the maximum swollen point in preparation, it does not change after the preparation, because MCC particles cannot be fully dispersed without any agitation. When the dispersion liquid undergoes sufficient agitation in the preparation, the viscosity increases gradually after the preparation, reaching a plateau after 6 hours. This behaviour means that the suspension liquid comes into a region of stable dispersion levels. Effect of pH on viscosity In general, MCC aqueous dispersion remains stable against pH change; however, RC591, which contains CMC-Na, shows a slight increase in viscosity at pH4.5. In the case of RC-N81 and N30, the viscosity change is not observed in the range of pH above 4.

Fig. 19.8 Effect of dispersing rate on relation between apparent viscosity and dispersing time: AvicelÕ RC-591 2.0 %wt aqueous dispersion; dispersed at 6000, 8000 and 14,700 rpm by a Waring blender; viscosity was measured at 20ºC after 24 hrs.

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Effect of alcohol In a range of alcohol concentrations above 20%, RC-591 tends to aggregate and the viscosity decreases monotonically with an increase in alcohol concentration. Effect of temperature As shown in Fig. 19.4, the viscosity of an MCC aqueous suspension never decreases even if its temperature increases. In the case of aqueous polysaccharide solution, such as guar gum and locust bean gum, its viscosity reduces dramatically with an increase in its temperature. Relative viscosity of those gum solutions measured at 80ºC is less than 20% of that at 20ºC. AvicelÕ RCs contain aqueous polysaccharides like karaya gum, xanthan gum and CMC-Na, but temperature has very little effect on the viscosity of AvicelÕ RC dispersion. That is because its main component is MCC. This property is extremely important to stabilise the food systems that undergo high temperature and/or high shear conditions in their manufacturing and/or cooking process.

19.5 Application of colloidal type microcrystalline cellulose to food systems Table 19.2 shows examples of general applications of AvicelÕ and CeolusÕ to food systems.

19.5.1 Ground green tea milk a. Formulations Table 19.3 shows formulations of ground green tea milk. b. Procedure 1. After blending powder ingredients (except ascorbic acid), disperse them in warm water (70ºC) using an agitating mixer under appropriate conditions. 2. Seven to eight minutes after the beginning of the dispersing process, add ascorbic acid. 3. Homogenise twice using a Manton-Gaulin homogeniser: 1st pass at 150 kg/cm2, 2nd pass at 200 kg/cm2. 4. After filling the milk into heat-resistant glass bottles, pasteurise at 121ºC for 30 min. 5. Take out the bottles immediately after pressure is reduced to atmospheric pressure and cool the bottles quickly with running water for 30 min. 6. After cooling, shake the bottles up and down slowly three times. c. Evaluation 1. Dispersion stability: after settling for one night, measure the volume of the sedimentation layer (Vs, % by volume) and that of the dispersion layer (Vd, % by volume). Check the state of emulsification, i.e. the generation of syneresis, aggregates and sediments. 2. Redispersion: after evaluating the dispersion stability, shake the bottles up and down three times. Settle them for 10 minutes and then measure Vs and Vd. Check the state of emulsification, i.e. the generation of syneresis, aggregates and sediments. 3. Viscosity: measure viscosity by a Brookfield type viscometer at 20ºC. 4. Taste: taste the milk at 20ºC.

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Table 19.2 Applications of AvicelÕ RC, CeolusÕ SC, CeolusÕ Cream FP and CeolusÕ ST to food systems Standard conc./ %wt

Application

Benefits

Grade

Ice cream Milk shake Soft ice cream

Prevents whey separation without increasing mix viscosity. Gives refreshing texture. Prevents ice crystal growth Provides body and stabilises foam Improves extrusion and heat shock resistance

RC, SC, 0.2~0.6 FP

Whipped toppings Prevents creaming Coffee whitener Improves overrun control Improves form retention and stabilises while storing Stabilises emulsifying while sterilising

RC, SC, 0.2~0.5 FP

Chocolate drink Energiser Milk beverage Soup

RC, SC, 0.3~0.5 FP

Stabilises suspension while preventing viscosity increase Gives refreshing texture Prevents whey separation and oil-off Stabilises while in store

Jam, Paste, Improves spreadability and adds thixotropy Fillings, Puddings Improves heat resistance Improves form retention and gives cloudy effect

RC, SC, 0.5~1.5 FP

Dressings Sauces

Stabilises emulsion and suspension Provides yield value Improves heat resistance Creates small oil doublets Prevents texture, reduces calories

RC, SC, 0.5~3.0 FP

Frozen foods Batter coating

Gives crispy texture Reduces oil absorption Improves cling of batter

RC, SC, 0.5~1.0 FD

Prepared mustard Prepared wasabi

Improves extrusion Improves dispersion

RC, SC, 0.5~2.0 FP

Breads, Dough, Pancakes

Adds freezing resistance (especially to confectionery breads) Improves water holding ability (enhances moisture content) RC, SC, 0.3~1.0 Reduces senility FP Improves its stiffness

Shredded cheese Seasonings Powdered soup

Oil adsorption Prevents binding

FD

1.0~2.0

Tablet type foods

Provides compressibility

FD, ST

5.0~30.0

d. Result Table 19.4 summarises the results of evaluation in green tea milk with AvicelÕ RCs.

19.5.2 Calcium fortified milk a. Summary 1. By treating calcium fortified milk with AvicelÕ RC-N81, hard sedimentation of whey calcium was not formed even after 20h settlement. Although soft sedimentation was observed at the bottom, it was easily redispersed by gentle shaking. AvicelÕ

Microcrystalline cellulose: an overview Table 19.3

343

Formulations of ground green tea milk

Ingredients

RC-N81 Formulation

RC-N30 Formulation

RC-591 Formulation

Control

6.0% wt 3.35 0.4 0.2 0.02 0.4 – – balance

6.0 3.35 0.4 0.2 0.02 – 0.4 – balance

6.0 3.35 0.4 0.2 0.02 – – 0.25 balance

6.0 3.35 0.4 0.2 0.02 – – – balance

100

100

100

100

Sugar Whole powdered milk Green tea1 Emulsifier2 Ascorbic acid AvicelÕRC-N81 AvicelÕRC-N30 AvicelÕRC-591 Water Total

1. Uji ground green tea 2. Glycerine fatty ester (Kao, Atmos no. 150)

Table 19.4

Comparison of evaluation results: green tea milk

Evaluation item

RC-N30 Formulation

RC-591 Formulation

Blank

1. Dispersion stability: 38.4/0 Vd /Vs Colour tone of (% by volume) the dispersion layer was slightly dark compared with the upper layer

36.8/0 Same as left

100/0 A small amount of sedimentation was observed at the bottom

–/14.7 Colour tone of the sedimentation was different from that of RC formulation

2. Emulsification:

No oil ring No fat aggregation

Same as left

Same as left

Same as left

3. Re-dispersion: Vd/Vs (% by volume)

100/0 Thin sedimentation at the bottom

100/0 Same as left

100/0 Same as left

–/14.7 Uneven parts in the sedimentation layer

4. Viscosity: (mPa.s) at shear rate 15.2 (sÿ1) 5. Taste: at 20ºC

2.

RC-N81 Formulation

5.5

Refreshing texture Not watery

5.9

Slight thick texture Smooth and good mouthfeel

7.5

Smooth and good mouthfeel

4.2

Refreshing texture Watery and unsatisfied

RC-N81 prevents direct contact of whey calcium to form soft aggregates instead of hard ones. With AvicelÕ RC-N81, fine dispersion stability of whey calcium was achieved even after the sedimentation was redispersed.

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Fig. 19.9

Procedure for preparing calcium fortified milk.

b. Procedure Figure 19.9 shows the procedure for preparing calcium fortified milk. c. Evaluation 1. Redispersibility: count number of times necessary to turn the tube upside down before the sediment is completely dispersed. 2. Stability after redispersion: after redispersion, settle the sample for a certain period and measure the volume fraction of sediment (Vs in volume %). d. Results Table 19.5 shows results of redispersion stability in calcium fortified milk. 19.5.3 Jellies There are many gel foods with similar textures in the market. AvicelÕ is attracting attention as a unique agent providing new texture when it is combined with conventional gelling agents. When AvicelÕ is applied to gel foods such as jellies, puddings and mizuyokan (soft adzuki-bean jellies), the gel structure is changed, resulting in different textures in the gel foods.

Table 19.5

Comparison of redispersion stability: calcium fortified milk Concentration (%wt)

Redispersion stability Vs in volume %

Run no. 1 2

Whey calcium1

RC-N81

0.1 0.1

– 0.3

Redispersion (times)

15 min.

30 min.

60 min.

120 min.

38 3

0.5 < 0.1

0.8 < 0.1

1.0 < 0.1

1.0 < 0.1

1. Additional amount as calcium. (Calcium content of whey calcium = 18% wt) Normally calcium content in milk is 100mg/100g = 0.1% wt. In this examination, calcium content is doubled.

Microcrystalline cellulose: an overview Table 19.6

345

Contents of MCC and -carrageenan in jelly applications RC-N30

-carrageenan

– 0.5 1.0

0.6% wt 0.7 0.8

Control AvicelÕ 0.5% wt AvicelÕ 1.0% wt

a. Summary Gel foods with MCC show the following properties: 1. 2. 3. 4. 5.

Syneresis of gel can be prevented slightly. Smoothness of gel is improved. Fragility texture of carrageenan gel is improved. Clingability of gelatin gel is decreased. Decrease in crushing strength, elasticity and fragility of gel are prevented and good mouthfeel is provided even after the freeze-thaw process. Sedimentation and separation may occur when AvicelÕ is added directly in powder form. In this case, it is better to prepare aqueous dispersions of AvicelÕ using a home mixer or a homogeniser beforehand.

b. Formulation A standard formulation (control) of jelly is listed by weight % as follows: Gelling agent (-carrageenan) Sugar Water

0.6% wt 20% wt Balance

Table 19.6 shows contents of MCC and -carrageenan in jelly applications. AvicelÕ is pre-dispersed beforehand. c. Results Physical properties were measured using a rheometer. Although the crushing strength of jellies did not change by introducing MCC into the gels, maximum stiffness was observed earlier during the measurement in both MCC and carrageenan systems. Furthermore, the fragility of gels decreased and the resistance after crushing increased. In the MCC/ carrageenan system, consistency was imparted a little, together with lumpy texture.

19.6

Regulatory status

Numerous animal toxicological feeding studies support the safe use of microcrystalline cellulose and microcrystalline cellulose products are generally recognised as safe (GRAS) for food additive use.12 ADI (acceptable daily intake) as determined by JECFA (Joint FAO/WHO Experts Committee on Food Additives) for microcrystalline cellulose is ‘not specified’. Microcrystalline cellulose has been recognised as safe and has been in widespread use in foods for decades. It is listed in the National Formulary, the European Pharmacopeia, the Pharmacopeia of Japan and Food Chemical Codex.

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Regulatory information of MCC products:13 Cellulose is on the TSCA (Toxic Substance Control Act) inventory. All types of microcrystalline cellulose products do not contain any toxic chemicals subject to the reporting requirements of Section 313 of US SARA Title III. Concerning California Proposition 65, they do not contain any chemicals currently on the California list of known carcinogens and reproductive toxins. They are not a controlled product under the Canadian Workplace Hazardous Materials Information System (WHMIS).

19.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

References and E. J. MCGINLEY ‘Microcrystalline Cellulose Technology’ in Polysaccharide Association Structures in Food Marcel Decker, Inc., New York, NY, 1998, pp. 169– 205. A. ISOGAI ‘Allomorphs of cellulose and other polysaccharides’, Cellulosic Polymers, Blends and Composites, ed. R. D. Gilbert, Carl Hanser Verlag, New York, NY, 1994, pp. 1–24. M. SUGIYAMA, K. HARA, N. HIRAMATSU and H. IIJIMA ‘Small-angle neutron scattering observation of aqueous suspension of microcrystalline cellulose’ Jpn. J. Appl. Phys, 1998 37 (Pt.2, No. 4A) L404–5. M. SUGIYAMA, K. HARA, N. HIRAMATSU and H. IIJIMA ‘SANS studies of aqueous suspension of microcrystalline cellulose’ in Hydrocolloids 1: Physical chemistry and industrial application of gels, polysaccharides, and proteins, ed. K. Nishinari, Amsterdam, Elsevier, 2000, pp. 277–81. J. SCHURZ and H. KLAPP ‘Untersuchungen an Mikrokristallinen und Mikrofeinen Cellulosen’ Das Papier, 1976 30 510–12. J. SOLTYS, Z. LISOWSKI and J. KNAPCZYK ‘X-ray diffraction study of the crystallinity index and the structure of the microcrystalline cellulose’ Acta Pham Technol, 1984 30 (2) 174–80. T. PESONEN and P. PARONEN ‘Evaluation of a new cellulose material as binding agent for direct compression of tablets’ Drug Dev Ind Pharm, 1986 12 (11–13) 2091–111. Y. NAKAI, E. FUKUOKA and S. NAKAJIMA ‘Crystallinity and physical characteristics of microcrystalline cellulose’ Chem. Pharm Bull, 1977 25 (1) 96–101. K. OBAE, H. IIJIMA and K. IMADA ’Morphological effect of microcrystalline cellulose particles on tablet tensile strength’ Int J Pharm, 1999 182 (2) 155–64. K. NISHINARI, E. MIYOSHI and T. TAKAYA ‘Rheological studies of aqueous dispersions of microcrystalline cellulose’ in Spec Publ R Soc Chem, 218 (Gum and Stabilisers for the Food Industry 9, ed. P. A. Williams and G. O. Phillips, The Royal Society of Chemistry, London, UK), 1998, pp. 16–25. M. J. FALKIEWICZ ‘Rheology. Fundamental principles in product development’ Soap Cosmet Chem Spec, 1980 56 (10) 46, 48, 51, 54, 70. UNITED STATES FOOD AND DRUG ADMINISTRATION GRAS (Generally Recognized As Safe) Food Ingredients – Cellulose and Derivatives, PB 221–8 (1972). MSDS of AvicelÕ PH, RC; CeolusÕ Cream FP-03 (Asahi Chemical Industry Co., Ltd.). G. S. BULIGA, G. W. AYLING, G. R. KRAWCZYK

20 Gums for coatings and adhesives A. Nussinovitch, The Hebrew University of Jerusalem

20.1

Introduction

Water-soluble gums are beneficial in many fields, including adhesives, agriculture, biotechnology, ceramics, cosmetics, explosives, food, paper, textiles and texturisation, among many others. Two fields in which many novel utilisations of gums have been developed and much progress has been recently made are coatings and adhesives. In foods, hydrocolloids are used to produce thin layers of edible materials on surfaces or between food components. Such films serve as inhibitors of moisture, gas, aroma and lipid migration. They can include antioxidants, preservatives or other additives to improve food mechanical integrity, handling and quality, and to change surface gloss.1 In addition to foods, gums are also used in coatings for seeds, fibreglass, fluorescent lamps, glass, metals, optical products, paper products, latex and textiles.2 Hydrocolloid glues are distinguished from most organic-based adhesives by their hydrophilic, nontoxic nature, and are used at many different concentrations, viscosities, and molecular weights. Gums are inflammable and possess good wettability properties which enhance their penetration into porous substrates. The development of synthetic hydrocolloids throughout this century has broadened their uses with such commodities as paper, wood, textiles, leather, food, cosmetics and medicine. 3

20.2

Today’s edible protective films

The use of edible protective films has risen rapidly due to the search for improved food protection to prevent moisture loss, retard bacterial growth, eliminate oxidative rancidity, and improve appearance and food handling.1 Many of today’s food coatings are similar to those used in the past. Alginate-based coatings have been used to coat meats,4–8 and are good oxygen barriers.9 Calcium-alginate coating of lamb carcasses helped reduce surface microbial growth and achieve a faster chill rate.10 A number of hydrocolloids, namely alginates, carrageenans, cellulose ethers, pectin and starch derivatives, have been used to improve stored meat quality. Such coatings serve as sacrificing agents, i.e. moisture loss

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is delayed until it evaporates from the film.11 Meat, poultry, seafood, dough products, vegetables, extruded foods and cheeses coated with calcium-alginate exhibit reduced shrinkage, and decreased oxidative rancidity, moisture migration and oil absorption during processing.12 Alginates, carrageenans, cellulose ethers, pectin and starch derivatives have been reported to improve stored meat quality.11 Alginate coatings have been used to retard the development of oxidative off-flavours in precooked meat patties. The shelf-lives of frozen shrimp, fish and sausages were extended by use of alginate coatings.13, 14 Other sodium-alginate and gellan-based coatings have been investigated.15, 16–18 As a barrier to moisture loss, these hydrocolloid coatings postponed the drying of mushroom tissue, thereby preventing changes in its texture during short periods of storage. Coated mushrooms had a better appearance and gloss, as did garlic.19 Incorporation of ingredients found naturally in garlic skin or which are chemically similar to these into the gum solution before coating improves film adhesion to the surface of the coated commodity (Figs 20.1 and 20.2). Gellan-based films can be used to reduce oil absorption. Such coatings have been used for several years in Japan and other Asian countries with tempura-type fried foods.20 LMP (low-methoxyl pectin), which resembles alginate in its cross-linking mechanism, can be used for coating purposes. This film, coated with lipids, increases resistance to water-vapour transmission.21 An edible coating system called Fry Shield, developed and patented by Kerry Ingredients (Beloit, WI) and Hercules (Wilmington, DE), is based on calcium-reactive pectin and reduces fat intake during frying. French fries treated with pectin took up half the usual amount of oil.20 Gum arabic with or without gelatin has been used to produce protective films for chocolates, nuts, cheeses and pharmaceutical tablets,22 and has also been reported to inhibit darkening of cooked potatoes.23 A composite film of gum acacia and glycerolmonostearate was reported to have good water-vapour barrier properties.24 Chitosan, which has yet to be approved as a food ingredient in the United States, produces films which are clear, tough and flexible, and are good oxygen barriers.25–26

Fig. 20.1

Coated garlic in cross-section. The coating glued to the top of the garlic epidermis is strong and transparent (adapted from Nussinovitch and Hershko19).

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Fig. 20.2 Gellan-sitosterol-coated garlic. The distance between the film and the garlic is equal to or smaller than 10 m.

Water loss and browning of cut apple slices were inhibited by coatings of chitosan and lauric acid.27 The chitosan derivative NOCC (N, O-carboxymethyl chitosan) selectively forms permeable nontoxic films which are used as a postharvest edible coating for fresh foods.28–30 Synthetic polymers can modify the permeation response of a chitosan membrane to O2 and CO2.31 Extracellular microbial polysaccharides, such as pullulan, levan and elsinan, can be used to produce edible and biodegradable films. Pullulan films are clear, odourless and tasteless and act as efficient oxygen barriers.25 Hydroxypropylated amylomaize starch has been used to produce transparent watersoluble films, with low permeability to oxygen, increased bursting strength and elongation, and reduced tensile strength.32 Other components helped to develop coatings of this nature for prunes, raisins, dates, figs, nuts and beans. The National Starch and Chemical Co (West Bridgewater, NJ) has developed starch formulations for a variety of different products, for example starch hydrolysates to coat dried apricots, almonds and apple slices.33 Edible coatings based on modified food starches serve as adhesives for seasoning and are applied to the surface of oil-roasted and dry-roasted peanuts. Investigations of edible film properties have progressed appreciably during the last century; the results are expected to be applied to a wide variety of food and other applications.34–35 The carrageenan-based coatings Soageena and Soafil (edible polysaccharides) were developed by Mitsubishi International Corp. (Tokyo, Japan).36 Aside from their designation for fresh produce, no information is available.37 Other carrageenan coatings have been used to retard moisture loss from coated foods.38 A carrageenan-based coating applied to cut grapefruit halves was successful in decreasing shrinkage and taste deterioration in storage at 4.5ºC for 2 weeks.39 Carrageenan (Fig. 20.3), gellan and alginate have been used to coat fresh, soft-brined cheeses.40 Cellulose can be used following chemical modifications to produce edible and biodegradable films. Methyl cellulose (MC) (manufactured by Dow Chemical Co., Midland, MI), hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC)

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Fig. 20.3

Carrageenan-coated soft-brined white cheese (courtesy of N. Kampf).

and carboxymethyl cellulose (CMC) are examples of raw materials for the production of films with moderate strength, resistance to oils and fats, flexibility and transparency. Other desired properties include lack of odour and taste, and the ability to serve as a moderate barrier to moisture and oxygen.41–43 Information on MC and HPMC can be found elsewhere.44 MC films are good barriers to oil and fat migration.45 MC and HPMC can be used to create composite films with solid lipids.46–49 Such films can be produced from ethanol solutions of fatty acids and cellulose. Bilayer films consisting of a solid lipid and a layer of MC or HPMC have been used to reduce the migration of water in model foods.50 Nonionic cellulose ethers are capable of producing tough, transparent and flexible films that are both water-soluble and fat- and oil-resistant.51 MC and HPMC films are effective in reducing oil absorption by french fries, onion rings, and other fried, processed products.52 In addition to decreasing oil penetration and absorption by dry foods, they can reduce weight loss and improve the adhesion of batter to products.53 MC films prevent lipid migration.45 HPMC and a bilayer film consisting of stearic-palmitic acid slowed moisture transfer from tomato paste to crackers.54 Formulations involving MC, HPMC and HPC delayed browning and increased volatile flavour components.55 HPC films retard oxidative rancidity and moisture absorption in nut meats, coated nuts and candies.51, 56 A few water-soluble composite coatings composed of CMC with fattyacid-ester emulsifiers are used for fruits, such as pears and bananas.57–62 In the latter, delayed ripening and changes in internal gas levels are observed.58, 59, 63 This type of commercial coating was first called Tal Pro-long (Courtaulds Group, London) and later, Pro-long. It increases resistance to some types of fungal rot in apples, pears and plums but is not effective in decreasing respiration rate or water loss in tomato or sweet pepper.64, 65 Valencia oranges coated with Tal Pro-long have a better flavour and lower ethanol levels than controls.66 Another coating with a similar composition, Semperfresh (United Agriproducts, Greely, CO), contains a higher proportion of short-chain unsaturated fattyacid esters in its formulation.57 These coatings retarded colour development and retained acids and firmness as compared to controls when tested on apples.60 Semperfresh also extended the storage life of citrus.67 The addition of waxes to Semperfresh gives fruit with a better shine and higher turgidity, less decay and good flavour. On the other hand, Semperfresh is not effective at retarding water loss in melons.68

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20.3 Parameters to be considered before, during and after food coating The coating process involves wetting the food surface with the coating gum solution, possible penetration of the solution through the food surface69 and possible adhesion between these two commodities. The wetting stage (spreadability) is the shortest and most important; if the compound used for spreading is ideally suited to the food to be coated, spreadability is spontaneous.70 However, it is almost impossible to find hydrocolloid solutions or their combinations that are ideally suited to the surface properties of the object, i.e., surface tension and polarity. Thus the closest possible combination should be determined to achieve compatibility. Effecting a successful, tailored coating that adheres to a food surface requires estimation of the interfacial tension between the coating solution and the surface. It depends on the surface tension of the surface, the surface tension of the coating solution and the contact angle between the two.71 To gauge a solid’s surface tension, the critical surface tension first needs to be calculated. Prior to this, the critical surface tension of the surface to be coated should be derived from Zisman plots followed by extrapolation. These plots are obtained by calculating the cosine of each measured contact angle of the pre-chosen liquids on this surface and plotting it against already-known surface-tension values of the solvents being used.72–73 The detection of a suitable coating gum solution can be very complicated because coating solutions are water based, i.e. they contain mostly water with a surface tension of 72.8 dyne/cm, and lower surface-tension values for many solid surfaces must be taken into account. In general, hydrocolloids have the potential to lower the surface tension of solutions designated for use as coating agents.74 The lower the surface or interfacial tensions of a gum solution, the higher its surface or interfacial activity.74 Considering that efficient coating involves compatibility between liquid and solid surface tensions,75 it is logical to reduce the surface tension of the coating solutions to conform to the lower surface tension of the food surface, and to lower the interfacial tension to improve adhesion.75 In our previous studies,15 we observed that the addition of sterols effectively yields better adhesion between fruit and vegetable surfaces and coating films due to better compatibility with respect to the hydrophobicity of the two adhered surfaces.19 Another aspect to consider is spreading which is enhanced by a food’s rough surface for coating solutions having contact angles smaller than 90º76 and inhibited by rough surfaces for solutions with contact angles greater than 90º. Surface roughness has been defined as the ratio of the true area of the solid to the apparent area. Wenzel77 was the first to propose a relationship between the contact angle of a liquid on a rough surface to its contact angle on an ideally smooth surface. Surface roughness can be evaluated with a roughness tester78 or from image-processing of atomic force microscopy (AFM) micrographs.69 Roughness decreases the interfacial tension as a result of improved spreadability.76 The importance of the magnitude of the interfacial tension is well recognised by the polymer coating industry. Better compatibility between the coated object and the coating films can be achieved by incorporating surface-active agents within the coating gum solution. It can be concluded from the compatibility requirements that tailor-made hydrocolloid coatings for different food materials can be achieved only by further determination of the chemical and physical properties of the coating solutions and the coated objects.

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20.4

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Hydrocolloid non-food coatings

Seed coating is an important application of polymers, whereby the seed surface is coated with a hydrophilic polymer. After planting, the polymer absorbs water and the rate, as well as the chances of germination, increase. Because coating composition and manufacture can be controlled, they can be designed to delay germination, inhibit rot, control pests, fertilise or bind the seed to the soil. Seed coatings have been composed of agar, alginates, various water-soluble cellulose ethers and hydrolysed starch-gpolyacrylonitrile copolymers (e.g., HSPAN). Many seeds have been coated, among them soybean, cotton, corn, sorghum, sugar beet and those of a number of vegetables. As a result, cotton and soybean yields increased 20–30%. The poorer the growing conditions, the more pronounced the advantage of the coated seeds vs. controls.79, 1 Although most of the reports on seed coatings deal with alginate coatings, reports on other gels can also be found.80 Many hydrocolloids are used to obtain effective paper coatings. Agar has been found suitable for use in photographic papers when esterified with succinic or phthalic anhydride and after enzymic hydrolysis. Agar can also be used as an adhesive in the gloss-finishing of paper products. HPC is used for coating, due to its solvent solubility and thermoplasticity. HPC serves as an oil and fat barrier and is responsible for thermoplastic coating. Polyethylene oxide (PEO) is used for paper coating and sizing. As a processing additive, it is used as a fibre-formation aid.1 The associative behaviour of CMCs, hydroxy ethyl celluloses (HECs) and hydrophobically modified cellulosic thickeners (HMCTs) was determined in clay-based coatings and their effects on coating rheology and coated-paper properties identified. The anionic CMCs were less adsorbed to kaolin clay than the nonionic HECs and HMCTs. Coatings containing less clay-adsorbent CMCs were less resistant to flow under high shear than those containing the clayadsorbent HECs, but they appeared to have a weaker after-blade structure. Coatings thickened with HMCT had both low flow resistance under high shear and a strong afterblade structure.81 Polyvinyl alcohol (PVA; a synthetic resin) is used in paper sizings and coatings. PVP (polyvinylpyrrolidone) is used in all types of paper manufacture, mostly as an economical fluidiser and antiblocking agent in paper coating. The starches used in coating colours are enzyme-converted starches, thermochemically converted starches, oxidised starches, dextrins, hydroxyethyl starch ethers and starch acetates.2 The use of polyacrylic acid (PAA) as a film-former has many applications. One example is the suggested use of the sodium salt of PAA as the major component of nonglare coatings for headlights. Antifogging coatings of glass and transparent plastics for optical use have been made by cross-linking PAA with aminoplast resins to produce scratch- and water-resistant coatings. These polymer acids have been used as release coatings for polymerisation moulds, as parting agents, and as soil-release coatings for hard surfaces subject to spattering by printing ink.2 In the manufacture of fluorescent lights, phosphor bonding to the glass tube is enhanced by using polyethylene oxide resin as a temporary binder in combination with barium nitrate as a bonding agent. The coating is usually applied by preparing a suspension of the phosphor, binder and dispersant. The suspension is used to flush the glass tube, leaving a thin coating of phosphor on the inside. Subsequent drying and lehring removes water, solvents and organic additives and leaves the phosphor coating, bonded to the inside of the tube. The advent of water-based latex paints raised the need for speciality CMC types to meet the rheology requirements of the flow point under the roller or brush. The

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availability of property variations during the manufacture of CMC to meet the specific coating-system requirements has been greeted with acceptance by the coating industry. For the manufacture of coatings, paints, foams or adhesives, xanthan gum is compatible with the common types of latex emulsions, making it effective as a stabiliser, thickener and modifier of rheological properties.

20.5

Film-application techniques and stages

Films can be applied by either dipping or spraying, the latter being better suited to planar surfaces.82 Brushes, falling-film enrobing technique, panning or rollers can also be used to apply films to the surfaces of the coated objects.83 Foods to be coated are dipped in a hydrocolloid solution followed by draining and drying. The coating of a surface with a gelling agent that requires subsequent cation cross-linking involves a process that can be theoretically divided into four successive steps and time stages: immersion of fresh produce in a hydrocolloid solution (not including the cationic cross-linking agent) can take between 15 and 120 s for a complete coating. Duration depends on wettability, the concentration and viscosity of the hydrocolloid solution, as well as the surface roughness, and possible penetration of the coating solution into the biological specimen.84 At this point, the coating solution can be dried to achieve a dry film adhering to the food surface; thus a film that did not pass through a gel state is formed. A second immersion of the hydrocolloid-coated food in a cross-linking bath to induce gel formation (i.e., in alginate, LMP, - or -carrageenan or gellan) takes between 30 s and ~2 min. depending on the concentration and temperature of the cross-linking agent, the thickness of coating gum solution and the geometric complexity of the coated object. (It is important to note that a food coated with a thin layer of gel that will be further dried into a coating film can be produced in one step if gelling agents that gel directly on the food surface such as agar are used.) The third stage of producing a cross-linked coating is the continuous strengthening of the gel coating layer at high relative humidity. The fourth stage, namely drying, can result in different dry-film textures and structures, depending on the duration of drying. When the coated object is dried, after a short or long gelation process or none at all, texture and structure of the dried film will vary according to the time required for the gelled film layer to lose its inherent moisture. The properties of the dried films also depend on properties of the drying equipment and thickness and composition of the coating film. A casting technique is used in many laboratories to produce flat films of nearly constant thickness for an easy determination of their mechanical properties (by puncture and tensile tests) and permeabilities. Thus, the peeling of films produced on foods is avoided. The gel or hydrocolloid solution is poured into a ‘sandwich’ prepared from glass plates. After the gels are cast, they are left to equilibrate under high humidity at room temperature. Next, the films are dried with warm air until a predetermined amount of moisture remains within them. They are then ready for testing.

20.6

Methods for testing coatings

Gas permeability of packaging films can be measured in several ways.85–87 Many devices for measuring film permeability to oxygen are on the market.88,89 Water-vapour transmission rates through dried coatings can be determined by ASTM E96-93 (standard

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test methods for water-vapour transmission of materials). Peel testing, i.e. the force necessary to peel the coating, is used to estimate the film’s degree of adhesion to a surface. The coating is peeled at 90º from the substrate, and the adhesion strength is estimated by the force per unit width necessary to peel the coating. It is important to study surface wetting and adhesion properties of coated commodities to obtain ‘good’ coatings. As discussed, the roughness of products and of film surfaces can be studied using a roughness tester, and electron and AFM (the latter for finer mapping of surface roughness). Parameters which can be studied include Rt, the distance between the highest peak and the deepest valley of the roughness profile within an evaluation length of the tested surface. Other important parameters, such as Ra, the arithmetic mean of the absolute values of the roughness profile’s deviations from the centre line within the evaluation length can also be determined. The surface tension of gelling and inducing solutions, and their contact angles on fruit and vegetable surfaces, can be studied with surface-tension instruments (maximum adhesion requires a contact angle of 0º). It is important to note that ‘if the coating does not spread spontaneously over the substrate surface, so that there is intermolecular contact between the substrate surface and the coating, there cannot be interactions and hence no contribution to adhesion.’90

20.7

Adhesives: non-food uses and applications

The adhesive properties of many hydrocolloids (gums) have been known for centuries. The word ‘gum’ means a sticky substance, and was previously defined as such by the Egyptian term qemai or kami, referring to the exudate of the acanthus plant and its adhesive capacity.91 A large number of hydrocolloids have been mentioned in the literature as adhesive agents.92, 93 They include gum talha (similar to gum arabic), gum ghatti, gum karaya, gum tragacanth, arabinogalactan, dextran, pectin, tapioca-dextrin, CMC, MC, HPC, HPMC, carbopol, polyethylene oxide, PVP, PVA, retene, pullulan, and chitosan. Some lesser-known water-soluble gums which possess wet-adhesive bonding properties include: gum angaco, brea gum and psylium seed gum,94 gum cashew,95 gum damson, jeol, myrrh96 and scleroglucan.91 Bioadhesive applications of hydrocolloids in medicine and cosmetics have been widely explored. A few examples are adhesive biodelivery systems,97–102 adhesive bioelectrodes,103 cosmetic preparations,104 pressure-sensitive adhesives,105, 106 ostomy rings,91 adhesive ointments,107 and dental adhesives.108 The use of hydrocolloids in the paper industry began in ancient Egypt when starch was used to adhere papyrus sheets.109 Now starch, as well as dextrin, gelatin, PVA, gum arabic and cellulose derivatives make up the major portion of the glue market for the paper industry. Synthetic glues are based on polyvinyl acetate, ethylene-vinyl acetate, acrylic acid polyurethane and latex, among many others.110 Hydrocolloid glues are used for paper lamination, to prepare cardboard.111 Special methods (e.g., the ‘Stain Hall’ procedure) are used to prepare starch adhesives for paperboards.92, 112 For envelopes, stamps, wallpapers and similar products, special dry hydrocolloid glues that regain their adhesive properties upon wetting are used. They are based on gum arabic, PVA, dextrins and other gums.113–114 For paper bags, mostly starch or modified starch and dextrin are used.92 Glue’s resistance to water is increased by including PVA and/or polyvinyl acetate in its composition, as well as urea-formaldehyde at 5–15% of the gum’s weight in the formulation.115 Paper glues can be used to attach paperboard to gypsum sheets. Such glue can be composed of calcium carbonate, mica, attapulgite

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clay, HPMC, ethylene glycol, preservative, anti-foaming agent and latex. Tobacco is sometimes packaged in paper cylinders that are glued together with starch or gum tragacanth.110 In ancient times, gum arabic and gelatin were used by the Egyptians in furniture manufacturing.116 The use of hydrocolloid glues in the modern wood industry began at the turn of the century. Until 1912, gelatin and starch were the most commonly used adhesives. Today, many other glues are also in use. They include polyamide, neoprene, polyvinyl acetate, urea-formaldehyde, phenol-formaldehyde, melamine-urea-formaldehyde, recorcinol-phenol-formaldehyde, epoxy ethylene vinyl acetate, and others. Gelatin is still used, especially for gluing cloth to wood.117 CMC, at ~30% of the dry solids in the glue, is used to retain moisture better in the recipe, without influencing adhesive strength. Synthetic PVA is used as an additive in urea-formaldehyde glues to improve the strength of the wet glue and its stability over time. Chitosan has been used to glue pine-tree plates, in a mixture with water and acetic acid.118 HEC, as part of a glue composed of bentonite, asbestos, coquina, and glycerol, was used to fill cracks in wood boats. Another hydrocolloid used in the preparation of elastic and strong glues for woods is fonuran, combined with carbon disulfide. The preparation involves mixing with sulfuric acid and later adding sodium hydroxide, heating to 50ºC for 10 min followed by heating for 3 h at the same temperature after adding the carbon disulfide.119 Hydrocolloid glues are used as a tool in drying leather. The leather is smeared with a polymeric glue and attached to glass, aluminum, stainless-steel or porcelain plates. The plates move perpendicularly through a drying tunnel at ~93ºC for several hours. The better the adherence of the leather to the plate, the less shrinkage occurs. The glues are composed mainly of MC, CMC and PAA. MC is the most efficient hydrocolloidal ingredient due to its tendency to gel when heated and to maintain its elasticity during drying, resulting in less leather shrinkage during the process. In addition, the gel can be easily removed at the end of drying without damaging the leather’s fibre structure. CMC can be substituted for MC.120,121 PAA can also be used in combination with starch or CMC.122 In ancient times, mummies were wrapped in bandages glued with gum arabic.109 Starch and dextrins are used for fibre strengthening, as are HEC and CMC. A typical formula for such glues includes cornstarch, sulfonated oil, kerosene and water. Special starches, following acetate modification, are also applicable. Oxidised starch is also used for printing and finishing.123

20.8

Adhesives: food uses and applications

A simple way of achieving different textures and tastes in the same bite is to build a food product composed of layers with different properties. A few multilayered food products are already available, e.g., crunchy wafers, which include a sweet vegetable-fat-based chocolate- or vanilla-flavoured filling between brittle wafers. For children, a multilayered, sweetened agar- or starch-based three-colour confection can also be found, in which the texture of the layers is similar, but the tastes and colours differ. In the Orient, where the awareness of different gel textures is much more developed than in the West, a curdlan-based, sweetened, multilayered gel has been developed.124–126 In this case, all layers are built from the same hydrocolloid, because two types of curdlan gel can easily be prepared from its powder by heating the suspension to different temperatures. Multilayered foods based on hydrocolloids are important in the framework of foods of the future.

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Recently, multilayered gels (composed of different combinations of agar, four galactomannans, xanthan, carrageenan and konjac mannan), and gelled texturised fruits (based on banana, apple, kiwi and strawberry pulps and agar-LBG combinations), glued together by three different adhesion techniques, were studied.127 The gluing techniques consisted of pouring the hot hydrocolloid solution onto the gelled layer, using melted agar as a glue between already gelled layers, or simultaneously pouring pre-gelled (gum solution before setting) hydrocolloid solutions. The compressive deformabilities of these gels were predicted. Two assumptions were made: that the normal force in the layers is the same, and that the deformations are additive. The effects of lateral stresses were considered negligible. The calculation was performed using a mathematical model previously developed for double-layered curdlan gels.128 The model constants were determined from the behaviour of the individual layers. Good agreement was found between experimental and fitted results over a considerable range of strains. Thus the model’s applicability to a given gel system was demonstrated, suggesting a very convenient tool for analysing and predicting the compressive behaviour of any number of arrays with different layer combinations.127

20.9

Bioadhesives: uses and applications

Hydrocolloids are used in biodelivery adhesive systems to carry fluoride,99 benzylamineHCl or other drugs to cure gingivitis. The hydrocolloids are part of both the biodelivery and protective layers. The first is prepared, for example, from HPC, HPMC, gum karaya, propylene glycol alginate 400 and the drug. The second is composed of ethyl cellulose, sodium CMC and HPC, and is not adhesive. The whole preparation is dried and compressed to a thickness of ~100 m.129 Many other adhesive biodelivery systems have been described.130, 131 Other additional gums can be used in these preparations, such as PVP, PVA and carbopol. Agar is frequently used for the intermediate, non-adhesive layer. Many hydrocolloids have been used as biological glues to mucus, with PC and HPC being preferred. One of the earliest discussions on hydrocolloids’ ability to serve as glues in biological systems was published 30 years ago.93 These authors also presented results of in-vitro tests comparing adhesion characteristics. Nearly 15 years later, Smart et al.102 examined the mean adhesive forces of many hydrocolloids to mucus. They found that sodium CMC produces the strongest adhesive force, followed by carbopol and gum tragacanth. Similar results were obtained by Robinson et al.100 Other research130 estimated the minimal amount of polymer needed for the glue to stay in position for about the same duration. The following order of efficiency was found: HEC > HPC > PVP and PVA. Other scientific approaches can be found in the literature calculating adhesion energy of different formulations attached to biological membranes.97 It is interesting to note that although locust bean gum (LBG)-xanthan systems were evaluated as mucosal glues and found unsatisfactory, they are nevertheless used due to their good mouthfeel and for health safety reasons.129 Biological hydrocolloid glues for the vaginal area and eyes, and in sprays for the nose and other mucosal areas, have been developed.99, 129 Chitosan, pullulan, gum tragacanth, HPC, starch and carbopol have been suggested for these preparations. Adhesive dressings contain many hydrocolloids in their compositions, such as PVP, PEO and PVA. Adhesive bioelectrodes use PVA, hydroxyethyl methacrylate, gum karaya, agar, MC or CMC in their formulations.103 Other biological applications of hydrocolloids are the use of dextran in cosmetic preparations, pullulan and CMC as part of medicinal adhesive tapes, gum karaya for ostomy rings, and a wide range

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of hydrocolloids such as gum karaya, gum arabic, cellulose derivatives, polyox and others for dentistry.

20.10

Hydrocolloid adhesion tests

A relatively large number of tests have been proposed to evaluate adhesive-bonding strength. They include peeling at 90º (Fig. 20.4), and tensile-bond and lap-shear tests (Figs 20.5 and 20.6). The bond-strength value measured by a specific test is not just an inherently fundamental property of the type of adhesive; it also depends on other factors. Many experimental procedures, using biological or other hydrocolloid adhesives, have been conducted to test different important variables such as crosshead speed at debonding,101 adhesive-layer thickness,102 water-holding capacity of a specimen,93, 107 length of contact,93, 102 the effect of molecular weight102 and the type of adhesive.93

20.11

Hydrocolloids as wet glues

Recently, 26 hydrocolloids were tested for their ability to produce wet glues, i.e., they were studied for their ability to create very thick suspensions with ‘good’ adhesive

Fig. 20.4

Specimen mounted on the Instron universal testing machine (UTM) during the 90º peel test.

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Fig. 20.5

Specimen mounted on the Instron UTM prior to application of the tensile load.

properties at predetermined gum concentrations ranging from 10–75% by weight. The hydrocolloids were: gum talha, gum ghatti, gum arabino-galactan (AG), gum karaya, gum tragacanth, dextran, apple pectin, CMC, HPMC, tapioca-dextrin, carbopol-934, HPC, MC, gelatin, casein, starch, LBG, guar gum, alginate, -carrageenan, tara gum, fenugreek gum, konjac mannan, xanthan gum, gellan, and curdlan. The hydrocolloids (at different concentrations) were added in powdered form to double-distilled water and mixed with a standard dough mixer for at least 15 min. until a thick, uniform and smooth paste-wet glue was obtained.131,132 Preliminary tests revealed that only 13 of these, namely: gum talha, gum ghatti, gum karaya, gum tragacanth, AG, dextran, pectin, tapioca-dextrin, CMC, MC, HPC, HPMC and carbopol, can serve as bioadhesives in hydrophilic systems. Wet glues were produced from these hydrocolloids and tested over a wide range of concentrations, i.e., 10–75% by weight, and the colour and pH of each glue was determined. All preparations were tested immediately following their production. Paste temperature was taken at the end of mixing, and 5 minutes later had risen by 0.970.14ºC. The pH of the wet glues ranged from 1.2 with carbopol to ~9.6 with AG. pH may be an important factor in the utilisation of bioadhesive materials. Studies have shown108 that gum karaya (pH ~3.6) may cause allergic reactions such as hives and angioneurotic edema. Paste colour may be a factor in choosing an ointment or bioadhesive for a particular application. A variety of different colours could be found among the wet glues, ranging from off-white to yellowish or dark-brown.132

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Fig. 20.6

359

Specimen mounted on the Instron UTM prior to application of the lap-shear test.

Once in very thick paste form, the 13 hydrocolloids were smeared homogeneously onto two different substrates: (a) cellulose-acetate film, normally used for dialysis, and (b) a skin-surface model (SSM), proposed131 for testing the adhesion of medical adhesives. The water content in the SSM was approximately 1%. The three aforementioned mechanical tests, i.e. peel, tensile and lap-shear tests, were performed to check the properties of the wet glues.131 Seven typical hydrocolloids (gum ghatti, AG, pectin, tapioca-dextrin, dextran, HPMC and carbopol-934) were chosen for an evaluation of their physical properties as representatives of tree and shrub exudates, tree extracts, fruit extracts, grains, exocellular polysaccharides, cellulose derivatives and petrochemicals, respectively.131 Typical curves for the 90º peel, tensile-load and lap-shear tests are presented in Fig. 20.7. Two common types of curves were obtained from the peel test. When a sample is pulled apart at a constant crosshead speed, the measured force should ideally be constant after reaching a steady-state condition. In practice, however, this is not always the case. There is evidence in the literature that when such results are reported, the mean values (averages) of the curve’s ruggedness (deviations from a smooth line after reaching a steady-state condition) can be calculated and observed. In some instances, as observed macroscopically during testing, the rupture process occurs abruptly, sample failure propagates faster than the rate at which the sample is pulled apart, and failure is initiated periodically. The force has been claimed to go through welldefined maxima and minima, and the distance between two minima or maxima is

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Fig. 20.7 Typical curves for the 90º peel test: (a) pectin, 25%; (b) gum talha, 70%; (c) typical curve for the tensile-bond test; (d) typical curve for the lap-shear test (HPMC, 20%). (Courtesy of O. Ben-Zion.)

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independent of testing rate.133 This author also mentions that the variability of the force is due to sample imperfections, that all the points on such a curve can be considered a statistical population, and that their frequency distribution is Gaussian.133 Tensile load (Fig. 20.7(c)) and lap shear (Fig. 20.7(d)) were applied and plotted as load (g force cmÿ2) vs. displacement (cm) curves.132 Tensile-bond strength increased in parallel to increases in deformation, until the beginning of failure. Tensile-bond tests are commonly used to analyse various adhesives, ranging from those for wood to those for metal (ASTM D-897 and ASTM D-2094, respectively134). Lap-shear strength decreased linearly as deformation increased. Lap-shear tests are used to examine adhesion when the samples are relatively easy to construct and closely resemble the geometry of many practical joints (ASTM D-1002; ASTM D-3528). Data were collected continuously as the specimens were stretched uniaxially to rupture. The maximal force was recorded in both cases.132 It was difficult to run comparative, economic or practical analyses of the various hydrocolloids tested in this study, since the concentrations and viscosities differed, and formation of the desired suspension did not necessarily require the same time or energy. Therefore, in choosing a hydrocolloid for a particular application, such as wet biological adhesives or ointments, one needs to consider, in addition to the aforementioned factors, clinical or medicinal aspects such as the ability of the gum to perform as a medium for different electrolytes that could cause changes in viscosity and thus changes in adhesion potential (e.g., bioelectrodes). Also important are the wet glue’s potential to cause dermatological irritation or allergic reactions, and its cost.132 The previous reference gave experimental values of tensile-bond and lap-shear strengths for seven hydrocolloids (representing seven different origins of natural and synthetic gums), tensile-bond vs. 90ºpeel adhesive strengths, the influence of deformation rate on the peel-bond-strength values, the dependence of peel-bond strength on adhesive-layer thickness, the influence of water absorbance on peel-bond strength and the influence of molecular weight on peelbond strength. In general, adhesion and bioadhesion studies on hydrocolloids reveal that compounds capable of inducing good wet adhesion have high molecular weights. Thus, chain length may contribute heavily to adhesive strength.

20.12

Coatings and adhesion: future prospects

Although food coatings have been used from the 12th century, not too many hydrocolloid formulations are in use today, especially not when producing a gel coating over an object before drying is involved. The reason for this is that most of the experimental work in foods has been empirical, and properties of surface and wetting solutions, as well as the relationships between them, have not been investigated to a large extent or understood. It was concluded135 that much of the edible film and coating work reported in the literature is of limited value owing to the lack of quantitative data on the barrier characteristics of the coatings. It is only through the compilation of barrier properties and their correlation with edible polymer structure and composition that generalised theories can be formulated to explain, for example, oxygen and aroma mass transfer behaviour to solve food-packaging problems.135 In addition, known approaches from other fields of coating science are not always used. More basic research into the chemical and physical properties of coating solutions and coated objects is needed to tailor hydrocolloid coatings to different vegetative materials136 or other food products. With regards to nonfood coatings, many new ways of measuring adhesion have been documented in the

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literature for films and coatings. However, there is perennial discord among the people working in this field as to what exactly is measured when one of these techniques is used.137 In around 1930, water-based glues made up ~90% of the adhesives market. From 1930 to 1986, this figure decreased to ~60%. However, an average annual increase of ~3.9% in the last 10 years in these glue types, due to renewed interest by the packaging, construction and medicinal industries, suggests that nature-based products will still make up 48% of the market in the near future, whereas early synthetic adhesives, including PVA, PVP and PAA, will take 16% of the market and new synthetic adhesives ~36%.138, 139

20.13

References

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26. SANDFORD, P. A. ‘Chitosan: commercial uses and potential applications’ in Chitin and Chitosan: Sources Chemistry, Biochemistry, Physical Properties and Applications. (eds Skjak Braek, T. Anthonsen and P. Sandford), NY, Elsevier Applied Science, 1989, pp. 51–69. 27. PENNISI, E. ‘Sealed in edible film’ Science News, 1992 141 12. 28. EL GHAOUTH, A., ARUL, J., PONNAMPALAN, R. and BOULET, M. ‘Chitosan coating effect on storability and quality of fresh strawberries’ J. Food Sci, 1991 56 1618. 29. MEHERIUK, M. and LAU, O. L. ‘Effect of two polymeric coatings on fruit quality of Bartlett and d’Anjou pears’ J. Ameri Soc Hort Sci, 1988 113 (2) 222–6. 30. MEHERIUK, M. ‘Skin color in Newton apples treated with calcium nitrate, urea, diphenylamine and a film coating’ Hortscience, 1990 25 (7) 775–6. 31. BAI, R., HUANG, M. and JIANG, Y. ‘Selective permeabilities of chitosan-acetic acid complex membrane and chitosan-polymer complex membranes for oxygen and carbon dioxide’ Polymer Bulletin, 1988 20 83–8. 32. ROTH, W. B. and MEHLTRETTER, C. L. ‘Some properties of hydroxypropylated amylomaize starch films’ Food Technol, 1967 21 72–4. 33. MURRAY, D. G. and LUFT, L. R. ‘Low D.E. corn starch hydrolysates’ Food Technol, 1973 27 (3) 32–40. 34. HERSHKO, V. and NUSSINOVITCH, A. ‘Relationships between hydrocolloid coating and mushroom structure’ J. of Agric and Food Chem, 1998 46 (8) 2988–97. 35. KROCHTA, J. M. and DE MULDER-JOHNSTON, C. ‘Edible and biodegradable polymer films’ Food Technol, 51 (2), 61–73. 36. IFT New from Mitsubishi, Annual Meeting and Food Expl. Program and Exhibit Directory, Dallas Convention Center, Chicago, IL, 1991. 37. BALDWIN, E. A. ‘Edible coatings for fresh fruits and vegetables: past, present and future’ in Edible Coatings and Films to Improve Food Quality (eds J. Krochta, E. Baldwin and M. Nisperos-Carriedo), Basel, Switzerland, Technomic Publishing Co., 1994. 38. TORRES, J. A., MOTOKI, M. and KAREL, M. ‘Microbial stabilization of intermediate moisture food surfaces. Control of surface preservative concentration’ J Food Proc Pres, 1985 9 75. 39. BRYN, D. S. US Patent 3,707,383, 1972. 40. KAMPF, N. and NUSSINOVITCH, A. ‘Hydrocolloid coating of cheeses’ Polymer Networks, Trondheim, Norway, 1998. 41. ANON. A Food Technologist’s Guide to Methocell Premium Food Gums, Midland, MI, The Dow Chemical Co., 1990. 42. HAGENMAIER, R.D and SHAW, P. E. ‘Moisture permeability of edible films made with fatty acid and (hydroxypropyl) methylcellulose’ J Agric. Food Chem 1990 38 1799–803. 43. NISPEROS-CARRIEDO, M. O. ‘Edible coatings and films based on polysacharides’ in Edible Coatings and Films to Improve Food Quality (eds J. M. Krochta, E. A. Baldwin and M. O. Nisperos-Carriedo), Basel, Switzerland, Technomic Publishing Co., 1994, pp. 305–36. 44. VOJDANI, F. and TORRES, J. A. ‘Potassium sorbate permeability of methylcellulose and hydroxypropyl methylcellulose coatings: effect of fatty acids’ J Food Sci, 1990 55 (3) 841. 45. NELSON, K. L and FENNEMA, O. R. ‘Methylcellulose films to prevent lipid migration in confectionery products’ J Food Sci, 1991 56 504–9. 46. DEBEAUFORT, F., MARTIN-POLO, M. and VOILLEY, A. ‘Polarity homogeneity and structure affect water vapor permeability of model edible films’ J Food Sci, 1993 58 426–34. 47. KOELSCH, C. M. and LABUZA, T. P ‘Packaging, waste disposal and food safety. II: Incineration or degradation of plastics and a possible integrated approach’ Cereal Foods World, 1991 36 284–98. 48. MARTIN-POLO, M., MAUGUIN, C. and VOILLEY, A. ‘Hydrophobic films and their efficiency against moisture transfer. Influence of the film preparation technique’ J Agric Food Chem, 1992 40 407–12. 49. PARK, J. W., TESTIN, R. F., PARK, H. J., VERGANO, P. J. and WELLER, C. L. ‘Fatty acid concentration effect on tensile strength, elongation and water vapor permeability of laminated edible films’ J Food Science, 1994 59, 916–19. 50. RICO-PENA, D. C. and TORRES, J. A. ‘Edible methylcellulose-based films as moisture impermeable barriers in sundae ice cream cones’ J Food Sci, 1990 55 1468–9. 51. KRUMEL, K. L. and LINDSAY, T. A. ‘Nonionic cellulose ethers’ Food Technol, 1976 30 (4) 36–43. 52. SANDERSON, G. R. ‘Polysaccharides in foods’ Food Technol 1981 35 (7) 50–7. 53. DZEIZAK, J. D. ‘Special Report: A focus on gums’ Food Technol, 1991 45 (3) 116–32. 54. KAMPER, S. L. and FENENEMA, O. R. ‘Use of edible films to maintain water vapor gradients in foods’ J. Food Sci, 1985 50 382–4. 55. NISPEROS-CARRIEDO, M. O., BALDWIN, E.A. and SHAW, P. E. ‘Development of edible coating for extending postharvest life of selected fruits and vegetables’ Proc Florida State Horticultural Society, 1992 104 122–5. 56. GANZ, A. J. ‘CMC and hydroxypropylcellulose versatile gums for food use’ Food Prod Dev, 1969 3 (6) 65. 57. DRAKE, S.R., FELLMAN, J. K. and NELSON, J. W. ‘Postharvest use of sucrose polyesters for extending the shelf life of stored Golden Delicious apples’ J. Food Sci, 1987 52 1283–5. 58. BANKS, N. H. ‘Some effects of Tal Pro-long coating on ripening bananas’, J Experimental Bot, 1984 35 (150) 127–37. 59. BANKS, N. H. ‘Studies of the banana fruit surface in relation to the effects of Tal Pro-long coating on

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gaseous exchange’ Scientific Hort, 1984 24 279–86. 60. SMITH, S. M. and STOW, J. R. ‘The potential of a sucrose ester coating material for improving the storage and shelf-life qualities of Cox’s Orange Pippin apples’ Ann Appl. Biol, 1984 104 383–91. 61. MITSUBISHI-KASEI RYOTO Sugar Ester, Technical Information, Mitsubishi-Kasei, 1989, pp. 1–20. 62. UKAI, N. T., TSUTSUMI, T. and MARAKAMI, K. US Patent 3,997,674, 1975. 63. BANKS, N. H. ‘Evaluation of methods for determining internal gases in banana fruit Musa acuminata Musa ballisiana’, J Experimental Bot, 1983 34 (144) 871–9. 64. LOWINGS, P. H. and CUTTS, D. G. ‘The preservation of fresh fruits and vegetables’ in Proc. Inst. Food Sci. Tech. Ann. Symp., Nottingham, UK, 1982, p. 52. 65. NISPEROS-CARRIEDO, M. O. and BALDWIN, E.A. ‘Effect of two types of edible films on tomato fruit ripening’ in Proc. Fla. State Horticulture Society, 1988 101 217–20. 66. NISPEROS-CARRIEDO, M. O., SHAW, P. E. and BALDWIN, E.A. ‘Changes in volatile flavor components of pineapple orange juice as influenced by the application of lipid and composite film’ J Agric and Food Chem, 1990 38 1382–7. 67. CURTIS, G. J. ‘Some experiments with edible coatings on the long-term storage of citrus fruits’ in Proc. 6th Int. Citrus Congress 3, 1988, pp. 1514–20. 68. EDWARDS, M. E. and BLENNERHASSETT, R. W. ‘The use of postharvest treatments to extend storage life and to control postharvest wastage of Honey Dew melons (Cucumis melo L. var. inodorus Naud.) in cool storage’ Aust J Exp Agric, 1990 30 693–7. 69. HERSHKO, V. WEISMAN, D. and NUSSINOVITCH, A. ‘Methods for studying surface topography and roughness of onion and garlic skins for coating purposes’ J Food Sci, 1998 63 (2) 317–21. 70. MITTAL, K. L. ‘The role of the interface in adhesion phenomena’ Polymer Engineering and Science, 1997 17 (7) 467–73. 71. ADAMSON, A. W. Physical Chemistry of Surfaces, 3rd edn NY, John Wiley and Sons, 1976, pp. 1–43. 72. DANN, J. R. ‘Forces involved in the adhesive process. Critical surface tensions of polymeric solids as determined with polar liquids’ J of Colloid Interface Sci, 1970 32 (2) 302–19. 73. BARTON, A. F. M. CRC Handbook of Solubility Parameters and Other Cohesion Parameters, US, CRC Press Inc., 1983, pp. 427, 441. 74. GAONKAR, A. G. ‘Surface and interfacial activities and emulsion of some food hydrocolloids’, Food Hydrocolloids 1991 5 (4) 329–37. 75. WU, M. T. and SALUNKHE, D. K. ‘Control of chlorophyll and solanine synthesis and sprouting of potato tubers by hot paraffin wax’ J of Food Sci, 1972, 37 629–30. 76. OLIVER, J. F. and MASON, S. G. ‘Microspreading studies of rough surface by scanning electron microscopy’, J. Colloid Interface Sci, 1977 60 (3) 480–7. 77. WENZEL, R. N. ‘Surface roughness and contact angle’ Industry Engineering Chemistry, 1936 28 988–93. 78. WARD, G. and NUSSINOVITCH, A. ‘Gloss properties and surface morphology relationships of fruits’ J. Food Sci, 1996 61 (5) 973–7. 79. MARK, H. F., OTHMER, D. F., OVERBERGER, C.G. ET AL., Kirk-Othmar Encyclopedia of Chemical Technology, 3rd edn, vol. 20, NY, Wiley-Interscience, 1985, pp. 219–20. 80. FERRARIS, R. ‘Gel seeding of sorghum into Mywybilla clay’ in Proc Aust Sorghum Workshop, Toowoomba, Queensland (eds Foale M. A., Hare B. W. and Henzell R. G.), Australian Institute of Agricultural Science, Brisbane, Australia, 1989. 81. YOUNG, T. S. and FU, E. ‘Associative behavior of cellulosic thickeners and its implications on coating structure and rheology’ TAPPI J. 74 (4) 197–207. 82. KROCHTA, J. M., BALDWIN, E. A. and NISPEROS-CARRIEDO, M. Edible Coatings and Films to Improve Food Quality, Basel, Switzerland, Technomic Publishing Co., 1994. 83. GUILBERT, S. ‘Technology and application of edible protective films’ in Food Packaging and Preservation Theory and Practice (ed. M. Mathlouthi), London, Elsevier Applied Science Publishing Co., 1986, p. 371. 84. HERSHKO, V., KLEIN, E. and NUSSINOVITCH, A. ‘Relationships between edible coatings and garlic skin’ J. Food Sci 1966 61 (4), 769–77. 85. KARAL, M. ‘Protective packaging of foods’ in Principles of Food Science (ed. O Fennema), NY, Marcel Dekker, 1975, pp. 399–464. 86. AYDT, T. P., WELLER, C. L. and TESTIN, R. F. ‘Mechanical and barrier properties of edible corn and wheat protein films’ Amer Soci of Agricult Engineers, 1991 34 (1) 207–11. 87. STERN, S. A., SINCLAIR, T. P. and GAEIS, T. P. ‘An improved permeability apparatus of the variable-volume type’ Mod. Plastics, 1964 10 50–3. 88. QUAST, D. G. and KAREL, M. ‘Technique for determining oxygen concentration within packages’ J Food Sci, 1972 37 490–1. 89. LANDROAC, A. H. and PROCTOR, B. E. ‘Gas permeability of films’ Mod Packaging, 1952 25 (10) 131–5, 199–201. 90. WICKS, Z. W., JONES, F. N. and PETER-PAPPAS, S. Organic Coatings, Science and Technology, NY, John Wiley and Sons Inc., 1994. 91. GLICKSMAN, M. Food Hydrocolloids, vol. 3, Boca Raton, FL, CRC Press Inc., 1982, p. 176. 92. BAUMAN, M. G. D. and CONNER, A. H. ‘Carbohydrate polymers as adhesives’ in Handbook of Adhesive Technology (ed. A. Pizzi), NY, Marcel Dekker Inc., 1994, pp. 299–313.

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93. CHEN, J. L. and CYR, G. N. ‘Compositions producing adhesion through hydration’ in Adhesion in Biological Systems (ed. Manly, R. S.), NY, Academic Press, 1970, pp. 163–81. 94. MANTELL, C. L. Water-Soluble Gums, NY, Reinhold Pub. Corp., 1947, pp. 48, 71, 72. 95. HOWES, F. N. Vegetable Gums and Resins, Waltham, MA, Chronica Botanica Comp., 1949, pp. 56–8, 61–2. 96. SMITH, F. and MONTGOMERY, R. The Chemistry of Plant Gums and Mucilages, NY, Reinhold Pub. Corp., 1959, pp. 15–20, 199, 404–5. 97. BOTTENBERG, P. CLEYMAET, R. DE-MUYNCK, C. ET AL. ‘Development and testing of bioadhesive fluoride containing slow-release tablets for oral use’ J Pharm Pharmacol, 1991 43 457–64. 98. BOUCKAERT, S. and REMON, J. P. ‘In-vitro bioadhesion of a buccal, miconazole slow-release tablet’, J Pharm Pharmacol, 1993 45 504–7. 99. IRONS, B. K. and ROBINSON, J. R. ‘Bioadhesives in drug delivery’ in Handbook of Adhesive Technology (ed. A. Pizzi), NY, Marcel Dekker Inc, 1994, pp. 615–27. 100. ROBINSON, J. R., LONGER, M. A. and VEILLARD, M. ‘Bioadhesive polymers for controlled drug delivery’ in Controlled Delivery of Drugs (ed. R. L. Juliano), Ann Arbor, MI, Acad. Sci. 1987 507 307–14. 101. SMART, J. D. ‘An in-vitro assessment of some mucosa dosage forms’ Int J Pharm, 1991 73 69–74. 102. SMART, J. D., KELLAWAY, I. W. and ORTHINGTON, H. E. C. ‘An in-vitro investigation of mucosa-adhesive materials for use in controlled drug delivery’ J. Pharm Pharmacol, 1984 36 295–9. 103. KEUSCH, P. and ESSMYER, J. L. US Patent 4,684,558, 1987. 104. TOULMIN, H. A. US Patent 2,749,277, 1956. 105. KIYOSI, O. and YASUO, S. ‘Pullulan-containing adhesive tapes, sheets and labels’ Chem Abstr, 1986 106. 106. PIGLOWSKI, J. and KOZLOWSKI, M. ‘Rheological properties of pressure sensitive adhesives: polyisobutylene/sodium carboxymethylcellulose’ Rheol Acta, 1985 24 519–24. 107. KANIG, J. L. and MANAGO-ULGADO, P. ‘The in-vitro evaluation of orolingual adhesives’ J Oral Therap Pharm, 1965 4 413–20. 108. SHAY, K. ‘Denture adhesives – choosing the right powders and pastes’ J Amer Dent Assoc, 1991 122 70–6. 109. LUCAS, A. ‘Adhesives’ in Ancient Egyptian Materials and Industries, London, Edward Arnold Pub. Ltd., 1962, pp. 8–14. 110. BRIEF, A. ‘The role of adhesives in the economy’ in Handbook of Adhesives (ed. I. Skeist), NY, Van Nostrand Reinhold, 1990, pp. 21–38. 111. TORREY, S. Adhesive Technology Developments Since 1977, NJ, Noyes Data Corporation, 1980, pp. 197– 200. 112. SADLE, A., NORRISTOWN, N. J. and PRATT, T. J. US Patent 4,157,318, 1979. 113. KIRBY, K. W. ‘Vegetable adhesives’ in Adhesion and Adhesives (ed. R. Howink), Amsterdam, Elsevier Pub. Comp., 1967, pp. 167–85. 114. SHARKEY, J. B. ‘Chemistry of stamps: dyes, phosphors, adhesives’, J. Chem Educ, 1987 64 195–200. 115. CAESAR, G. V. ‘Starch and its derivatives’ in Handbook of Adhesives (ed. I. Skeist), NY, Van Nostrand Reinhold, 1962, pp. 170–86. 116. KEIME, F. 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KRUGER, L. and LACOURSE, N. ‘Starch-based adhesives’ in Handbook of Adhesives (ed I. Skeist), NY, Van Nostrand Reinhold, 1990, pp. 153–66. 124. HARADA, T. ‘Curdlan: a gel forming -1, 3-glucan’ in Polysaccharides in Food (ed. J. M. V. Blanshard), London, Butterworths, 1979, p. 298. 125. MORRIS, O. US Patent 4,981,707, 1991. 126. HARADA, T. ‘Production, properties and application of curdulan’ in Extracellular Microbial Polysaccharides (ed. A. Sanford), Washington DC, ACS Symp. Ser. 1977, 265–83. 127. BEN-ZION, O. and NUSSINOVITCH, A. ‘Predicting the deformability modulus of multi-layered texturized fruits and gels’ Lebensm Wiss Technol, 1996 29 129–34. 128. NUSSINOVITCH, A., LEE, S. J. KALETUNC, G., ET AL. ‘Model for calculating the compressive deformability of double layered curdlan gels’ Biotechnology Progress, 1991 7 272–4. 129. NAGAI, T, and MACHIDA, Y. ‘Buccal delivery systems using hydrogels’ Adv Drug Delivery Rev, 1993 11 179–81. 130. 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fruits and gels’ Lebensm Wiss Technol, 1996 29 129–34. 132. BEN-ZION, O. and NUSSINOVITCH, A. ‘Hydrocolloid wet glues’ Food Hydrocolloids, 1997, 11 (4) 429–42. 133. GARDON, J. L. ‘Some destructive cohesion and adhesion tests’ in Treatise on Adhesion and Adhesives, vol 1 (ed. R. L. Patrick), Marcel Dekker, New York, 1966, pp. 286–323. 134. ASTM Annual Book of ASTM Standards, part 22, Philadelphia, PA American Society for Testing and Materials, 1982. 135. MILLER, K. S. and KROCHTA, J. M. ‘Oxygen and aroma barrier properties of edible films: A review’ Trends in Food Sci and Technol, 1997 8 228–38. 136. HERSHKO, V. and NUSSINOVITCH, A. ‘The Behavior of Hydrocolloid Coatings on Vegetative Materials’ Biotechnol Prog, 14 (5) 756–65. 137. MITTAL, K. L. Adhesion Measurement of Films and Coatings, The Netherlands, VSP, 1995. 138. HAGQIST, J., MEYER, K. F. and SANDRA, K. M. ‘Adhesives market and applications’ in Adhesives and Sealants (ed. C. A. Dostal), London, ASM International, 1990, London, pp. 87–8. 139. DARNAY, A. J. and REDD, M. A. Adhesives and Sealants, Detroit, MI, Market Share Reporter, Gale Research Inc. Staff, 1994, pp. 232–3.

21 Chitosan M. Terbojevich (University of Padua) and R. A. A. Muzzarelli (University of Ancona)

21.1

Introduction

At least ten gigatons (1013kg) of chitin are synthesised and degraded each year in the biosphere. Chitin, (1-4)-linked 2-acetamido-2-deoxy- -D-glucan, is widely distributed among invertebrates. It is found as -chitin in the calyces of hydrozoa, the egg shells of nematodes and rotifers, the radulae of molluscs and the cuticles of arthropods, and as -chitin in the shells of brachiopods and molluscs, the cuttlefish bone, the squid pen, and pogonophora tubes. Chitin is found in exoskeletons, peritrophic membranes and cocoons of insects. The chitin in the fungal walls varies in crystallinity, degree of covalent bonding to other wall components, mainly glucans, and its degree of acetylation. The reader is referred to a large body of information on chitin and chitosan available in a number of books: a selection is listed in the references1–21 at the end of this chapter. In the areas of fisheries, textiles, food and ecology, scientists and industry people were urged to upgrade chitin in order to exploit renewable resources and to alleviate waste problems. Today chitins and chitosans from different animals are commercially available. Chitin isolates differ from each other in many respects, among which: degree of acetylation, defined as the molar fraction of GlcNAc, typically close to 0.90; elemental analysis, with nitrogen content typically close to 7%, and N/C ratio 0.146 for fully acetylated chitin; molecular size and polydispersity. The average molecular weight of chitin in vivo is probably in the order of at least one million Daltons, but chitin isolates have lower values due to partial random depolymerisation occurring during the chemical treatments and depigmentation steps. Isolated chitin is a highly ordered copolymer of 2-acetamido-2-deoxy- -D-glucose, major component, and 2-amino-2-deoxy- -D-glucose. As a point of difference from other abundant polysaccharides, chitin contains nitrogen. The solubility of chitin is remarkably poorer than that of cellulose, because of the high crystallinity of chitin, supported by hydrogen bonds mainly through the acetamido group. Chitosan indicates a family of deacetylated chitins. In general, chitosans have nitrogen content higher than 7% and degree of acetylation lower than 0.40. The removal of the acetyl group from chitin is a harsh treatment usually performed with concentrated NaOH.

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Protection from oxygen, with a nitrogen purge or by addition of sodium borohydride to the alkali solution, is necessary in order to avoid undesirable reactions such as depolymerisation and generation of reactive species. Commercial chitosans may contain insoluble highly acetylated fractions that come from the core of the granules submitted to heterogeneous deacetylation. The acetyl groups in the acid-soluble fractions are randomly distributed, whilst the insoluble fractions contain relatively long sequences of acetylated units. The presence of a prevailing number of 2-amino-2-deoxyglucose units in a chitosan allows the polymer to be brought into solution by salt formation. Chitosan is a primary aliphatic amine that can be protonated by selected acids, the pK of the chitosan amine being 6.3. The following salts, among others, are water soluble: formate, acetate, lactate, malate, citrate, glyoxylate, pyruvate, glycolate and ascorbate. Under particular conditions chitin and chitosan can give hydrophilic highly water swellable hydrogels: gel formation is also promoted by crosslinking agents or organic solvents, particularly for chitosan derivatives. Chemical and physical gels are produced, thermally reversible and not reversible. Chitin and chitosan are not present in the human tissues, but acetylglucosamine and chitobiose are found in glycoproteins and glycosaminoglycans. Since chitosan is biodegradable, nontoxic, nonimmunogenic and biocompatibile in animal tissues, much research has been directed toward its use in medical applications such as drug delivery, artificial skin and blood anticoagulants. Chitosan has also been suggested for use as flocculant, food thickener, paper and textile adhesive, membrane and a chelating agent for metals.

21.2

Chitosan chemistry

21.2.1 Chemical structure and molecular characterisation The degree of acetylation for commercial samples is about 0.20. Experimental chitosan samples can be almost completely deacetylated, giving poly-GlcN. The evaluation of DA can be carried out by a number of techniques, depending on the amount of acetylated units. In this regard, a practical guide and an useful reference source is represented by the Chitin Handbook.3 In particular, high-resolution 1H and 13C NMR spectroscopy can provide information not only on DA in solution and solid state, but also on the sequence structure:22 random and block arrangements of GlcNAc and GlcN residues are found. In order to evaluate the molecular weight of chitosan chains, viscometric and gel permeation chromatographic (GPC) techniques are used;23 at variance with static light scattering (LS) method, which gives absolute values for molecular weight, the above techniques are empirically related to chain length and require calibration curves. On the other hand, LS measurements are difficult to perform and sometimes the data are not easy to interpret, in the presence of aggregation and/or association. As a conclusion, the molecular weight characterisation of chitosan is a problem to solve: probably the viscometric method, easy and rapid to perform, and not greatly affected by the presence of negligible amounts of very high molecular weight polymer, is the best choice, provided that the Mark-Houwink equation [] = K.ma is correctly used. A number of sets of a and K values are reported in the literature,24 for various aqueous salt systems, in which the viscometric measurements are performed. Average molecular weight for chitosans can reach values of 5105 or more. GPC methods are available to evaluate the molecular weight distribution of chitosan samples: calibration is performed by means of commercial standards (pullulan, for

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instance) or chitosan samples.25 A good improvement is obtained by using a low-angle laser light scattering instrument as an on-line detector.26 Samples of chitosan with polydispersity index about 1.2–1.5 were obtained by preparative GPC;27 no variation in the degree of acetylation related to molecular weight was found. Attempts to perform molecular weight fractionation of commercial chitosans by selective precipitation with ethanol or acetone from aqueous solutions or by ionexchange absorption were only partially successful. The reasons for this are depolymerisation of chitosan chains during the experiments, and incomplete recovery of the polymeric material from the chromatographic column. Chitosan chains can be chemically depolymerised by cleavage of the glycosidic bond catalysed by acids and, to a lesser extent, by bases. The rate of depolymerisation depends on the type and the concentration of the acid and on the temperature: the extent of depolymerisation can be calculated following the equations reported in the literature.28,29 Because the reactivity of a glycosidic bond close to the acetamido group is greater than the reactivity of a glycosidic bond close to a protonated amino group, the depolymerised sample, especially at DA > 0.1, does not present the most probable MD, which requires that all glycosidic bonds have the same probability of being hydrolysed. Much more selective is the reaction in the presence of nitrous acid:30 indeed nitrosating species attack the glucosamine, but not the N-acetylglucosamine moieties and a 2,5–anhydro-Dmannose unit is formed at the reducing end of the cleaved polymer. As a consequence, after the depolymerisation, the samples have not only lower molecular weight than starting chitosans, but also different composition and sequence arrangement. Hydrogen peroxide can also be conveniently used to depolymerise chitosan. As for enzymatic depolymerisation, many commercial enzyme preparations exert hydrolytic activity on chitosans, that appear to be unexpectedly vulnerable to a range of hydrolases: several proteases such as pepsin, bromelain, ficin and pancreatin display lytic activities towards chitosans that surpass those of chitinases and lysozyme. Cellulases, hemicellulases, lipases and pectinases are also effective.31 This is possibly due to the simplicity of the enzymatic mechanism.

21.2.2 Chitosan production The chemical composition and the distribution of different units along the chitosan chains are strictly related to the polymer preparation; therefore there is an important relationship between production and properties of chitosans, both in solution and in the solid state. The existence of chitosan in nature was unknown until 1954, when it was discovered in the yeast Phycomyces blakesleeanus.32 Chitosan occurs as the major structural component of the cell walls of certain fungi, mainly of the Zygomycetes species.33 However, to date, chitosans have been produced commercially by alkaline deacetylation of crustacean chitins.

21.2.3 Chitosan derivatives Chitosan amino and hydroxyl groups are reacted to modify the structural units under conditions mild enough to prevent glycosidic linkage hydrolysis. An important aspect is the uniformity of reaction when heterogeneous conditions have to be used, along with the degree of substitution (DS) of the modified samples. A number of derivatisation/ functionalisation reactions useful to enhance the chitosan properties and to impart new ones are reported in the literature.34 For instance, the chemical modifications of chitosan

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yield more soluble polymers. The latter have higher biodegradability in animal bodies and physical properties of interest for applications in the solid state or in solution. Chitosan can be efficiently N-acylated without concomitant hydroxyl modifications, using anhydrides (2–3 fold excess) in organic media; quantitative or near quantitative acylations are accomplished at room temperature within a few minutes. Further improvements in chitosan reaction rate are obtained using a number of pretreatment methods. Mixed N- and O-acylated products are prepared under similar conditions, using 10-fold excess anhydride.35 The Schiff reaction between chitosan and aldehydes and ketones gives the corresponding aldimines and ketimines, which can be converted to N-alkylderivatives on hydrogenation with cyanoborohydride. A wide range of aliphatic and aromatic carbonyl compounds have been used, including formaldehyde, unsaturated alkyl aldehydes, aldehydo and keto acids, carbonyl-containing carbohydrates and dialdehydes, such as glutaraldehyde. In particular, N-carboxymethylchitosan was obtained in a watersoluble form by a proper selection of the reactant ratios, i.e. using equimolecular quantities of glyoxylic acid and amino groups;36 the reaction with 2-oxoglutaric acid under reducing conditions gave glutamate glucan. The attachment of various reducing mono-, oligo- and polysaccharides to chitosan, under mild conditions and in the presence of sodium cyanoborohydride, affords products with DS values of 0.54–0.97 in high yields (70–100%, depending on the residue length). Reductive alkylations were also performed with other types of saccharides, such as fructose, streptomycin sulfate and selectively oxidised -cyclodextrin.35

21.3

Properties of chitosans and derivatives

21.3.1 Solubility Chitosan is insoluble in organic solvents, in acids at high concentrations and in alkali; it is also insoluble in aqueous solution at pH 6, except for low molecular weight samples. Chitosan is soluble in aqueous acidic media, following protonation of amino groups in the repeating unit; this polycationic structure is unique, other polysaccharides being usually neutral or anionic. As reported for polyelectrolyte chains, intrinsic viscosity values, [], of chitosan samples inpaqueous solutions depend upon the ionic strength (I) of the medium: plots of [] vs. 1/ I are linear and the [] decrease with increasing salt concentration of the system is due to the shielding effect of the counterions. The chitosan molecule is rather stiff, less than DNA and more than polyacrylate; increasing DA values lead to a more extended conformation and an even stiffer chain.37 It was suggested that this is due to intra-residue hydrogen-bonding between the carbonyloxygen of the N-acetyl group and H6 in the following unit. A persistence length of 220Aº has been found, which is lower than that of chitin (350Aº) and the existence of a cholesteric mesophase has been demonstrated.38 Hydrophobic derivatives of chitosan, containing a small number of hydrophobic side chains, can be obtained from long chain acyl chlorides or anhydrides. A series of Nalkylchitosans (C3–C12) was obtained by reacting chitosan acetate in an ethanol-water mixture with the desired aldehyde and reducing the Schiff base with sodium cyanoborohydride (3 moles per mole of amine, 24 h).39 In aqueous solution, above a certain polymer concentration, intermolecular hydrophobic interactions lead to the formation of polymolecular associations. As a consequence, these copolymers exhibit

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thickening properties equivalent to those observed for higher molecular weight homopolymers and play important roles as viscosity modifiers in a variety of waterborne technologies including paints, inks and cosmetics. The water-solubility of chitosans at neutral pH increases with increasing DA, and a chitosan with DA 0.6 is fully water-soluble at all pH values.40 The dependence of solubility upon DA may be explained by a decrease in the apparent pK0-value with DA,41 or by a decreased possibility of aligning polymer chains when increasing the amount of randomly distributed GlcNAc units.40 The selective introduction of saccharide residues at the chitosan amine function facilitates the conversion of the water-insoluble chitosan into various soluble, branched derivatives. The branched chitosan derivatives are soluble in either neutral or slightly acidic (pH 5–6) aqueous medium, with solubility being attained for some products at DS as low as 0.14.35 Chitosan can be reacetylated with acetic anhydride to obtain also watersoluble partially reacetylated chitin.42 The formation of derivatives suitable for industrial applications with good solubility in various organic solvents can be effected through the introduction of hydrophobic substituents by acylation with long chain fatty acyl halides or anhydrides.

21.3.2 Gelation Gel materials are utilised in a variety of technological applications and are currently investigated for advanced exploitations such as the formulation of ‘intelligent gels’ and the synthesis of ‘molecularly imprinted polymers’. A typical simple example of gel formation was provided with chitosan tripolyphosphate and chitosan polyphosphate gel beads. pH-responsive swelling ability, drug-release characteristics, and morphology of the chitosan gel bead depends on polyelectrolyte complexation mechanism and molecular weight of the enzymic hydrolysed chitosan.43 The complexation mechanism of chitosan beads gelled in pentasodium tripolyphosphate or polyphosphoric acid solution was ionotropic crosslinking or interpolymer complex, respectively. The chitosan-polyphosphoric acid gel bead is a better polymer carrier for the sustained release of anticancer drugs in simulated intestinal and gastric juice medium than the chitosan-tripolyphosphate gel beads. Pet foods based on these gels have been developed. Acylation One of the simplest ways to prepare a chitin gel is to treat chitosan acetate salt solution with carbodiimide to restore acetamido groups. Thermally not reversible gels are obtained by N-acylation of chitosans: N-acetyl-, N-propionyl- and N-butyryl chitosan gels are prepared using 10% aqueous acetic, propionic and butyric acid as solvents for treatment with appropriate acyl anhydride. Both N- and O-acylation are found, but the gelation also occurs by selective N-acylation in the presence of organic solvents, as methanol, formamide, ethylene glycol. The importance of many variables has been studied by determining their effects on the gelation, such as chitosan and acyl anhydride concentrations, temperature, molecular weight of acylating compound, and extent of Nacylation. Probably the gelation is due to the aggregation of chitosan chains through hydrophobic bonding. Applications for N-acylchitosan gels are reported in the literature.24 Chitosan gels can also be prepared by using large organic counter ions. The process involves mixing heated solutions of chitosan acetate and of the sodium salt of either 1naphthol-4-sulphonic acid (NSA) or 1-naphthylamine-4-sulphonic acid, the mixture

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gelling on cooling: the chitosan concentration required for gel formation is low, about 2–5 g/l, and similar to the concentrations used for gel formation with other polysaccharides such as the carrageenans. Gel-like properties were found in N-carboxymethyl chitosan: this behaviour was explained in terms of association of ordered chains into a cohesive network, analogous to that in normal gels but with weaker interactions between associating chains, i.e. a weak gel.44 Chitosan gel beads could be prepared45 in amino acid solutions of about pH9, despite the requirement for a pH above 12 for gelation in water. This phenomenon was observed not only in amino acid solutions but also in solutions of compounds having amino groups. A solute concentration of more than 10% was required for preparation of gel beads at pH9. Gelation of the chitosan beads required about 25–40 minutes depending on the species of amino acid. Novel pH-sensitive hydrogels were synthesised46, 47 by grafting D,L-lactic acid onto the amino groups in chitosan without a catalyst; polyester substituents provide the basis for hydrophobic interactions that contribute to the formation of hydrogels. The swelling mechanisms in enzyme-free simulated gastric fluid (SGF, pH2.2) or simulated intestinal fluid (SIF, pH7.4) at 37ºC were investigated. The crystallinity of chitosan gradually decreased after grafting, since the side chains substitute the -NH2 groups of chitosan randomly along the chain and destroy the regularity of packing between chitosan chains. Water uptake of the hydrogels was investigated as a function of side-chain length and degree of substitution. The influence of pH and salt concentration on the swelling behaviour of the hydrogels was determined and interpreted. Enzymatic reactions leading to gels Stable and self-sustaining gels were obtained from tyrosine glucan (a modified chitosan synthesised by reaction of chitosan with 4-hydroxyphenylpyruvic acid) in the presence of tyrosinase that oxidises the phenol to quinone, thus starting cross-linking with residual free amino groups. Gels were also obtained with 3-hydroxybenzaldehyde, 4hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde.48, 49 As an extension of these works, a mushroom tyrosinase was observed to catalyse the oxidation of phenolic moieties of the synthetic polymer poly(4-hydroxystyrene) (PHS) in water-methanol. Although oxidation was rapid, in the order of minutes, only a small number of phenolic moieties of the PHS polymer (1–2%) underwent oxidation. Enzymatically oxidised PHS was observed to undergo a subsequent non-enzymatic reaction with chitosan. Ultraviolet spectra of this chitosan film suggested oxidised PHS was grafted onto chitosan.50, 51 Further progress52, 53 led to internally skinned polysulphone capillary membranes coated with a viscous chitosan gel and useful as an immobilisation matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors were then used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilised polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. Reactions with aldehydes A very popular crosslinking agent for chitosan is glutaraldehyde, as proposed by Muzzarelli et al.54 Chitosan networks were obtained by reaction with glutaraldehyde in

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lactic acid solution (pH4–5). The rheology of the chitosan-glutaraldehyde gel system was studied by Arguelles-Monal et al.55 By reaction of chitosan with aldehydes, Nalkylidene- or N-aryldene chitosan gels are produced; the extent of modification of the amino groups is about 80% and the minimum amount of aldehyde required for gel formation increases by increasing the aldehyde molecular weight. The gels are colourless, rigid and infusible up to 200ºC. By using glutaraldehyde, chemical gels are produced following crosslinking interchains; the rate of gelation is proportional to both the chitosan and the glutaraldehyde concentrations, to the temperature and to addition of neutral electrolytes.56 In research on novel biocompatible hydrogels, based exclusively on polysaccharide chains, chitosan and the dialdehyde obtainable from scleroglucan by controlled periodate oxidation were linked together;57 the reaction takes place at pH10 and the reduction of the resulting Schiff base is performed with NaBH3CN. The swelling capacity of the hydrogel is remarkable, considering the highly hydrophilic character of both polysaccharides, and strongly depends on the pH of the bathing solutions. Semi-interpenetrating network was synthesised with chitosan, crosslinked by glyoxal, and polyethylene oxide.58 Chitosan was characterised by its degree of deacetylation determined by infra-red spectroscopy, and by its average viscosimetric molecular weight. Swelling studies were performed on the chitosan/polyethylene oxide semi-interpenetrating network and on the reference hydrogel (crosslinked chitosan) at pH1.2 and 7.2. The semi-interpenetrating network displayed a high capacity to swell, adjustable by pH. Rheological studies performed in simple shearing and in oscillation showed that the semiinterpenetrating network had elastic properties. Young’s modulus was determined by texture analysis, in uniaxial compression and indentation. The comparison between the semi-interpenetrating network and the reference gel, as regards the mechanical and swelling properties, demonstrates the interest of the addition of polyethylene oxide. Poly(ethyleneglycol) dialdehyde diethyl acetals of different molecular sizes were synthesised and used to generate in situ PEG dialdehydes for the crosslinking of partially reacetylated chitosan via Schiff reaction and hydrogenation of the aldimines. The watersoluble products obtained were instrumentally characterised. Upon freeze-drying, they aggregated to yield insoluble soft and spongy biomaterials, that swelled immediately upon contact with water. When exposed to papain and lipase, at physiological pH values, progressive dissolution of the biomaterials was observed, but no dissolution took place with lysozyme, collagenase and amylase. They were found to be biocompatible towards Caco-2 cells. These crosslinked partially acetylated chitosans seem suitable for medical items when prompt resorption is sought.59

21.3.3 Polyelectrolyte complex formation At high degree of protonation of the amine functions the cationic chitosan spontaneously forms macromolecular complexes by the reaction with anionic polyelectrolytes. These complexes are generally water insoluble and make hydrogel. Several reviews on polyelectrolytes60, 61 have been published so far. A variety of polyelectrolytes can be obtained by changing the chemical structure of component polymers, such as molecular weight, flexibility, functional group structure, charge density, hydrophilicity and hydrophobicity, stereoregularity, and compatibility, as well as changing reaction conditions, such as pH, ionic strength, polymer concentration, mixing ratio, and temperature. This, therefore, may lead to a diversity of physical and chemical properties of the complexes.

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Polyelectrolytes of chitosan with other polysaccharides, proteins, DNA and synthetic and inorganic polymers were investigated. A hydrogel with high sensitivity to the change in external pH was prepared between chitosan (DA = 0.18) and dextran sulfate:62 the maximum volume of the complex gel was observed in a dilute NaOH solution at pH10.5, and was about 300 times as large as the volume at pH values below 9. This behaviour, probably due to both equal and high densities of amino and sulfate groups and flexibility of anionic polymer chains, might be useful in various application fields.63 Microcapsules can be used for mammalian cell culture and the controlled release of drugs, vaccines, antibiotics and hormones. To prevent the loss of encapsulated materials, the microcapsules should be coated with another polymer that forms a membrane at the bead surface. A well-known system is the encapsulation of the alginate beads with poly-L-lysine. A covalently crosslinked superswelling sodium alginate gel type was also investigated64 for encapsulation of insulin producing cells. Because poly-L-lysine has some limitations due to the high cost and toxicity, systems of alginate beads coated with chitosan at different DA or its N-acyl derivates have been developed.65 For the application of the polyelectrolytes produced, the microencapsulation of guaifenesin was performed and the release of the drug was tested. The microcapsules were prepared in one step by the extrusion of a solution of the drug and sodium alginate into a solution containing calcium chloride and chitosan through interpolymeric ionic interactions. The drug release during the drug-loaded microcapsules storage in saline was found to depend on the pH where the microcapsules were formed and the kind of N-acyl groups introduced into the chitosan. Recently, calciuminduced alginate gel beads containing chitosan were prepared using nicotinic acid, in order to investigate the release of the vitamin and the uptake of bile acids into the complex, after oral administration.66 The possible applicability of chitosan treated alginate beads as a controlled release system of small molecular drugs with high solubility was investigated.67 The beads were prepared by the ionotropic gelation method and the effect of various factors (alginate, chitosan, drug and calcium chloride concentrations, the volume of external and internal phases and drying methods) on bead properties were also investigated. Spherical beads with 0.78–1.16 mm diameter range and 10.8–66.5% encapsulation efficiencies were produced. Higher encapsulation efficiencies and retarded drug release were obtained with chitosan-treated alginate beads. It appeared that chitosan-treated alginate beads may be used for a potential controlled release system of small molecular drugs with high solubility, instead of alginate beads. Various modifications of alginate-chitosan microcapsules were made by many authors among whom were Chandy et al.,68 such as the inclusion of polyethylene glycol and the use of crosslinkers such as carbodiimide and glutaraldehyde in the core and onto the microcapsule membrane surface. A characterisation of the modified microcapsules in terms of mechanical stability and albumin diffusion as well as their surface properties was performed. A mild glutaraldehyde treatment greatly enhanced the mechanical stability of the microcapsules, and this treatment did not affect the coating process of chitosan or polyethylene glycol. The biological response to such microcapsules was evaluated by microencapsulation of red blood cells and subsequent observation of their hemoglobin release. The encapsulated red blood cells in the polyethylene glycolglutaraldehyde coated microcapsules were found to be less hemolytic and had improved stability and biocompatibility. The results suggest the possibility of developing biological assist organs by microencapsulation of mammalian cells such as islets or liver cells in immunoisolative microcapsules in the near future.

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The conventional temporary wound dressing cannot be used in the presence of antimicrobial cream and ointment. In these cases, it is difficult for wound dressings to adhere to the wound surface due to their delayed wound healing. Patients feel pain because of the frequent replacement of the wound dressing. Therefore, the method of drug delivery system has been accepted for full-thickness skin wound care and antimicrobial agent-impregnated wound dressings are proven effective in controlling bacterial invasion through a porous matrix. In this regard, drug-impregnated polyelectrolytes wound dressing composed of chitosan and sodium alginate in sponge form were prepared;69 further advantages come from low toxicity of chitosan and its effect as a wound healing accelerator.1, 2 Alginate, as hydrophilic gel, provides a moist wound environment which promotes healing and epidermal regeneration, therefore it may be expected that wound healing of chitosan/alginate polyelectrolytes sponge will be promoted over chitosan or alginate itself. Chitosan alginate complexes might soon give way to oxychitin chitosan complexes that are now being developed based on the regiospecific oxidation of chitin at C6. These polyelectrolyte complexes are formed by polymers of opposite charges, both derived from the same parent polysaccharide, chitin.70,71 Complexes with type-B gelatin, -keratose and bovine atelocollagen are investigated, in function of pH, polymer ratio and ionic strength; complexation is often dependent on molecular weight of chitosan.72 It is well known that chitosan inhibits the growth of a wide variety of bacteria. In this respect, the potential of chitosan as an antifungal preservative for prolonging the storability of fresh produce was suggested.73 Chitosan, applied as a coating on the entire fruit, reduced the respiration rate of tomato, bell pepper, cucumber and strawberry fruits and the production of ethylene in tomato fruits. The film also delayed the ripening of tomato and strawberry fruits and reduced the desiccation of the fruits. This effect can be due to the penetration of chitosan into cells and its intracellular activity, following a complex formation with phosphate negative charges in the grooves of the DNA helix in the B form. Indeed, chitosan interacts very securely with DNA in solution and equivalent concentration of chitosan causes DNA to precipitate from solution.72 Chitosan/polyacrylic acid interpenetrating networks were prepared via radical polymerisation of acrylic acid, activated at low temperature, in an aqueous/alcoholic chitosan dispersion. Analytical data supported the existence of some PAA grafting on the reactive amine group of the chitosan. The degree of swelling of the membranes was highly dependent on pH and composition, showing a higher swelling of interpolyelectrolyte salt bonds.74

21.4

Conclusions

The good performance of chitosan itself and its derivatives in the dietary food area and in the pharmaceutical area, accompanied by the more thorough understanding of the chemistry of chitosan-based gels, leads one to expect that chitosan gels will have an expanding range of applications in the near future. This biopolymer, remarkable for its cationicity, has attained a unique position among the hydrocolloids.

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21.5 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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References (ed.), Chitosan per os: from Dietary Supplement to Drug Carrier. Atec, Grottammare, Italy, 2000. JOLLE`S, P. and MUZZARELLI, R. A. A. Chitin and Chitinases, Birkhauser Verlag, Basel, 1999. MUZZARELLI, R. A. A. and PETER, M. G. (eds), Chitin Handbook, Atec, Grottammare, Italy, 1997. JAPANESE SOCIETY FOR CHITIN AND CHITOSAN Chitin and Chitosan Handbook. Gihodo Shuppan Co, Tokyo, Japan, 1995. JEUNIAUX, C. Chitine et Chitinolyse. Masson, Paris, 1963. PETER, G. M., MUZZARELLI, R. A. A. and DOMARD, A. (eds) Advances in Chitin Science, Vol. 4, Univ. Potsdam, 2000. MUZZARELLI, R. A. A. Chitin, Pergamon, Oxford, 1977. MUZZARELLI, R. A. A. ‘Chitin Chemistry’ in: J. C. Salamone (ed.), The Polymeric Materials Encyclopedia, 312–314. CRC Press, Inc., Boca Raton FL, USA, 1996. MUZZARELLI, R. A. A. (ed.), Chitin Enzymology, Vol. 1, Atec Edizioni, Italy, 1993. MUZZARELLI, R. A. A. (ed.), Chitin Enzymology, Vol. 2, Atec Edizioni, Italy, 1996. MUZZARELLI, R. A. A. and MUZZARELLI, B. ‘Structural and functional versatility of chitins’ in S. Dumitriu (ed.), Structural Diversity and Functional Versatility of Polysaccharides. Marcel Dekker, New York, 1998. GOOSEN, M. F. A. (ed.), Applications of Chitin. Technomic, Lancaster USA, 1996. MUZZARELLI, R. A. A., STANIC, V. and RAMOS, V. ‘Enzymatic depolymerization of chitins and chitosans’ in C. Bucke (ed.), Carbohydrate Biotechnology Protocols, Humana Press, Totowa, 1999. MUZZARELLI, R. A. A. ‘Chitin’ in G. O. Aspinall (ed.), The Polysaccharides, Academic Press, New York, 1985, vol. 3. MUZZARELLI, R. A. A., JEUNIAUX, C. and GOODAY, G. W. (eds) Chitin in Nature and Technology, Plenum, New York, 1986. NEVILLE, A. C. Biology of the Arthropod Cuticle, Springer, Berlin, 1975. RICHARDS, A. G. The Integument of Arthropods, Univ. Minnesota Press, St. Paul, 1951. STEVENS, W. F., RAO, M. S. and CHANDRKRACHANG, S. Chitin and Chitosan. AIT, Bangkok, 1996. WOOD, W. A. and KELLOGG, S. T. (eds) Methods in Enzymology Vol. 161: Lignin, Pectin and Chitin, Academic Press, San Diego, 1988. ZIKAKIS, J. P. Chitin, Chitosan and Related Enzymes, Academic, London, 1984. DOMARD, A., JEUNIAUX, C., MUZZARELLI, R. A. A. and ROBERTS, G. A. F. (eds), Advances in Chitin Sciences, Jacques Andre´ Publ., Lyon, 1996. INOUE, Y. ‘NMR determination of the degree of acetylation’ in R. A. A. Muzzarelli and M. G. Peter (eds) Chitin Handbook, 133–136, Atec, Grottammare, Italy, 1997. TERBOJEVICH, M. and COSANI, A. ‘Molecular weight determination of chitin and chitosan’ in R. A. A. Muzzarelli and M. G. Peter (eds) Chitin Handbook, 87–101, Atec, Grottammare, Italy, 1997. ROBERTS, G. A. F. Chitin Chemistry, London, Macmillan Press, 1992. TERBOJEVICH, M., COSANI, A., FOCHER, B. and MARSANO, E. ‘High-performance gel-permeation chromatography of chitosan samples’, Carbohydr Res, 1993 250 301–14. BERI, R. G., WALKER, J., REESE, E. T. and ROLLINGS, J. ‘Characterization of chitosans via coupled sizeexclusion chromatography and multiple-angle laser light-scattering technique’ Carbohydr Res, 1993 238 11–26. OTTOY, H., VARUM, K. M., CHRISTENSEN, B. E., ANTHONSEN, M. W. and SMIDSROD, O. ‘Preparative and analytical size-exclusion chromatography of chitosans’ Carbohydr Polym, 1996 31 253–61. TERBOJEVICH, M., COSANI, A., FOCHER, B., NAGGI, A. and TORRI, G. ‘Chitosans from Euphausia superba. 1: Solution properties’ Carbohydr Polym, 1992 18 34–42. ALLAN, G. G. and PEYRON, M. ‘Molecular weight manipulation of chitosan. II. Prediction and control of extent of depolymerization by nitrous acid’ Carbohydr Res, 1995 277 273–82. ALLAN, G. G. and PEYRON, M. ‘Molecular weight manipulation of chitosan. I. Kinetics of depolymerization by nitrous acid’ Carbohydr Res, 1995 277 257–72. YALPANI, M. and PANTALEONE, D. ‘An examination of the unusual susceptibility of aminoglicans to enzymatic hydrolysis’ Carbohydr Res, 1994 256 (1) 159–75. KREGER, D. R. ‘Observations on cell walls of yeasts and some other fungi by x-ray diffraction and solubility tests’ Biochim Biophys Acta, 1954 13 1–9. DAVIES, L. L. and BARTNICKI-GARCIA, S. ‘Chitosan synthesis by the tandem action of chitin synthetase and chitin deacetylase from Mucor rouxii’ Biochemistry, 1984 23 (6) 1065–73. PETER, M. G. ‘Applications and environmental aspects of chitin and chitosan’ J Macromol Sci Pure & Appl Chem, 1995 A32 629–40. YALPANI, M. (ed.), Polysaccharides: Synthesis, Modifications and Structure/Property Relations, Amsterdam, Elsevier, 1988. MUZZARELLI, R. A. A. ‘Carboxymethylated chitins and chitosans’ Carbohydr Polym, 1988 8 (1) 1–21. ANTHONSEN, M. W., VARUM, K. M. and SMIDSROD, O. ‘Solution properties of chitosans: conformation and chain stiffness of chitosans with different degrees of N-acetylation’ Carbohydr Res, 1994 256 (1) 159–75. MUZZARELLI, R. A. A.

Chitosan 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67.

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and BIANCHI, E. ‘Chitosan: chain rigidity and mesophase formation’ Carbohydr Res, 1991 209 251–60. DESBRIE`RES, J., MARTINEZ, C. and RINAUDO, M. ‘Hydrophobic derivatives of chitosan: characterization and rheological behaviour’ Int J Biol Macromol, 1996 19 (1) 21–8. VARUM, K. M., OTTOY, M. H. and SMIDSROD, O. ‘Water-solubility of partially N-acetylated chitosans as the function of pH. Effect of chemical composition and depolymerization’ Carbohydr Polym, 1994 25 (2) 65– 70. RINAUDO, M. and DOMARD, A. ‘Solution properties of chitosans’ in G. Skjak-Braek, T. Anthonsen and P. Sandford (eds), Chitin and Chitosan, 71–86, London, Elsevier, 1989. HIRANO, S. and HORIUCHI, K. ‘Chitin gels’ Int J Biol Macromol, 1989 11 253–5. MI, F. L., SHYU, S. S., KUAN, C. Y., LEE, S. T., LU, K. T. and JANG, S. F. ‘Chitosan-polyelectrolyte complexation for the preparation of gel beads and controlled release of anticancer drug. I. Effect of phosphorous polyelectrolyte complex and enzymatic hydrolysis of polymer’ J Appl Polym Sci, 1999 74 1868–79. LAPASIN, R., STEFANCIC, S. and DELBEN, F. ‘Rheological properties of emulsions containing soluble chitosan’ Agro-food Ind High Tech, 1996 7 12–17. KOFUJI, K., SHIBATA, K., MURATA, Y., MIYAMOTO, E. and KAWASHIMA, S. ‘Preparation and drug retention of biodegradable chitosan gel beads’ Chem Pharm Bull, 1999 47 1494–6. QU, X., WIRSEN, A. and ALBERTSSON, A. C. ‘Synthesis and characterization of pH-sensitive hydrogels based on chitosan and D,L-lactic acid’ J Appl Polym Sci, 1999 74 3193–202. QU, X., WIRSEN, A. and ALBERTSSON, A. C. ‘Structural change and swelling mechanism of pH-sensitive hydrogels based on chitosan and D,L-lactic acid’ J Appl Polym Sci, 1999 74 3186–92. MUZZARELLI, R. A. A. and ILARI, P. (1994) ‘Chitosans carrying the methoxyphenyl function typical of lignin’ Carbohydr Polym, 1994 23 (3) 155–60. MUZZARELLI, R. A. A., ILARI, P., XIA, W., PINOTTI, M. and TOMASETTI, M. ‘Tyrosinase-mediated quinone tanning of chitinous materials’ Carbohydr Polym, 1994 24 294–300. SHAO, L. H., KUMAR, G., LENHART, J. L., SMITH, P. J. and PAYNE, G. F. ‘Enzymatic modification of the synthetic polymer polyhydroxystyrene’ Enzyme Microbial Technol, 1999 25 660–8. KUMAR, G., SMITH, P. J. and PAYNE, G. F. ‘Enzymatic grafting of a natural product onto chitosan to confer water solubility under basic conditions’ Biotechnol Bioeng, 1999 63 (2) 154–65. EDWARDS, W., LEUKES, W. D., ROSE, P. D. and BURTON, S. G. ‘Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation’ Enzyme Microbial Technol, 1999 25 769–73. EDWARDS, W., BOWNES, R., LEUKES, W. D., JACOBS, E. P., SANDERSON, R., ROSE, P. D. and BURTON, S. G. ‘A capillary membrane bioreactor using immobilized polyphenol oxidase for the removal of phenols from industrial effluents’ Enzyme Microbial Technol, 1999 24 (3–4) 209–17. MUZZARELLI, R. A. A., BARONTINI, G. and ROCCHETTI, R. ‘Immobilization of enzymes on chitosan columns: alpha-chymotrypsin and acid phosphatase’ Biotechnol Bioeng, 1976 18 1445–54. ARGUELLES-MONAL, W., GOYCOOLEA, F. M., PENICHE, C. and HIGUERA-CIAPARA, I. ‘Rheological study of the chitosan glutaraldehyde chemical gel system’ Polym Gels Networks 1998 6 (6) 429–40. ROBERTS, G. A. F. and TAYLOR, K. E. ‘Chitosan gels. 3. The formation of gels by reaction of chitosan with glutaraldehyde’ Makrom Chem, 1989 180 (5) 951–60. CRESCENZI, V., IMBRIACO, D., VELASQUEZ, C., DENTINI, M. and CIFERRI, A. ‘Novel types of polysaccharidic assemblies’ Macromol Chem Phys, 1995 196 (9) 2873–80. KHALID, M. N., HO, L., AGNELY, F., GROSSIORD, J. L. and COUARRAZE, G. ‘Swelling properties and mechanical characterization of a semi-interpenetrating chitosan/polyethylene oxide network – Comparison with a chitosan reference gel’ STP Pharma Sci, 1999 9 359–64. DAL POZZO, A., VANINI, L., FAGNONI, M., GUERRINI, M., DEBENEDITTIS, A. and MUZZARELLI, R. A. A. ‘Preparation and characterization of poly(ethyleneglycol)-crosslinked reacetylated chitosans’ Carbohydr Polym, 2000 42 201–6. TSUCHIDA, E. and ABE, K. ‘Interactions between macromolecules in solution and intermacromolecular complexes’ Adv Polym Sci, 1982 45 83–213. KUBOTA, N. and KIKUCHI, Y. ‘Macromolecular complexes of chitosan’ in S. Dumitriu (ed.), Polysaccharides Structural Diversity and Functional Versatility, 595–628, New York, Dekker, 1999. SAKIYAMA, T., TAKATA, H., KIKUCHI, M. and NAKANISHI, K. ‘Polyelectrolyte complex gel with high pHsensitivity prepared from dextran sulfate and chitosan’. J Appl Polym Sci, 1999 73 (11) 2227–33. JIANG, H., SU, W., BRANT, M., DE ROSA, M. E. and BUNNING, T.J. ‘Chitosan-based hydrogels: A new polymerbased system with excellent laser-damage threshold properties’ J. Polym Sci Part B-Polym Phys, 1999 37 (8) 769–78. OTTOY, M. H. and SMIDSROD, O. ‘Swelling of poly-L-lysine and chitosan-coated superswelling sodiumalginate beads’ Polym Gels Networks, 1997 5 307–14. LEE, K. Y., PARK, W. H. and HA, W. S. ‘Polyelectrolyte complexes of sodium alginate with chitosan or its derivatives for microcapsules’ J Appl Polym Sci, 1997 63 (4) 425–32. MURATA, Y., TONIWA, S., MIYAMOTO, E. and KAWASHINA, S. ‘Preparation of alginate gel beads containing chitosan nicotinic acid salt and the functions’, Eur J Pharm Biopharm, 1999 48 (1) 40–52. SEZER, A. D. and AKBUGA, J. ‘Release characteristics of chitosan treated alginate beads: II. Sustained TERBOJEVICH, M., COSANI, A., CONIO, G. MARSANO, E.

378 68. 69. 70. 71. 72. 73. 74.

Handbook of hydrocolloids release of a low molecular drug from chitosan treated alginate beads’, J Microencaps, 1999 16 687–96. CHANDY, T., MOORADIAN D. L. and RAO, G. H. R. ‘Evaluation of modified alginate-chitosan-polyethylene glycol microcapsules for cell encapsulation’ Artific Organs, 1999 23 894–903. KIM, H., LEE, H., OH, J., SHIN, B., OH, C., PARK, R., YANG, K. and CHO, C. ‘Polyelectrolyte complex composed of chitosan and sodium alginate for wound dressing application’ J Biomater Sci Polymer Edn, 1999 10 (5) 543–56. MUZZARELLI, R. A. A., MUZZARELLI, C., COSANI, A. and TERBOJEVICH, M. ‘6-Oxychitins, novel hyaluronanlike polysaccharides obtained by regioselective oxidation of chitins’ Carbohydr Polym, 1999 39 361–7. MUZZARELLI, R. A. A., MILIANI, M., CARTOLARI, M., GENTA, I., PERUGINI, P., MODENA, T., PAVANETTO, F. and CONTI, B. ‘Pharmaceutical use of the 6-oxychitin-chitosan polyelectrolyte complex’. STP Pharma Sciences, 2000 10 51–6. KUBOTA, N. and KIKUCHI, Y. ‘Macromolecular complexes of chitosan’ in S. Dumitriu (ed.), Polysaccharides Structural Diversity and Functional Versatility, 595–628, New York, Dekker, 1999. ARUL, J. and EL GHAOUTH, A. ‘Preservation of fresh fruit and vegetables with chitosan’ in Domard, A., Jeuniaux, C., Muzzarelli, R. A. A. and Roberts G. A. F. (eds), Advances in Chitin Sciences, 372–80, Jacques Andre´ Publ, Lyon, 1996. PENICHE, C., ARGUELLES-MONAL, W., DAVIDENKO, N., SASTRE, R., GALLARDO, A. and SAN ROMAN, J. ‘Selfcuring membranes of chitosan/PAA IPNs obtained by radical polymerization: preparation, characterization and interpolymer complexation’ Biomaterials, 1999 20 (20) 1869–78.

22 Alginates K. I. Draget, Norwegian University of Science and Technology

22.1

Introduction

As structural components in marine brown algae (Phaeophyceae) and as capsular polysaccharides in soil bacteria, alginates are quite abundant in nature. The industrial production is roughly 30,000 metric tons annually, being probably less than 10% of the annually bio-synthesised material in the standing macroalgae crops. As macroalgae also may be cultivated – as in mainland China – and as production by fermentation is technically possible (although not economically feasible at the moment), the sources for industrial production of alginate may be regarded as unlimited even for a steadily growing industry. As already mentioned, the biological function of alginate in brown algae is generally believed to be as a structure-forming component. The intercellular alginate gel matrix gives the plants both mechanical strength and flexibility.1 This relation between structure and function is reflected in the compositional difference of alginates in different algae or even between different tissues from the same plant (see Section 22.3.2). In Laminaria hyperborea, an algae which grows in very exposed coastal areas, the stipe and holdfast have a very high content of guluronic acid, giving high mechanical rigidity (see Section 22.6.1). The leaves of the same algae which float in the streaming water have an alginate characterised by a lower G-content giving a more flexible texture. The biological function of alginate in bacteria is not fully understood. It has been shown2 that alginate production is required for cyst formation in Azotobacter vinelandii. Cysts are metabolic dormant cells, characterised by having several layers of polysaccharide material around the cell. This polysaccharide coating protects the cells from desiccation and mechanical stress. Under favourable conditions, including the presence of water, the polysaccharide coating will swell and the cysts germinate, divide and regenerate to vegetative cells.2 The structural significance of alginates in the formation of microcysts by A. vinelandii does not explain the abundant production of exopolymer by vegetative cells under conditions not favouring cyst formation, neither does it explain the role of alginate in Pseudomonades.3 It is therefore reasonable to believe that alginate (as with other microbial exo-polysaccharides) has no single function

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for the vegetative cells itself, but rather provides the cells with a multitude of protective properties under various environmental conditions. Pharmaceutical, food and technical applications (such as in print paste for the textile industry) are the quantitative main market areas for alginates. There is also a large and growing potential for alginate in biotechnological applications. The latter is mostly connected to high value applications such as encapsulation of living cells for in vitro or in vivo use.4, 5 This type of application has been a driving force for research aimed at understanding structure-function relationships in alginates at an increasingly detailed level. Basic knowledge gained from biotechnological activities has made alginate one of the best characterised and well understood gelling polysaccharides. Focus in this chapter will be put on giving an overview of the understanding of structure-function relationships of the alginate system with emphasis on existent and potential gelling methodology and how to control these. As any potential application will have to fit within a framework with boundaries given by alginate functionality, some focus has also been put on chemical and physical limitations of alginates such as solubility and stability.

22.2

Manufacture

Alginate was first described by the British chemist E. C. C. Stanford in 1881,6 and exists as the most abundant polysaccharide in the brown algae comprising up to 40% of the dry matter. It is located in the intercellular matrix as a gel containing sodium, calcium, magnesium, strontium and barium ions.7 It is because of its ability to retain water, and its gelling, viscosifying and stabilising properties, that alginate is widely used industrially. Several bacteria also produce alginate exocellularly,3, 8, 9 and Azotobacter vinelandii has been evaluated as a source for industrial production. But at present, all commercial alginates are extracted from algal sources. The extraction of alginate from algal material is schematically illustrated in Fig. 22.1. Because alginate is insoluble within the algae with a counterion composition determined by the ion exchange equilibrium with seawater, the first step in alginate production is an ion-exchange with protons by extracting the milled algal tissue with 0.1–0.2 M mineral acid. In the second step, the alginic acid is brought into solution by neutralisation with alkali such as sodium carbonate or sodium hydroxide to form the water soluble sodium alginate. After extensive separation procedures such as sifting, floatation, centrifugation and filtration to remove algal particles, the soluble sodium alginate is precipitated directly by alcohol, by calcium chloride or by mineral acid, converted to the sodium form if needed and finally dried and milled. Besides Na-alginate, other soluble alginates are produced such as the potassium and ammonium salts. The only derivative of alginates today having a commercial value, is the propylene glycol alginate (PGA). This product is processed by an esterification of alginate with propylene oxide. PGA is used in beers and salad dressings due to its higher solubility at low pH. Following the increased popularity of alginate as an immobilisation matrix, Pronova Biomedical A/S now commercially manufactures ultrapure alginates highly compatible with mammalian biological systems. These qualities are low in pyrogens, and facilitate sterilisation of the alginate solution by filtration due to low content of aggregates.

Alginates

Fig. 22.1

22.3

381

Principal scheme for the isolation of alginate from seaweeds.

Chemical and physical properties

22.3.1 Composition and sequence Alginate is a family of unbranched binary copolymers of (1!4) linked -D-mannuronic acid (M) and -L-guluronic acid (G) residues (see Fig. 22.2(a) and (b)) of widely varying composition and sequence. The first information about the sequential structure of alginates came from the work by Haug et al.7, 10–13 By partial acidic hydrolysis and fractionation, they were able to separate alginate into three fractions of widely differing composition. Two of these contained almost homopolymeric molecules of guluronic and mannuronic acid, respectively, while a third fraction consisted of nearly equal proportions of both monomers, and was shown to contain a large number of MG dimer residues. It was concluded that alginate was a true block copolymer composed of homopolymeric regions of M and G, termed M- and G-blocks, respectively, interspersed with regions of alternating structure (MG-blocks; see Fig. 22.2(c)). In a series of papers14–16 it was shown that alginates have no regular repeating unit. Furthermore, the distribution of the monomers along the polymer chain cannot be described by Bernoullian statistics. Hence, knowledge of the monomeric composition is not sufficient to determine the sequential structure of alginates. By simulating random depolymerisation and comparing the oligomer distribution with experimental data,15 the results indicated that a second order Markov model seems to be required for a general description of monomer sequence in alginates. More detailed information about the structure became available following the introduction of high resolution 1H and 13C NMR-spectroscopy17–20 in the sequential analysis of alginate. These powerful techniques have made it possible to determine the monad frequencies FM and FG, the four nearest neighbouring (diad) frequencies FGG, FMG, FGM, FMM, and the eight next nearest neighbouring (triad) frequencies. Knowledge

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Fig. 22.2

Structural characteristics of alginates: (a) alginate monomers, (b) chain conformation, (c) block distribution.

of these frequencies enable, for example, the calculation of the average G-block length larger than 1: NG > 1 = ( FGÿFMGM) / FGGM. This value has been shown to correlate well with gelling properties (see Section 22.6.1). It is important to realise that in an alginate chain population, neither the composition nor the sequence of each chain will be alike. This results in a composition distribution of a certain width.

22.3.2 Source dependence Commercial alginates are produced mainly from Laminaria hyperborea, Macrocystis pyrifera, Laminaria digitata, Ascophyllum nodosum, Laminaria japonica, Eclonia maxima, Lessonia nigrescens, Durvillea antarctica and Sargassum spp. Table 22.1 gives some sequential parameters (determined by high field NMR-spectroscopy) for samples of these alginates. The composition and sequential structure may, however, vary according to seasonal and growth conditions.7, 21 Generally, a high content of -L-guluronic acid is found in alginate prepared from stipes of old Laminaria hyperborea plants. Alginates from A. nodosum, L. japonica and Macrocystis pyrifera are characterised by a low content of G-blocks and a low gelstrength (see Section 22.6.1). It is interesting to see how nature can tailor-make alginate to give different strengths and required flexibility to different plants and tissues.22 Alginates with more extreme compositions can be isolated from bacteria23 which can contain up to 100% mannuronate. Bacterial alginates are also commonly acetylated. Alginate with a very high content of guluronic acid can be prepared from special algal tissues such as the outer cortex of old stipes of L. hyperborea (see Table 22.1), by chemical fractionation13, 24 or by enzymatic modification in vitro using mannuronan C-5 epimerases from A. vinelandii.23 This family of enzymes is able to epimerise M-units into G-units in different patterns from almost strictly alternating to very long G-blocks. The

Alginates Table 22.1

383

Composition and some sequential parameters of algal alginates

Source

FG

FM

FGG

FMM

FGM,MG

Laminaria japonica L. digitata L. hyperborea, leaf L. hyperborea, stipe L. hyperborea, outer cortex Lessonia nigrescens Ecklonia maxima Macrocystis pyrifera Durvillea antarctica Ascophyllum nodosum, fruiting body Ascophyllum nodosum, old tissue

0.35 0.41 0.55 0.68 0.75 0.38 0.45 0.39 0.29 0.10 0.36

0.65 0.59 0.45 0.32 0.25 0.62 0.55 0.61 0.71 0.90 0.64

0.18 0.25 0.38 0.56 0.66 0.19 0.22 0.16 0.15 0.04 0.16

0.48 0.43 0.28 0.20 0.16 0.43 0.32 0.38 0.57 0.84 0.44

0.17 0.16 0.17 0.12 0.09 0.19 0.32 0.23 0.14 0.06 0.20

epimerases from A. vinelandii have been cloned and expressed, and they represent at present a powerful new tool for tailoring of alginates. It is also obvious that commercial alginates with less molecular heterogeneity, with respect to chemical composition and sequence, can be obtained by a treatment with one of the C-5 epimerases.23

22.3.3 Molecular weight Alginates, like polysaccharides in general, are polydisperse with respect to molecular weight. In this aspect they resemble more synthetic polymers than other biopolymers like proteins and nucleic acids. This may result from two different causes: (a) polysaccharides are not coded for in the DNA of the organism, but are synthesised by polymerase enzymes, and (b) during extraction there is a substantial depolymerisation of the polymer. Due to this polydispersity, the ‘molecular weight’ of an alginate becomes an average over the whole distribution of molecular weights. There are several methods for averaging the molecular weight, the two most common are the number-average, M n (which weighs the polymer molecules according to the number of molecules in a population having a specific molecular weight), and the weightaverage, M w (which weighs the polymer molecules in a population according to the weight of molecules having a specific molecular weight). The fraction M w =M n is called the polydispersity index (P.I.). A P.I. of less than 2.0 suggests that some fractionation has occurred during the production process. Precipitation, solubilisation, filtration, washing or other separating procedures may have caused loss of the high or the low molecular weight tail of the distribution. A P.I. of more than 2.0 indicates a wider distribution. This suggests mixing of products of different molecular weights to obtain a sample of a certain average molecular weight (viscosity) or that a non-random degradation of the polymer has occurred during the production process or in the raw material prior to extraction. Mixing, or more precisely ‘blending’, is a common method for alginate (and polysaccharides in general) manufacturers to reach a viscosity targeted product. In extreme cases, this implies that virtually no molecules in an alginate blend have the average molecular weight obtained from viscosity experiments; only higher and lower. The molecular weight distribution can have implications for the uses of alginates, as lowmolecular weight fragments containing only short G-blocks may not take part in gel network formation and consequently not contribute to the gel strength. Also, in some high-tech applications, the leakage of mannuronate-rich fragments from alginate gels

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may cause problems,25, 26 and a narrow molecular weight distribution is advantageous (see Section 22.5).

22.3.4 Selective binding of ions The ion-binding properties of alginates represent the basis for their gelling properties. Alginates show characteristic ion-binding properties in that their affinity for multivalent cations depends on their composition.7 The characteristic affinities are a property exclusive to polyguluronate; polymannuronate is almost without selectivity. The affinity of alginates for alkaline earth metals increase in the order Mg << Ca < Sr < Ba; a property unique for alginates compared to other polyanions. The only polyanion resembling alginates in this fashion is pectic acid, whose affinity follows the scheme Mg << Ca, Sr < Ba. From experiments involving equilibrium dialysis of alginate,7, 27–30 it was again found that the selectivity for alkaline earth metals and transition elements increased markedly with increasing content of -L-guluronate residues in the chains, and that polymannuronate blocks and alternating blocks were almost without selectivity. The high selectivity between as similar ions as the alkaline earth metals indicate that the mode of binding cannot be by non-specific electrostatic interaction only, but that some chelation caused by structural features in the G-blocks have to be responsible for the selectivity. This characteristic property was eventually explained by the so-called ‘egg-box’ model,31 based upon the linkage conformations of the guluronate residues. Although other, more accurate steric arrangements have been suggested, supported by Xray diffraction32 and NMR spectroscopy,33 the simple ‘egg-box’ model still persists, as it may be regarded as giving an intuitive understanding of the characteristic chelate-type ion-binding properties of alginates. The simple dimerisation in the ‘egg-box’ model is, however, at present under scrutiny. This is partly based on data from small-angle X-ray scattering on alginate gels suggesting lateral association far beyond a pure dimerisation with increasing [Ca2+] and G-content of the alginate,34 and partly from the fact that isolated and purified G-blocks (totally lacking elastic segments; typically DP = 20) are able to act as gelation modulators when mixed with a gelling alginate (see Section 22.6.1).

22.4

Limitations

22.4.1 Solubility In general, there are three essential parameters which determine and limit the solubility of alginates in water. The pH of the solvent is important due to the necessity of electrostatic charges on the uronic acid residues. The ionic strength of the medium also plays an important role (salting-out effects of non-gelling cations). And, obviously, the context of gelling ions in the solvent also limits the solubilisation of alginates. In the latter context, the ‘hardness’ of the water (i.e. the content of Ca2+ ions) is most likely to be the main problem. By potentiometric titration7 it was found that the dissociation constants for mannuronic and guluronic acid monomers were 3.38 and 3.65 respectively. The pKa value of the alginate polymer differs only slightly from those of the monomeric residues.7 Alginate solutions may behave in two fundamentally different ways when the pH in the solution is lowered. An abrupt decrease in pH causes a precipitation of alginic acid molecules, while a slow and controlled release of protons may result in formation of an

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385

‘alginic acid gel’ (see Section 22.6.2). Precipitation of alginic acid molecules has been extensively studied,7, 35–37 and addition of acid to an alginate solution leads to a precipitation within a relative narrow pH range. This pH range depends on the molecular weight of the alginate but definitely also on the chemical composition and sequence. For example, will alginates containing more of the ‘alternating’ structure (MG-blocks) precipitate at lower pH values compared to the alginates containing more homogeneous block structures (poly-M and poly-G)? The presence of homopolymeric blocks thus seems to favour precipitation by formation of crystalline regions stabilised by hydrogen bonds. By increasing the degree of ‘disorder’ in the alginate chain, like in alginates isolated from Ascophyllum nodosum (see Table 22.1), these crystalline regions are not formed as easily. A certain alginate fraction from A. nodosum is soluble at all pH values down to pH1.4.36 As already mentioned, propylene glycol alginate (PGA) is applied as a food stabiliser under acidic conditions. Generally, any change of ionic strength in an alginate solution has a profound effect on polymer behaviour, especially on polymer chain extension and hence also the solution viscosity. At high ionic strengths, also the solubility is affected. Alginate may be precipitated and fractionated to give a precipitate enriched in mannuronate residues by high concentrations of inorganic salts like potassium chloride.38, 39 Salting-out effects like this exhibit large hysteresis in the sense that less than 0.1M salt is necessary to slow down the kinetics of the dissolution process and hence limit the solubility.40 The drive of the dissolution process of alginate in water is most probably the gradient in the chemical potential of water between the bulk solvent and the solvent in the alginate particle due to a very high counter-ion concentration in the particle. This drive becomes severely reduced when alginate is attempted to be dissolved in an aqueous solvent already containing ions. If alginates are to be applied at high salt concentrations, the polymer should first be fully hydrated in pure water followed by addition of salt under shear. For the swelling behaviour of dry alginate powder in aqueous media with different concentrations of Ca2+, there seems to be a profound limit at approximately 3mM free calcium ions (unpublished results). Below 3mM Ca2+, almost all of the alginate is found within the supernatant, whereas almost no alginate (1–3%) is found in solutions above 3mM free calcium ions. This limit is not likely to depend on alginate concentration since the system under consideration is swelling of dry alginate powder. Alginate can be solubilised at [Ca2+] above 3mM by adding complexing agents, like polyphosphates or citrate, prior to adding the alginate powder.

22.4.2 Stability Dry powder of pure sodium alginate may have a shelf life of several months provided it is stored in a dry, cool place without exposure to sunlight. In the deep freezer, sodium alginate may be kept for several years without significant reduction of molecular weight. In contrast, dried alginic acid has a very limited stability at ordinary temperatures due to an intramolecular acid catalysed degradation.41 For many uses, it is important to be aware of the factors which determine and limit the stability of aqueous alginate solutions and the chemical reactions responsible for the degradation. The relative viscosity of an alginate solution may be severely reduced over a short period of time under conditions favouring degradation. The glycosidic linkages are susceptible to both acid and alkaline degradation and oxidation by free radicals. And because alginates are natural products, many micro-organisms digest alginates. Extracellular alginate lyases have been isolated from different marine and soil bacteria.42

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As function pH, the degradation is at its minimum around neutrality and increases in either direction. The increased instability at pH values less than 5 can be attributed to proton catalysed hydrolysis, whereas the reaction responsible for the degradation at pH10 and above is the -alkoxy-elimination.43, 44 An esterified alginate (like propylene glycol alginate) will have degradation rates 104–105 times that of the unsubstituted alginate because an esterification increases the electron-attracting effect of the carbonyl group at C-6 which in turn increases the rate of removal of H-5, the first step of the -elimination reaction. Any physical property depending directly on molecular weight will be more severely affected by chain breaks the higher the molecular weight of the starting material. The alginate producers are aware of this connection and will recommend higher concentrations of medium to low viscosity (molecular weight) products if an application involves the risk of polymer degradation. Degradation can also be significant even at pH values around neutrality. Most brown algae contain varying amounts of phenolic compounds which are extracted together with the alginate and represent a contamination in most commercial alginates. The amount will vary with the algal species, e.g. L. digitata alginates contain very small amounts of these phenolic compounds compared to A. nodosum alginates. The degradation in solutions of alginates can be diminished by reducing the content of phenols by making the phenols insoluble in the algae by crosslinking with formaldehyde prior to extraction.45 The degradation effect of phenolic compounds belongs to the class of ORD reactions (Oxidative-Reductive Depolymerisation). This effect is a result of autooxidation of reducing compounds with a subsequent formation of peroxides. This autooxidation increases with the amount of dissolved oxygen and with the pH in the solution. Degradation is caused by free radicals, probably hydroxyl radicals, formed in reactions between the reducing compound and peroxides.46–48 It is important to be aware of the potential impact reducing compounds may have at physiological conditions where polysaccharides and other natural polymers are regarded as being completely stable. For alginates, it is important to know that the phenolic content of a commercial alginate varies with the algal source from which the alginate is isolated and the isolation procedure. It is also possible to remove most of the phenolics with simple purification procedures.49 Sterilisation of alginate solutions and powder almost always causes depolymerisation, loss of viscosity and possibly also reduced gel strength. It has been shown50 that sterilisation processes as diverse as heating (autoclaving), ethylene oxide treatment and

-irradiation all lead to polymer breakdown. Heating of an alginate solution will inevitably enhance polymer breakdown because this process increases the reaction rate of all the depolymerisation processes outlined above. In addition, thermal degradation (homolysis) may take place at high temperatures. For immobilisation purposes, sterilefiltering has been recommended rather than autoclaving to preserve the alginate solution’s original viscosity.51 The effect of -irradiation on both solutions and dry powder of alginate is often disastrous. It has been observed that an applied standard sterilisation dose of 2.32Mrad from a Co-60 source on dry alginate powder reduces the relative viscosity of the resulting solution by 95% with an almost complete loss of gelling capacity.50 It is generally believed that under these conditions Oÿ is formed which in turn can lead to the formation of the very 2_ reactive OH free radical.52 The O2 level gets depleted very quickly under irradiation, and when the period of irradiation extends up to 1.5 hours, additional O2 may diffuse into the sample and increase the amount of free radicals. An electron accelerator could be an alternative to Co-60 as irradiation source because the dose is applied very quickly

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387

(typically within 1 minute) compared to 1.5 hours for the Co-60 source. Hence, the amount of free radicals can be dramatically reduced. It can be deduced that sterilisation of alginate solutions and dry powder is not straightforward. But if it is known which kind of effects the chosen sterilisation method gives and which properties the sterile alginate or alginate product should have, it is possible to overcome this problem by choosing a high viscosity (high Mw) alginate sample which retains functional properties after sterilisation.

22.5

Properties and applications of alginate in the liquid phase

Due to the unique cold setting property of alginate gels, one generally tends to look upon applications of alginate as being mainly in the gel state. This is only partly true; there is, for example, a quantitatively important market for alginate as a shear-thinning viscosifier in printing paste for the textile industry. There are also abundant uses of alginate in liquid foods where properties like viscosity enhancement and stabilisation of aqueous mixtures, dispersions and emulsions are exploited. Furthermore, it has recently been established that the alginate molecule itself, with its high variety of possible chemical compositions and molecular weights, has different effects on e.g. biological systems and gives different technological properties in the liquid phase. A biological effect of alginate was indeed suggested in the first animal transplantation trials of encapsulated Langerhans islets for diabetes control. Here, overgrowth of alginate capsules by phagocytes and fibroblasts, resembling a foreign body/inflammatory reaction, was reported.53 Induction of tumour necrosis factor (TNF) and interleukin 1 (IL-1), both cytokines having pro-inflammatory activities, in bioassays showed that the inducibility depended upon the content of mannuronate in the alginate sample.54 This result also directly explained the observed capsule overgrowth; mannuronate-rich fragments, not taking part in the gel network, leached out of the capsules and directly triggered an immune response.55 The presence of immunologic response can, at least partly, be linked to (1!4) glycosidic linkages since also other homopolymeric di-equatorial polyuronates, like D-glucuronic acid (C6-oxidised cellose), also exhibit this feature.56 In vivo animal models have now revealed the immunologic potential of polymannuronate in such diverse areas as protection against lethal bacterial infections and irradiation, and for increasing non-specific immunity.56

22.6

Gels and gelling technologies

In contrast to most gelling polysaccharides, alginate gels have the particular feature of being cold setting. In principle, this implies that alginate gels set more or less independent of temperature. The kinetics of the gelling process may, however, be strongly modified by a change in temperature. The properties of the final gel will also change if gelation occurs at different temperatures. This is due to alginates being nonequilibrium gels and thus being dependent upon the history of formation.29 Another implication of thermoirreversibility is that alginate gels are heat stable. In practice, this means that alginate gels can be heat treated without melting. This is the reason why alginates are used in baking creams. It should, however, be kept in mind that alginates, as outlined above, are subject to chemical degrading processes. A prolonged heat treatment at low or high pH may thus destabilise the gel.

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Fig. 22.3

Parameters controlling the kinetics and final properties of alginate gels.

One should generally look into the application of alginate gels as consisting of two stages with different optimisation criteria. First, the gelling kinetics should be considered and optimised against the manufacturing process. Since alginate gelation cannot be controlled easily by temperature, the importance of other parameters must be evaluated. The most important factors would be alginate concentration and chemical composition and sequence, the ratio between gelling and non-gelling ions and the presence of complexing agents such as phosphates or citrate as illustrated in Fig. 22.3. It is also important to keep in mind that alginate is a poly-electrolyte, implying that under favourable conditions, alginates may interact electrostatically with other charged polymers (e.g. proteins) in mixed systems resulting in a phase transition or an increase in viscosity. These types of interactions can be utilised to stabilise and increase the mechanical properties of, e.g., some restructured foods. In studies involving gelling of bovine serum albumin (BSA) and alginate on both sodium and calcium form, a considerable increase in Young’s modulus was found within some range of pH and ionic strength.57, 58 These results suggest electrostatic interactions to be the main driving force for the observed strengthening effects. On the other hand, if the purpose is to avoid such electrostatic interactions, the mixing of alginate and protein should take place at relatively high pH where most proteins have a net negative charge. Secondly, the properties of the resulting gel are to be optimised against the desired specifications of the product. This includes parameters such as modulus, elasticity, brittleness and syneresis (‘ageing’ of the gel).

22.6.1 Ionic crosslinking Due to the very rapid and irreversible binding reaction between multivalent cations and alginates, a direct mixing of these two components rarely produces homogeneous gels. The most likely result of such mixing is a dispersion of gel lumps (‘fish-eyes’). The only exception is if a low molecular weight alginate is mixed with low amounts of crosslinking ions at high shear. This produces a weak gel which may be applied in some food products.

Alginates

Fig. 22.4

389

The two principal methods for the manufacture of alginate gels: (a) diffusion setting, (b) internal gelling.

For most gelling purposes, ability to control the introduction of the crosslinking ions is essential. This control is possible by the two fundamentally different methods of preparing an alginate gel: the diffusion method and the internal setting method. The diffusion method is characterised by letting a crosslinking ion (e.g. Ca2+) diffuse from an outer reservoir into an alginate solution (Fig. 22.4(a)). Internal setting (sometimes also referred to as in situ gelation) differs from the former in that the Ca2+ ions are released in a controlled fashion from an inert calcium source within the alginate solution (Fig. 22.4(b)). Controlled release is usually obtained by a change of pH and/or by a limited solubility of the calcium salt source. Ionic cross-linking always implies an ion exchange process; the starting point is a water soluble alginate with, e.g., sodium or potassium ions and these counter-ions must be exchanged with Ca2+ to give a sol/gel transition. It can be observed that, under some given circumstances, gelling kinetics and gel properties may depend on the type of counterion. In one study, the effect of different monovalent alginate counterions was studied in sol/gel transition and at apparent equilibrium for Ca-alginate gels.59 At

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identical conditions, K-alginates were generally found to undergo faster sol/gel transitions compared to Na-alginates, and at apparent equilibrium and at low Ca2+ concentration, Na-alginate gels showed reduced elastic moduli compared to the Kalginates. The relative differences both in sol/gel transition kinetics and modulus at apparent equilibrium increased with increased content of guluronic acid residues in the alginate sample. Furthermore, increasing the amount of internally released Ca2+ resulted in equal elastic properties at apparent equilibrium, suggesting that dissimilarities in ion exchange reactions between Ca2+/Na+ and Ca2+/K+ is the cause of the differences at low Ca2+ levels.59 Another and rather new discovery is that if highly purified G-blocks are removed from a ‘normal’ alginate molecule by acid hydrolysis and subsequently refined by fractionation methods, these molecules (typically DPn = 20) are able to modify both gelling kinetics and apparent equilibrium properties when mixed with an ordinary alginate.60, 61 The observed effects were higher gel strengths at high Ca2+ concentrations, reduced sol/gel transitional kinetics and a considerably reduced degree of syneresis. These modifications are obtained without rendering the viscosity of the solution prior to gelling since these low DP molecules will not contribute to viscosity. Higher gel strengths are rather unexpected since these molecules do not have elastic segments. A reasonable explanation is that the pure G-blocks act as some kind of glue between sub-optimally located Gblocks within the gelling alginate. This new method suggests control of alginate-based gelling systems on a new level. Diffusion setting This method has gained great popularity as an immobilisation technique, and is used in several methods for the restructuring of foods such as artificial berries, pimiento strips and onion rings.62 Diffusion setting is characterised by rapid gelling kinetics, and this high-speed setting is indeed utilised for immobilisation purposes where each droplet of alginate solution makes one single gel bead entrapping the active agent. Rapid gelling is also beneficial in the restructuring of foods when a given size or shape is to be achieved. It has been shown that as long as alginates with an average molecular weight above 100kDa are used, the molecular weight dependence in this system is negligible.30 The resulting gel strength is, however, strongly dependent on chemical composition and sequence. As already mentioned, a good correlation is found between gel strength and the average G-block length larger than 1 (NG > 1) as shown in Fig. 22.5. An important feature in the diffusion setting method is that the final gel can exhibit an inhomogeneous distribution of alginate, with the highest concentration at the surface and gradually decreasing towards the centre of the gel. Extreme alginate distributions have been reported63 with a fivefold increase at the surface (from the concentration in the alginate solution prior to gelation) and virtually zero concentration in the centre. This result has been explained by the fact that diffusion setting will create a sharp gelling zone moving from the surface towards the centre of the gel. The activity of alginate (and of the gelling ion) will equal zero in this zone, and alginate molecules will diffuse from the internal, non-gelled part of the gelling body towards the zero activity region.63, 64 It is important to know that homogeneity can be controlled and the parameters governing the final alginate distribution. Maximum inhomogeneity is reached by letting a low molecular weight alginate gel at low concentration of the gelling ion and in the absence of non-gelling ions. Maximum homogeneity is reached by gelling a high molecular weight alginate at high concentrations of both gelling and non-gelling ions.65 The importance of the non-gelling ions in alginate gelling systems is also observed in the

Alginates

Fig. 22.5

391

Elastic properties of alginate gels as function of average G-block length.

stability of the gels. It has been shown that alginate gels start to swell markedly when the ratio between non-gelling and gelling ions becomes too high, and that the observed destabilisation increases with decreasing FG.66 Internal setting As outlined earlier, this system is based on an addition of an inactive form of the crosslinking ion into an alginate solution. In the case of calcium the insoluble CaCO3 or the slightly soluble CaSO4 may be used, or the Ca2+ ions may be complexed in a chelating agent (EDTA, citrate, etc.). The activation of the crosslinking ions is usually linked to a change in pH caused by the addition of organic acids or lactones. Lowering of the pH readily releases Ca2+ from CaCO3 and complexing compounds. Chelating agents do, however, have discrete pH ranges where the complexed ions are released; in the case of EDTA, pH has to be lowered to around 4.0 to obtain a release of calcium ions. By using salts like CaCO3 and CaSO4, gels can be prepared over a much wider pH range.67 The main difference between internal and diffusion setting is the gelling kinetics. With internal setting, the tailor-making of an alginate gelling system towards a given manufacturing process is possible. For example, in the alginate/CaCO3/D-glucono-lactone (GDL)-system, reducing the average particle size of the carbonate, and thereby increasing the total surface available to the acid, reduces the transition time.67 The modulus of the final gel, however, approaches the same value independent of gelling kinetics. Other mediators can be necessary for the control of gelling kinetics. In the case of CaSO4, the solubility is so high that gelation would occur spontaneously if complexing agents such as polyphosphates were not present. Internal setting almost always gives homogeneous gels. An exception is when large particle size calcium salt is used in combination with a low molecular weight alginate. In a combination like this, inhomogeneities can be observed due to salt sedimentation in a low-viscosity solution.67 The gel strength of internally set alginate gels depends more on molecular weight than diffusion set gels.68 Whereas gels made by the latter method describe almost a step function where gel strength gets independent of molecular weight at around 100kDa30

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(weight average degree of polymerisation, DPw 500), the internally set gels still depend on molecular weight even at 300kDa (DPw 1500). This is, at least partly, due to the fact that the internally set gels are more calcium limited compared to the gels made by diffusion, which means that the non-elastic fraction (sol and loose ends) will be higher in the internally set gels at a given molecular weight. Observations have shown that the internally set gels are more exposed to syneretic effects than gels set by diffusion. As a rule of the thumb, [Ca2+] = 0.5[G] represents the limit at which syneresis becomes prominent in internally set gels.67 There is no detailed understanding of this difference at the moment, but part of the explanation is certainly due to the different modes of gelation. As outlined earlier, diffusion setting gives a gelling zone which moves towards the centre of the gelling body. Here, the alginate molecules become saturated with Ca2+ and their activity drops towards zero. Internal setting implies a process where gelation starts simultaneously at a large number of locations. This puts some topological strains on the alginate molecules, but their activity and translational mobility do not equal zero. One can therefore imagine that after the primary gel network has been formed, there will still exist elastic segments with free Gblocks that could create new junction zones given the proximity of another free G-block and the presence of calcium ions. If the concentration of Ca2+ increases, a second class of junction zones may therefore be formed which will contract the gel network resulting in volume reduction.

22.6.2 Alginic acid gels It has been known and utilised for several decades that alginates precipitate at pH below the pKa value.7 In fact, the discovery that alginates were block co-polymers originated from the discovery that the different types of blocks had different solubilities at low pH.10 It is also well known that, under controlled conditions, alginates may form acid gels at these low pH. These acid gels are, however, far less studied than the ionically crosslinked alginate gels, and with the exception of some pharmaceutical recipes, the number of applications is rather limited. The preparation of an alginic acid gel has to be performed with care. Direct addition of a mineral acid to, e.g., a Na-alginate solution leads to a an instantaneous precipitation rather than a gel. pH must therefore be lowered in a controlled fashion, and this is carried out most conveniently by the addition of slowly hydrolysing lactones like D-glucono--lactone (GDL). It has been shown69 that gel strength of acid gels prepared by this method becomes independent of pH below 2.5. GDL is added as dry powder, and a sol/gel transition is observed within 30–60 minutes, depending on the chemical composition and molecular weight of the alginate. Alginic acid gels can be prepared in two different ways; by a direct addition of GDL and by converting an ionically crosslinked gel to the acid form by mineral acid. It has been found69 that the strength of the acid gels is more or less independent of the method by which they are made; acid gels thus seem to be independent of the history of formation. An important difference between the acid gels and the ionically crosslinked gels seems therefore to be that the former behave more as being of an equilibrium nature. If acid gels are made from alginates with different chemical composition, it has been found that these gels resemble ionic gels in the sense that a high content of guluronate (high values of NG > 1) give the highest moduli.69 But in contrast to ionic gels, also polymannuronate sequences support acid gel formation. Poly-alternating sequences seem to perturb gel formation in both cases. The obvious demand for homopolymeric sequences in acid gel formation suggests cooperative processes to be involved just as in the case of

Alginates

393

ionic gels. A broad molecular weight dependence has been observed, and this dependence becomes more pronounced with increasing content of guluronic acid residues.69 The equilibrium properties of the alginic acid gels were confirmed in a study of the swelling and partial solubilisation at pH4.70 By comparing the chemical composition and molecular weight of the alginate material leaching out from the acid gels with the same data for the original alginate, an enrichment in mannuronic acid residues was found, a reduction in the average length of G-blocks and a lowering of the molecular weight.

22.7

Regulatory status

The safety of alginic acid and its ammonium, calcium, potassium, and sodium salts were last evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) at its thirty-ninth meeting in 1992. An ADI ‘not specified’ was allocated. JECFA allocated an ADI of 0–25mg/kg bw to propylene glycol alginate at its seventeenth meeting. In the US, ammonium, calcium, potassium, and sodium alginate are included in a list of stabilisers that are generally recognised as safe (GRAS). Propylene glycol alginate is approved as a food additive (used as an emulsifier, stabiliser or thickener) and in several industrial applications (coating of fresh citrus fruit, as an inert pesticide adjuvant, and as a component of paper and paperboard in contact with aqueous and fatty foods). In Europe, alginic acid and its salts and propylene glycol are all listed as EC approved additives other than colours and sweeteners. Alginates are inscribed in Annex I of the Directive 95/2 of 1995 and as such can be used in all foodstuffs (except those cited in Annex II and those described in art. II of the Directive) under the Quantum Satis principle in the EU.

22.8 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

References

and HEMMER, P. C. ‘Some biological functions of matrix components in benthic algae in relation to their chemistry and the composition of seawater’ ACS Symp Ser, 1977 48 361–81. SADOFF, H. L. ‘Encystments and germination in Azotobacter vinelandii’ Bacteriol Rev, 1975 39 516–39. LINKER, A. and JONES, R. S. ‘A new polysaccharide resembling alginic acid isolated from Pseudomonas’ J Biol Chem, 1966 241 3845–51. ˚ K-BRÆK, G. ‘Alginate as immobilization matrix for cells’ Trends Biotechnol, 1990 SMIDSRØD, O. and SKJA 8 71–78. ANDRESEN, I-L., SKIPNES, O., SMIDSRØD, O., ØSTGAARD, K.

˚ K-BRÆK, G., ESPEVIK, T., SOON-SHIONG, P., FELDMAN, E., NELSON, R., KOMTEBEDDE, J., SMIDSRØD, O., SKJA HEINTZ, R. and LEE, M. ‘Successful reversal of spontaneous diabetes in dogs by intraperitoneal

microencapsulated islets’ Transplantation, 1992 54 769–74. British patent no. 142. and properties of alginates’ Thesis, Norwegian Institute of Technology, Trondheim, 1964. GORIN, P. A. J. and SPENCER, J. F. T. ‘Exocellular alginic acid from Azotobacter vinelandii’ Can J Chem, 1966 44 993–8. SUTHERLAND, I. W. Surface carbohydrates of the prokaryotic cell, London, Academic Press, 1977, pp. 22– 96. HAUG, A., LARSEN, B. and SMIDSRØD, O. ‘A study of the constitution of alginic acid by partial hydrolysis’ Acta Chem Scand, 1966 20 183–90. HAUG, A. and LARSEN, B. ‘A study on the constitution of alginic acid by partial acid hydrolysis’ Proc Int Seaweed Symp, 1966 5 271–7. HAUG, A., LARSEN, B. and SMIDSRØD, O. ‘Studies on the sequence of uronic acid residues in alginic acid’ Acta Chem Scand, 1967 21 691–704. HAUG, A. and SMIDSRØD, O. ‘Fractionation of alginates by precipitation with calcium and magnesium ions’ Acta Chem Scand, 1965 19 1221–6. STANFORD, E. C. C., 1881 HAUG, A. ‘Composition

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Handbook of hydrocolloids and HAUG, A. ‘A computer study of the changes in composition-distribution occurring during random depolymerisation of a binary linear heteropolysaccharide’ Acta Chem Scand, 1968 22 1637–48. LARSEN, B., SMIDSRØD, O., PAINTER, T. J. and HAUG, A. ‘Calculation of the nearest-neighbour frequencies in fragments of alginate from the yields of free monomers after partial hydrolysis’ Acta Chem Scand, 1970 24 726–8. SMIDSRØD, O. and WHITTINGTON, S. G. ‘Monte Carlo investigation of chemical inhomogeneity in copolymers’ Macromolecules, 1969 2 42–4. 13 GRASDALEN, H., LARSEN, B. and SMIDSRØD, O. ‘ C-NMR studies of alginate’ Carbohydr Res, 1977 56 C11–C15. GRASDALEN, H., LARSEN, B. and SMIDSRØD, O. ‘A PMR study of the composition and sequence of uronate residues in alginate’ Carbohydr Res, 1979 68 23–31. PENMAN, A. and SANDERSON, G. R. ‘A method for the determination of uronic acid sequence in alginates’ Carbohydr Res, 1972 25 273–82. 1 GRASDALEN, H. ‘High-field H-nmr spectroscopy of alginate: Sequential structure and linkage conformations’ Carbohydr Res, 1983 118 255–60. ˚ K-BRÆK, G. ‘Characteristics of alginate from Laminaria digitata cultivated in a INDERGAARD, M. and SKJA high phosphate environment’ Hydrobiologia, 1987 151/152 541–9. ˚ K-BRÆK, G., SMIDSRØD, O. and LARSEN, B. ‘Tailoring of alginates by enzymatic modification in vitro’ SKJA Int J Biol Macromol, 1986 8 330–6. ˚ G, H. and SKJA ˚ K-BRÆK, G. ‘Genetics and biosynthesis of alginates’ Carbohydr Eur, VALLA, S., ERTESVA 1996 14 14–18. RIVERA-CARRO, H. D. ‘Block structure and uronic acid sequence in alginates’ Thesis, Norwegian Institute of Technology, Trondheim, 1984. ˚ K-BRÆK, G. ‘Distribution of uronate residues in alginate STOKKE, B. T., SMIDSRØD, O., BRUHEIM, P. and SKJA chains in relation to alginate gelling properties’ Macromolecules, 1991 24 4637–45. ˚ K-BRÆK, G., SMIDSRØD, O., SOON-SHIONG, P. and ESPEVIK, T. ‘Induction OTTERLEI, M., ØSTGAARD, K., SKJA of cytokine production from human monocytes stimulated with alginate’ Int J Immunother, 1991 10 286– 91. SMIDSRØD, O. and HAUG, A. ‘Dependence upon uronic acid composition of some ion-exchange properties of alginate’ Acta Chem Scand, 1968 22 1989–97. HAUG, A. and SMIDSRØD, O. ‘Selectivity of some anionic polymers for divalent metal ions’ Acta Chem Scand, 1970 24 843–54. SMIDSRØD, O. ‘Some physical properties of alginates in solution and in the gel state’ Thesis, Norwegian Institute of Technology, Trondheim, 1973. SMIDSRØD, O. ‘Molecular basis for some physical properties of alginates in the gel state’ J Chem Soc Faraday Trans, 1974 57 263–74. GRANT, G. T., MORRIS, E. R., REES, D. A., SMITH, P. J. C. and THOM, D. ‘Biological interactions between polysaccharides and divalent cations: The egg-box model’ FEBS Lett, 1973 32 195–8. MACKIE, W., PEREZ, S., RIZZO, R., TARAVEL, F. and VIGNON, M. ‘Aspects of the conformation of polyguluronate in the solid state and in solution’ Int J Biol Macromol, 1983 5 329–41. STEGINSKY, C. A., BEALE, J. M., FLOSS, H. G. and MAYER, R. M. ‘Structural determination of alginic acid and the effects of calcium binding as determined by high-field nmr’ Carbohydr Res, 1992 225 11–26. STOKKE, B. T., DRAGET, K. I., YUGUCHI, Y., URAKAWA, H. and KAJIWARA, K. ‘Structural studies of homogeneous alginate gels’ The Wiley Polym Network Group Rev Ser, 1998 1 119–28. HAUG, A. and LARSEN, B. ‘The solubility of alginate at low pH’ Acta Chem Scand, 1963 17 1653–62. MYKLESTAD, S. and HAUG, A. ‘Studies on the solubility of alginic acid from Ascophyllum nodosum at low pH’ Proc Int Seaweed Symp, 1966 5 297–303. HAUG, A., MYKLESTAD, S., LARSEN, B. and SMIDSRØD, O. ‘Correlation between chemical structure and physical properties of alginate’ Acta Chem Scand, 1967 21 768–78. HAUG, A. and SMIDSRØD, O. ‘Precipitation of acidic polysaccharides by salts in ethanol-water mixtures’ J Polym Sci, 1967 16 1587–98. HAUG, A. ‘Fractionation of alginic acid’ Acta Chem Scand, 1959 13 601–3. HAUG, A. ‘Ion exchange properties of alginate fractions’ Acta Chem Scand, 1959 13 1250–1. SMIDSRØD, O., HAUG, A. and LARSEN, B. ‘The influence of pH on the rate of hydrolysis of acidic polysaccharides’ Acta Chem Scand, 1966 20 1026–34. GACESA P, CASWELL, R. C. and KILLE, P. ‘Bacterial alginases; Pseudomonas aeruginosa infection’ Antibiot Chemoter, Basel, Karger, 1989 42 67–71. HAUG, A., LARSEN, B. and SMIDSRØD, O. ‘The degradation of alginates at different pH values’ Acta Chem Scand, 1963 17 1466–8. HAUG, A., LARSEN, B. and SMIDSRØD, O. ‘Alkaline degradation of alginate’ Acta Chem Scand, 1967 21 2859–70. SMIDSRØD, O. ‘Structure and properties of charged polysaccharides’ Int Congr Pure Appl Chem, 1980 27 315–27. SMIDSRØD, O., HAUG, A. and LARSEN, B. ‘Oxidative-reductive depolymerization: a note on the comparison PAINTER, T. J., SMIDSRØD, O.

Alginates 47. 48. 49. 50. 51. 52. 53.

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of degradation rates of different polymers by viscosity measurements’ Carbohydr Res, 1967 5 482–5. SMIDSRØD, O., HAUG, A. and LARSEN, B. ‘The influence of reducing substances on the rate of degradation of alginates’ Acta Chem Scand, 1963 17 1473–4. SMIDSRØD, O., HAUG, A. and LARSEN, B. ‘Degradation of alginate in the presence of reducing compounds’ Acta Chem Scand, 1963 17 2628–37. ˚ K-BRÆK, G., MURANO, E. and PAOLETTI, S. ‘Alginate as immobilization material. II: Determination of SKJA polyphenol contaminants by fluorescence spectroscopy, and evaluation of methods for their removal’ Biotechnol Bioeng, 1989 33 90–4. LEO, W.J., MCLOUGHLIN, A. J. and MALONE, D. M. ‘Effects of sterilization treatments on some properties of alginate solution and gels’ Biotechnol Prog, 1990 6 51–3. ˚ K-BRÆK, G. and ØSTGAARD, K. ‘Regeneration, cultivation and differentiation DRAGET, K.I., MYHRE, S., SKJA of plant protoplasts immobilized in Ca-alginate beads’ J Plant Physiol, 1988 132 552–6. PARSONS, B. J., PHILLIPS, G. O., THOMAS, B., WEDLOCK, D. J. and CLARK-STURMAN, A. J. ‘Depolymerization of xanthan by iron-catalysed free radical reactions’ Int J Biol Macromol, 1985 7 187–92. ˚ K-BRÆK, G., SMIDSRØD, O., HEINTZ, R., LANZA, R. P. and ESPEVIK, T. ‘An SOON-SHIONG, P., OTTERLEI, M., SKJA immunologic basis for the fibrotic reaction to implanted microcapsules’ Transplant Proc, 1991 23 758–9.

54.

˚ KSOON-SHIONG, P., FELDMAN, E., NELSON, R., HEINTZ, R., YAO, Q., YAO, T., ZHENG, N., MERIDETH, G., SKJA BRÆK, G., ESPEVIK, T., SMIDSRØD, O. and SANDFORD, P. ‘Long-term reversal of diabetes by the injection of

55.

STOKKE, B. T., SMIDSRØD, O., ZANETTI, F., STRAND, W.

56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70.

immunoprotected islets’ Proc Natl Acad Sci, 1993 90 5843–7. and SKJA˚K-BRÆK, G. ‘Distribution of uronate residues in alginate chains in relation to gelling properties 2: Enrichment of -D-mannuronic acid and depletion of -L-guluronic acid in the sol fraction’ Carbohydr Polym, 1993 21 39–46. ˚ K-BRÆK, G. ‘Application of alginate gels in biotechnology and biomedicine’ ESPEVIK, T. and SKJA Carbohydr Eur, 1996 14 19–25. NEISER, S., DRAGET, K. and SMIDSRØD, O. ‘Gel formation in heat-treated bovine serum albumin – sodium alginate systems’ Food Hydrocolloids, 1998 12 127–32. NEISER, S., DRAGET, K. I. and SMIDSRØD, O. ‘Interactions in bovine serum albumin – calcium alginate gel systems’ Food Hydrocolloids, 1999 in press. ˚ G, K., ONSØYEN, E. and SMIDSRØD, O. ‘Na- and K-alginate; effect on Ca2+-gelation’ DRAGET, K. I., STEINSVA Carbohydr Polym, 1998 35 1–6. DRAGET, K. I., ONSØYEN, E., FJÆREIDE, T., SIMENSEN, M. K., HJELLAND, F. and SMIDSRØD, O. ‘Procedure for producing uronic acid blocks from alginate’ Intl Pat Appl no. PCT/NO98/00142, 1998. DRAGET, K. I., ONSØYEN, E., FJÆREIDE, T., SIMENSEN, M. K. and SMIDSRØD, O. ‘Use of G-block polysaccharides’ Intl Pat Appl no. PCT/NO97/00176, 1997. ONSØYEN, E. ‘Commercial applications of alginates’ Carbohydr Eur, 1996 14 26–31. ˚ K-BRÆK, G., GRASDALEN, H. and SMIDSRØD, O. ‘Inhomogeneous polysaccharide ionic gels’ Carbohydr SKJA Polym, 1989 10 31–54. MIKKELSEN, A. and ELGSÆTER, A. ‘Density distribution of calcium-induced alginate gels – a numerical study’ Biopolymers, 1995 36 17–41. ˚ K-BRÆK, G., GRASDALEN, H., DRAGET, K. I. and SMIDSRØD, O. ‘Inhomogeneous calcium alginate beads’ SKJA in Biomedical and biotechnological advances in industrial polysaccharides New York, Gordon and Breach, 1989, pp. 385–98. ˚ K-BRÆK, G. and SMIDSRØD, O. ‘Alginate as immobilization material: I. Correlation MARTINSEN, A., SKJA between chemical and physical properties of alginate gel beads’ Biotechnol Bioeng, 1989 33 79–89. DRAGET, K. I., ØSTGAARD, K. and SMIDSRØD, O. ‘Homogeneous alginate gels: a technical approach’ Carbohydr Polym, 1991 14 159–78. DRAGET, K. I., SIMENSEN, M. K., ONSØYEN, E. and SMIDSRØD, O. ‘Gel strength of Ca-limited alginate gels made in situ’ Hydrobiologia, 1993 260/261 563–5. ˚ K-BRÆK, G. and SMIDSRØD, O. ‘Alginic acid gels; the effect of alginate chemical DRAGET, K. I., SKJA composition and molecular weight’ Carbohydr Polym, 1994 25 31–8. ˚ K-BRÆK, G., CHRISTENSEN, B. E., GA ˚ SERØD, O. and SMIDSRØD, O. ‘Swelling and partial DRAGET, K. I., SKJA solubilization of alginic acid gel beads in acidic buffer’ Carbohydr Polym, 1996 29 209–15.

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23 FrutafitÕ-inulin Sensus Operations CV, The Netherlands

23.1

Introduction

FrutafitÕ-inulin is a naturally occurring storage carbohydrate, present in numerous plants. It is built up of 2–60 fructose-units with one terminal glucose molecule (Fig. 23.1). At Sensus inulin is extracted as a fructo-oligosaccharide from chicory roots using hot water. After purification, the solution is spray dried. The resulting product is a white odourless, easily dispersable powder with a neutral taste. At certain levels FrutafitÕ-inulin is soluble in water. Its solubility increases with the temperature. At room temperature, inulin solutions up to 7.5% are completely clear. When concentrations exceed 15%, insulin has the ability to form a gel or cream, showing an excellent fat-like texture. This inulin gel is a perfect fat replacer offering various opportunities in a wide range of foods. Upon digestion, inulin is not broken down in the mouth, stomach and small intestines. It arrives unchanged in the large colon, where it is anaerobically fermented by the microflora. Biochemical calculations and studies in rats and humans, point toward a calorific value for FrutafitÕ-inulin close to 6kJ/g ( = 1.5kcal/g). Regulations differ from country to country. In general FrutafitÕ-inulin is considered as a food ingredient, so it has no E-number and it can be used ad libitum in all food categories. FrutafitÕ-inulin has the status of dietary fibre in almost every country.

23.2

FrutafitÕ-inulin and hydrocolloids

The function of hydrocolloids, which are also known as gums, thickeners, gelling agents, stabilisers, texturisers, is to thicken or gel aqueous systems. In doing so, they provide texture, body and mouthfeel to food products. Most of the hydrocolloids are polysaccharides which are high molecular weight molecules consisting of polymers of sugar-building units. The water-binding property of these polymers is basically due to the fact that these macromolecules can form junction zones and so enclose large amounts of water. Because of this high water binding effect they can be used at low dosage levels. Proteins like gelatin also have the ability to form gels.

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Fig. 23.1 Inulin molecule.

Hydrocolloids can be divided in three groups: emulsifying/stabilising agents, thickening agents and gelling agents. Thickeners are used to thicken and increase viscosity of aqueous systems while gelling agents are able to convert water to a demouldable solid or gel.

23.3

FrutafitÕ-inulin

FrutafitÕ-inulin has different thickening and stabilising properties from hydrocolloids. The FrutafitÕ-inulin molecules are much smaller and the water-binding ability is low compared to hydrocolloids. When concentrations exceed 15%, inulin has the ability to form a gel or cream. Below this concentration low viscous aqueous solutions are obtained. Gel formation is also different compared to hydrocolloids. FrutafitÕ-inulin forms particle gels whereas the increase of viscosity through most hydrocolloids is obtained by weak or strong bonds between chains.

23.4

Interactions of FrutafitÕ-inulin

Although FrutafitÕ-inulin does not act not like a thickener or gelling agent, in combination with hydrocolloids it influences the behaviour of these polysaccharides. In this way, FrutafitÕ-inulin can optimise the rheological properties of products. The inulin product is highly polydisperse. It has significant influence (with its Hbinding efficiency and consequent solubility (gel formation)) on various high waterbinding ingredients such as guar gum, xanthan gum, carrageenans, alginates, pectins, locust bean gum, maltodextrin and starch. By adding inulin to thickeners the viscosity (decrease or increase) and flow characteristics of the aqueous solution can be affected (Fig. 23.2).

FrutafitÕ-inulin

Fig. 23.2

399

Inulin and starch viscosity vs. temperature.

In the same way the gel structure (brittleness, elasticity) and gel strength of gelling agents can be influenced by adding inulin. Synereses can also be reduced.

23.5

FrutafitÕ-inulin as a gelling agent

Preparation of an inulin gel is straightforward. FrutafitÕ-inulin is dissolved/dispersed in water to produce a watery fluid. When the fluid is cooled the molecules start to precipitate and crystallise. The rate of precipitation or crystallisation and consequently the size of the inulin particles depends on temperature, concentration and cooling process. The submicron-sized crystals go on to form aggregates which become interlinked to form a network. Within a few hours the free water is captured in the network of crystallised inulin particles, resulting in a gel structure.

23.6

Parameters affecting gel characteristics

The character of an inulin gel can be influenced by the following parameters: • • • • •

chain length inulin inulin concentration preparation temperature shear treatment use of seeding process.

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23.6.1 Chain length As has been mentioned before, the formation of an inulin gel is based on the precipitation and crystallisation of inulin molecules. Research has shown that only the longer inulin molecules (DP1 > 10) participate in the gel structure and the smaller molecules remain dissolved. It can be seen from Fig. 23.3, that inulin types with a higher average chain length, containing more long chain inulin molecules, will form firmer inulin gels.

23.6.2 Inulin concentration An inulin gel belongs to the group of particle gels. Firmness increases, with increasing concentration. See Fig. 23.3.

23.6.3 Preparation temperature Firmness of an inulin gel reaches its maximum when, during the manufacturing process, nearly all the inulin molecules become fully hydrated, with the exception of some seed crystals. These seed crystals are necessary to initiate the gelling process. The degree of hydration depends on the inulin concentration and temperature. Example: model system 25% FrutafitÕEXL-gel • • • • • •

dissolve 50 g FrutafitÕEXL in 150g tap water place the dispersion in a water bath at 72ºC heat the dispersion for 15 minutes while stirring remove the solution from the water bath place it in a cold room, 5ºC for at least 6 hours the gel is formed!

Fig. 23.3 Firmness of short- and long-chain inulin gels vs. the concentration in water.

FrutafitÕ-inulin

401

23.6.4 Shear treatment The application of a shear treatment immediately after pasteurisation or during cooling will have a positive effect on increasing the firmness of an inulin gel (Fig. 23.4).

23.6.5 Seeding process Maximum firmness can be achieved by using the so-called seeding process: an inulin solution is heated to a high temperature, so that all the inulin molecules are completely hydrated. Addition of seed crystals during cooling, in combination with a shear treatment will result in a firm inulin gel with perfect sensory properties (Fig. 23.4).

23.7

Applications

The rheological and sensory characteristics of inulin gels make them an excellent fat replacer in a wide range of foods.

23.7.1 Fat spreads and dairy spreads Addition of 7.5% FrutafitÕ HD to fat spreads with 20–40% fat (w/o emulsions) results in a product with a good structure and a perfect taste. The excellent spreadability and mouthfeel make it a perfect, low-fat substitute for sandwich margarines with 80% fat! An inulin gel based on 20% FrutafitÕEXL is the base for a very low-fat spread (o/w emulsions) with no more than 5% fat and for dairy spreads like cream and cheese spreads.

Fig. 23.4

The effect of shear treatment and seeding on the firmness of inulin gels.

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23.7.2 Low-fat mayonnaise/dressing Traditional problems with low-fat or no fat mayonnaise and dressings are a lumpy texture with an acidic taste and a dry and sour aftertaste. With the addition of inulin (5%) a stable low-fat mayonnaise or dressing with good flowing properties is achieved. Furthermore, inulin is capable of masking off the acidic/sour taste.

23.7.3 Low-fat yoghurt The preparation of low-fat no fat yoghurt is done by starting with skimmed milk. To obtain the same mouthfeel as traditional yoghurt, inulin is added. By adding inulin the viscosity of the yoghurt is increased and a similar mouthfeel to normal yoghurt is obtained. A problem which occurs in other fat-free yoghurts is that the taste is more acidic. FrutafitÕ-inulin is capable of masking off this effect.

23.7.4 Low-fat cake Partial replacement of the pastry margarine by an inulin gel results in a fat-reduced cake with perfect sensoric properties and a long shelf life. Research has also shown that the combination of FrutafitÕ-inulin with maltodextrin results in cakes with more volume.

23.7.5 Fillings The creamy structure of an inulin gel makes it a perfect base for fillings. Research resulted in biscuits with a filling in which 25% fat was replaced.

23.7.6 Wafers The most important characteristic of wafers is their crispness, because this influences the perception of freshness. They should not lose their crispness during storage and preferably they should also stay crisp when they are filled with ice cream and fruit or as a sandwich wafer. The final wafer product becomes more crisp when FrutafitÕ-inulin is used. The crispness of a product can be characterised by the sound during breaking of the product. The degree of crispness depends on several aspects. The density of the product has a major influence; the lower the density the easier the wafer breaks. At higher moisture contents the wafer will also not be crisp. Another aspect is the absorption profile of the starch used. The absorption profile is characterised by the relation between the water content and the water activity (aw). A product stays crisp until a water activity of 0.1–0.2. When the absorption profile line is steep (see Fig. 23.5, line 1) the product is crisp only until a water content of approximately 3%. With a less steep absorption profile the product stays crisp until a higher water content (Fig. 23.5, line 2). FrutafitÕ-inulin is able to bind water, so there will be less free water in the product. This means that a wafer with FrutafitÕ-inulin can contain more moisture.

23.7.7 Low-fat hazelnut spread Commercial hazelnut spreads are fat-based products. Dry ingredients are milled and finely dispersed in the fat phase to achieve the right mouthfeel. A structure based on 12% FrutafitÕ HD with addition of sugars, cocoa powder, milk proteins and hazelnut paste

FrutafitÕ-inulin

Fig. 23.5

403

Absorption profiles.

make it possible to produce a delicious low-fat (water continuous) hazelnut spread. Compared with the conventional fat continuous products a calorie reduction of 45% can be achieved.

23.8

Hydrocolloids

A positive influence is observed in the following applications by adding inulin. With thickeners (xanthan-, guar gum and pectins) stabilised beverage systems become more homogeneous when inulin is added. Due to H-binding influences, inulin provides the necessary flow properties and physiological effects. This is also particularly effective in gum-based fat-free dressings and sauce applications where fat-like flow and mouthfeel are of primary importance. The viscosity of starch is influenced because of the greater affinity for water of inulin compared to starch. A smoother flow is obtained which can be a benefit in starch-based sauces. A creamier mouthfeel is also obtained when inulin is added to dairy products due to interactions with the dairy components whey proteins and caseinate. This effect is even stronger when combined with -carrageenan as seen in flans and instant mousses. In dairy- and low-fat spreads, stabilised by gelatin or maltodextrin, the spreadability and mouthfeel is improved when inulin is added and a more fat-like structure is obtained.

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24 CRC emulsifying biopolymer: a new emulsifier for the soft-drinks industry A. Lane (Food Science Australia) and E. Chai (University of Melbourne)

24.1

Introduction

The Cooperative Research Centre for Industrial Plant Biopolymers1 has developed a cell culture fermentation process to produce a novel emulsifying biopolymer (the ‘CRC Biopolymer’) for use in the soft-drinks industry. This product is at least 20 times more effective than gum arabic on a weight basis in this application, concentrations of less than 1% (w/v) being needed to generate emulsions with 1m droplets which are stable for long periods (6 months) in soft-drinks formulations. The global soft-drinks market is currently estimated at 220 billion litres per year, and is growing strongly, both in its established markets and particularly in the potentially huge markets of the newly emerging industrialised nations. Approximately 66% of this market consists of carbonated beverages containing oil-based flavouring emulsions, based largely on gum arabic as the emulsifier of choice. Despite recent advances in the processing of gum arabic and the development of speciality products for specific applications, supplies and quality of raw gum arabic still remain dependent upon both weather and politics in the producing countries. Although there is currently (1999) an adequate supply and a reasonable stockpile of gum arabic, this has been the result of a succession of good seasons, so that the present situation will inevitably be followed once again by shortages and increased prices. The CRC Biopolymer offers the opportunity to overcome these difficulties, by providing a reliable supply of emulsifying product which has been manufactured under carefully controlled conditions, so has consistent price and performance characteristics. CRC Biopolymer can be used either alone, or as a supplement to the available supplies of gum arabic.

Many dedicated individuals and teams have contributed to the data presented in this report. On behalf of the CRC for Industrial Plant Biopolymers, the authors wish to thank all participants, past and present, for the commitment and expertise which has taken the project to a successful and exciting outcome. 1

Established under the Cooperative Research Centres Program of the Government of Australia.

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Fig. 24.1

24.2

Outline of stages of development of the CRC process.

The CRC process

An extensive screening programme covering more than 130 plant species resulted in a plant cell line which produces economically attractive yields of an emulsifier with applications similar to gum arabic, but with greatly enhanced efficacy. The stages in establishing the cell line, developing optimised medium and cultivation conditions and scaling the process up to a commercial operation are illustrated diagrammatically in Fig. 24.1. Detailed studies have optimised the culture medium, the conditions of cultivation and the operation of the fermenters to achieve maximum rates of production of emulsifier, and to recover the product from the culture filtrates in its fully functional and soluble form. One of the fermenters (capacity 10,000 litres) used in the manufacturing process is shown in Fig. 24.2. Harvested cell culture is pumped to a sanitised filtration system to separate the biomass from the culture broth. Immediately after filtering, the broth is pasteurised and stabilised to protect the emulsifying CRC Biopolymer, then the product is concentrated and purified by ultrafiltration and washing. The efficacy of the product is standardised by blending with a food-approved extender, after which it is finally dried and agglomerated to an ‘instantised’ powder. Due diligence has been exercised in drawing up a HACCP plan to analyse and control the manufacture of CRC Biopolymer and the process has been audited and approved for HACCP certification.

24.3

Product specifications for CRC Biopolymer

Synonyms Definition

None. CRC Biopolymer is the processed culture filtrate from selected suspension cultured plant cells, obtained using standard culturing and processing conditions.

CRC emulsifying biopolymer

407

Fig. 24.2 Fermenter (10,000-litre capacity) for producing CRC Biopolymer.

CAS number Description Function Solubility

Hydrolysis products Optical rotation Loss on drying Total ash Acid insoluble ash

Not yet allocated. Spray-dried powder, pale in colour, or pale straw coloured liquid, which flows readily. Emulsifier and stabiliser. As for gum arabic, one gram dissolves in 2 ml of water forming a solution that flows readily and is acid to litmus; insoluble in ethanol. Monosaccharides and amino acids. Solutions of CRC Biopolymer are dextrorotatory. 10% (105ºC, 4 h). As for gum arabic, 4%. As for gum arabic, 0.5%.

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Acid insoluble matter Arsenic Lead Starch or dextrin Tannin-bearing gums Microbiological criteria

2,4-D

24.4

As for gum arabic, 1.0%. As for gum arabic, 3 mg/kg. As for gum arabic, 5 mg/kg. As for gum arabic. As for gum arabic. Total plate count < 1000 cfu/g. Salmonella negative per test. E. coli negative in 1g. Liquid Biopolymer 20 ppb (20g/l). Spray-dried Biopolymer 1.2 ppm (1.2 mg/kg). Final soft drink 1210ÿ3 ppb (1210ÿ3g/l).

Functional properties and efficacy of CRC Biopolymer

CRC Biopolymer is highly effective at producing stable emulsions having the small median droplet size and narrow droplet size distribution required by the soft-drinks industry. Figure 24.3 shows the effect of emulsifier concentration on median droplet size for three representative batches of CRC Biopolymer, compared with that for gum arabic. The high efficacy of CRC Biopolymer is further illustrated by the data in Table 24.1, showing that it is between 20 and 49 times more effective than gum arabic on a weight basis at producing emulsions with a median droplet size in the range required for soft drinks (1.1–1.5 m). Figure 24.4 illustrates the droplet size distribution found in emulsions made with 1% (w/v) CRC Biopolymer and 20% (w/v) gum arabic, and shows that both median droplet size and droplet size distribution in both cases are similar.

Fig. 24.3

Semi-log plot comparing median droplet size vs. emulsifier concentration for three batches of CRC Biopolymer with that for gum arabic.

CRC emulsifying biopolymer

409

Table 24.1 Efficacy superiority of representative batches of CRC Biopolymer, compared with that of gum arabic Droplet size (m)

Concentration (% w/w)

GAE1

1

1.0 1.1 1.5

1.4 1.0 0.3

15.9 20.2 49.3

2

1.0 1.1 1.5

1.1 0.8 0.3

20.2 25.3 40.3

Gum arabic

1.0 1.1 1.5

22.2 20.2 14.8

1.0 1.0 1.0

Batch

1. GAE (gum arabic equivalent) = efficacy relative to gum arabic, weight for weight.

Fig. 24.4

24.5

Typical droplet size distribution achieved with 1% (w/v) CRC Biopolymer or 20% (w/v) gum arabic (pH4.0).

Emulsion stability

Soft drinks are prepared in two stages. First, concentrated ‘cloud base’ emulsion is prepared, typically using 20% (w/v) gum arabic and 26% (v/v) citrus oil. This cloud base is incorporated into bottling syrup, which is further diluted in carbonated water to make the final soft-drink beverage. The beverage industry requires both the cloud base emulsion and the final product to be shelf-stable for six months. In the soft-drinks industry, stability trials are therefore carried out over several months at ambient temperature, or in an accelerated version of the test, for shorter periods of time at 37ºC. Table 24. 2 compares the median droplet size during storage for six months at 20ºC, of both cloud base emulsion and soft drinks prepared from either 20% (w/v) gum arabic or 1% (w/v) CRC Biopolymer. A slight increase occurs in droplet size in all cases, but no appreciable difference is seen between the performance of the different emulsifiers. Similar results are obtained from the accelerated stability test carried out at 37ºC. As shown in Table 24.3, a slight increase in median droplet size is once again seen in all

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Table 24.2 Storage trials of emulsions and soft drinks at 20ºC, pH4.0: comparison of CRC Biopolymer (1% w/v) and gum arabic (20% w/v) Median droplet size (m) 1 day

1 month

2 months

4 months

6 months

Emulsions Gum arabic (20%) CRC Biopolymer (1%)

0.99 0.94

1.3 1.4

1.5 1.7

1.8 1.8

2.4 2.2

Drinks Gum arabic (20%) CRC Biopolymer (1%)

– –

1.4 1.5

1.5 1.4

1.5 1.5

1.6 1.7

Table 24.3 Storage trials of emulsions and soft drinks at 37ºC, pH4.0: comparison of CRC Biopolymer (1% w/v) and gum arabic (20% w/v) Median droplet size (m) 1 month

5 months

Emulsions Gum arabic (20%) CRC Biopolymer (1%)

1.3 1.4

3.1 3.3

Drinks Gum arabic (20%) CRC Biopolymer (1%)

1.6 1.4

1.4 1.4

preparations over the period of the test, but the performance of the CRC Biopolymer at a concentration of 1% (w/v) is indistinguishable from that of gum arabic at 20% (w/v). The droplet size distributions for 1.0% and 1.3% CRC Biopolymer and 20% gum arabic after three months storage at 37ºC are shown in Fig. 24.5. These data confirm both the efficacy advantage of the CRC Biopolymer and that the stability of CRC Biopolymer emulsions is indistinguishable from that of gum arabic emulsions.

24.6

Composition of CRC Biopolymer

CRC Biopolymer is composed principally of a mixture of polysaccharides similar to those commonly found in fresh fruit and vegetables, such as xyloglucans, arabinogalactan-proteins and pectic polysaccharides. In addition, it contains about 4% (w/w) protein and 4% (w/w) inorganic material (ash). The polysaccharide component is composed of the monosaccharides typically found in vegetable polysaccharides, with the following average relative proportions: galactose (35%), galacturonic acid (23%), glucose (17%), arabinose (11%), xylose (9%), with rhamnose, fucose and glucuronic acid each present at < 3%. The amino acid profile of the protein component is relatively evenly distributed, unlike that in gum arabic (which contains 40% hyp; 22% ser; 13% pro; 12% leu), indicating that, though arabinogalactan-protein is present, other proteins are present as well.

CRC emulsifying biopolymer

Fig. 24.5

411

Droplet size distribution in emulsions made with CRC Biopolymer (1.0 and 1.3% w/v) and gum arabic (20% w/v), after storage for 3 months at 37ºC (pH4.0).

The monosaccharide composition, protein content and amino acid profile of CRC Biopolymer are consistent from batch to batch within the range expected for biological materials, and are not influenced by the scale of the fermentation, all of which is evidence of a reliable and robust manufacturing process.

24.7

Patent status of CRC Biopolymer

The Intellectual Property of the CRC Biopolymer is protected by international patents PCT/AU88/0005; US415,263; 07/920,788; PCT/AU93/0037; 08/409,737. The patent protection provided by these can be summarised as follows: 1.

2.

3.

Australian food-use patent covering the use as texture-modifying agents in foods of all polysaccharide materials produced by cell culture, irrespective of the parent plant species. US and international food-use patents covering the use as texture-modifying agents in foods of polysaccharide materials produced by cell culture from a specified list of plant species. Non-food and industrial use patents covering a wide range of applications including cosmetics, personal care products, printing and lithography, agricultural sprays, textiles, ceramics and glass-making and petroleum oil recovery.

24.8

Contact details

Further details of the CRC Biopolymer and the CRC for Industrial Plant Biopolymers can be obtained through the office of the CRC for Industrial Plant Biopolymers, School of Botany, University of Melbourne, Parkville, Australia 3052, phone +61 3 9344 5041, fax +61 3 9347 1071. The Program Leaders, under whose direction the research was carried out, were:

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Professor Tony Bacic, Botany School, University of Melbourne, Parkville, Australia 3052; Dr David Dunstan, Department of Chemical Engineering, University of Melbourne, Parkville, Australia 3052; Dr David McManus, Tridan Ltd. – Albright & Wilson (Australia) Ltd. Partnership, Yarraville, Australia 3013; Dr Alan Lane, Food Science Australia, North Ryde, Australia 2113.

25 Konjac mannan S. Takigami, Gunma University

25.1

Introduction

Konjac (Lasioideae Amorphophallus) is a perennial plant and a member of the family of Araceae. The original home of the konjac plant is not certain, but is considered to be in Southeast Asia. There are many species of konjac plants in the Far East and Southeast Asia that belong to the Amorphophallus,1 for example, A. konjac K. Koch (Japan, China, Indonesia), A. bulbifer Bl. (Indonesia), A. oncophyllus Prain ex Hook. f. (Indonesia), A. variabilis Blume (the Philippines, Indonesia, Malaysia), etc. Only Amorphophallus konjac K. Koch grows in Japan. They contain konjac mannan in their tubers. Konjac mannan is a heteropolysaccharide consisting of -D-glucose (G) and -D-mannose (M), with a G/M ratio of 1 to 1.6. The konjac tuber grows in size year by year and three- to five-year-old plants bloom with purplish-red flowers in the spring. Konjac is an allogamous plant and plant breeding is performed by cross-fertilisation. Figures 25.1 and 25.2 show konjac plants and tubers. The main component of the konjac tuber is konjac mannan (KM), which varies in composition from 8–10% of a raw tuber. Starch, lipid and minerals are also present in the tuber. KM is accumulated in egg-shaped cells covered with scale-like cell walls2, 3 and the KM cells are observed within the parenchyma of the tuber. The size and number of the KM cells increase with distance from the epidermis, reaching ~650m at the central part of the tuber. Other types of organelles in the parenchyma surround the KM cells. Starch exists in spherical organelles as small particles. Bunches of needle-like crystals are also observed in the tuber and the size of a crystal is ca. 150m5m. Since a high content of calcium was detected in the crystal by energy dispersive X-ray (EDX) analysis, the needle-like crystal is considered to be calcium oxalate. The konjac tuber, unprocessed, has a harsh taste. This can be removed from the konjac flour by processing. Irreversible konjac mannan gel is prepared by alkali treatment of grated konjac tuber or konjac flour aqueous solution. KM has very small amount of acetyl groups and deacetylation occurs with the alkali treatment.4 It is considered that the gelation of konjac mannan is induced by deacetylation. The lowest critical concentration of konjac flour aqueous solution necessary for gel formation is about 0.5%.

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Fig. 25.1

Konjac plants.

The konjac gel (Kon-nyaku in Japanese) is classified as a dietary fibre and it has a chewy texture. The first description of konjac gel and its preparation process are found in an old Chinese poem composed by Zuo Shi and its annotation written in the third century.5 It is thought in Japan that the production method of konjac gel was introduced from Korea with Buddhism in the sixth century as a medicine. However, it took a long time before konjac gel became a popular food and this was due to two important investigations for the production process of konjac flour. T. Nakajima (1745–1826) developed a manufacturing technique to produce konjac flour by pulverising dried chips of konjac tuber (Arako). K. Mashiko (1745–1854) improved on this technique to obtain cleaner konjac flour (Seiko). He polished Arako using a mortar worked by a water wheel and separated impurities from the konjac flour by wind sifting. Nowadays, konjac flour is produced in very modern factories controlled by computer systems. However, the principle of the production manufacturing process is the same. It is well known that konjac mannan interacts synergistically with kappa carrageenan6 and xanthan gum7, 8 and forms elastic thermoreversible gels. These synergistic gels are major products in the food industry as new healthy gel foods, particularly in Japan. In the United States, the US Department of Agriculture recently accepted the use of konjac flour as a binder in meat and poultry products. Konjac flour is suitable for thickening, gelling,

Konjac mannan

Fig. 25.2

415

Two-year-old konjac tubers.

texturing, and water binding. It may be used to provide fat-replacement properties in fatfree and low-fat meat products.

25.2

Manufacture

25.2.1 Cultivation Only Amorphophallus konjac K. Koch grows in Japan and selective breeding of konjac plants has been carried out. Recently, five species of the A. konjac have been cultivated, namely, Zairai, Shina, Haruna-kuro, Akagi-ohdama and Miyogi-yutaka. The latter three species are improved breeds and Haruna-kuro and Akagi-ohdama account for more than 90% of the total tuber output. The cultivation process of the konjac tuber in Japan is as follows. Seed tubers (Kigo) and/or one-year-old tubers are planted in the spring. The tubers push out new shoots and are consumed completely. The konjac plants grow during the summer and have new tubers. In the late autumn, the plants die and new tubers are dug from the ground. The new tuber has seed tubers at the top of its suckers. The two-year-old tubers are used to produce konjac flour. One-year-old tubers and the seed tubers are kept in a storehouse with heating during the winter to avoid freezing. This cycle is repeated in the following spring. In China, there are six kinds of konjac plants containing konjac mannan and two species can be cultivated, namely, A. rivieri Duieu and A. aldus Lie et Chen. The selective breeding of konjac plants is also actively carried out.

25.2.2 Production process of konjac flour The two-year-old konjac tubers are brought to a storehouse in containers from farmhouses. The tubers are transported to a washing apparatus using conveyer belts and are washed with water, brushing away mud and epidermis and then distributed to

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each line. The washed konjac tubers are sliced into thin chips, and the chips are dried in a hot-air drier equipped with a heavy oil burner. This is because konjac flour contains a small amount of sulphur dioxide as an impurity. Sulphur dioxide bleaches konjac chips and for this reason the colour of lower-quality konjac flour is extremely white. The dried konjac chips are called Arako in Japanese. The dried chips are pulverised and konjac mannan (KM) particles (i.e., konjac flour) are obtained. Since the KM particles are very tough, they are polished after being produced to remove impurities surrounding the KM cells. Then konjac flour is separated by wind shifting. The polished konjac flour is called Seiko. Micro-fine powder obtained as a by-product is collected using a dust collector. The by-product is called Tobiko in Japanese, which literally means flying powder. The main components of Tobiko are starch and fine KM powder. Protein (ca. 24%) and ash (ca. 10%) are also included in Tobiko. The viscosity of konjac flour is dependent on the raw tubers and is controlled by the mixing of flours produced from different types of tubers. Then the konjac flour thus prepared is packed into the bags and is kept in a cool storehouse to avoid a change in quality.

25.2.3 Purification of konjac flour Commercial konjac flour (Seiko) is a light-coloured powder with fish-like smell and a slightly harsh taste. The current practice of several companies is to wash konjac flour with ethanol aqueous solution to remove the micro-fine powders remaining on the surface and the impurities trapped inside the konjac particles. The konjac flour is whitened by washing. Figures 25.3 and 25.4 show the SEM images of commercial konjac flour, with Fig. 25.4 being the one which has been highly purified.

Fig. 25.3

Scanning electron micrograph of konjac flour (Seiko).

Konjac mannan

Fig. 25.4

417

Scanning electron micrograph of purified konjac flour.

The surface of konjac flour shows scale-like patterns and seems to have been worn smooth (Fig. 25.3). After purification, the scale-like patterns are more clearly observed. Table 25.1 shows the composition of the various components in konjac flour before and after purification. Since the protein content was determined by nitrogen analysis, the value represents not only protein but also all nitrogen-containing substances. The carbohydrate content increased with washing but the concentration of the other components decreased by washing. The carbohydrate value parallels that of KM. The fish-like smell decreases remarkably by washing. It has been reported that alkali treated konjac gel contains trimethylamine and that the fish-like smell of the flour is caused by the amine.9, 10 Konjac flour with and without purification showed mass spectra attributable to nitrogen-containing substances, but they were not identical to trimethylamine.2 This demonstrates that konjac flour does not contain trimethylamine as an impurity. Trimethylamine should be separated from other nitrogen-containing substances by the alkali treatment. The purification of konjac flour is very effective in preventing the putrefaction of konjac gel prepared by alkali treatment and the syneresis of the mixed gels prepared by konjac mannan and other gums. Table 25.1

Analytical results of components in konjac flour before and after purification Contents (g/100g of sample)

Konjac flour Purified konjac flour

Water

Protein

Lipid

Carbohydrate

Fibre

Ash

7.2 7.5

2.2 0.8

2.3 0.9

82.6 88.6

0.5 0.5

5.2 1.7

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25.3

Structure

The main component of konjac flour is a glucomannan called konjac mannan (KM), whose main chain consists of D-glucose and D-mannose linked by -D-1,4 bonds. The ratio of glucose (G) to mannose (M) is reported to be 1 to 1.611–13 or 2 to 3.14, 15 Although the repeating structural unit of the main chain is still uncertain, typical proposals for the unit by research scientists are as follows: 1. 2. 3.

G-G-M-M-M-M-G-M or G-G-M-G-M-M-M-M11 M-M-M-G-G13 G-G-M-M-G-M-M-M-M-M-G-G-M.13, 16

It is also reported that KM has side chains and the branching position is considered to be the C3 position of mannose residues11, 17 or C3 positions on both glucose and mannose13 in the main chain. The degree of branching is estimated at approximately three for every 32 sugar units14 or at one for 80 sugar residues.11 The length of the branched chain was also evaluated as 11 to 16 hexose residues17 or as several hexose units.11 KM contains acetyl groups in the main chain. Figure 25.5 shows a Fourier transformation infra-red (FT-IR) spectrum of purified KM. An absorption due to stretching vibration of C = O group in acetyl group is observed at 1730 cmÿ1. The acetyl group content was estimated at one for 19 sugar residues.4 Figure 25.6 shows the chemical structure of KM proposed by Okimasu.1, 18 The crystalline form of KM was studied by the X-ray diffraction method.19 KM shows a different X-ray diffraction powder pattern from both crystalline polymorphs of other glucomannans (mannan I and mannan II) which have been studied. The fibre pattern of the annealed KM indicated that it exists in an extended two-fold helical structure. Since konjac flour forms very viscous solutions, measurement of the weight average molecular weight (Mw) and the mean square radius of gyration (<S2>1/2) of KM was carried out using partially methylated KM samples.20 The average values of Mw and <S2>1/2 were determined to be 10105 and 110nm. It was also reported that both Mw

Fig. 25.5

FT-IR spectrum of konjac mannan analysed by the attenuated total reflection (ATR) method.

Konjac mannan

Fig. 25.6

419

Chemical structure of konjac mannan.

and <S2>1/2 were found to be dependent on species of konjac plant, cultivation districts and preparation method. The authors21 measured molecular weight (Mw), molecular dispersity and root mean square (RMS) of KM (Akagi ohdama species obtained in Gunma prefecture, Japan) using the Dawn multi-angle laser light scattering method, associated with a gel permeation chromatographic (GPC) fractionation. The Mw, molecular dispersity and RMS were 13.2105, 2.1 and 130nm, respectively.

25.4

Technical data

The quality of commercial konjac flour is appraised by the size of KM particles, viscosity, whiteness, moisture and admixing of impurities such as pieces of scorched epidermis and denatured KM particles during the hot-air drying. Some kinds of bacteria are observed in konjac flour but these are not colon bacilli.22 They cause putrefaction of konjac gel and degradation of molecular weight of konjac mannan. The most important criterion of the quality of konjac flour is its high viscosity in aqueous solution, which in turn depends on the molecular weight of the polysaccharide. Table 25.2 shows typical technical data of two types of commercial konjac flours and purified flour of them. The data is kindly given from Ogino Shoten Co. Ltd. in Gunma Prefecture, Japan. The Chinese konjac flour is a bonded one and was pulverised by Ogino Shoten Co. Ltd. Konjac mannan is a water-soluble polymer but it needs a special technique to dissolve it in water completely. To dissolve at room temperature, konjac flour must be added to water with stirring until the powder is completely dissolved. It is important to stir the solution continuously so that the powder does not lump. Hot water is not effective to dissolve konjac flour. The relationship between viscosity of purified commercial konjac flour and stirring time is shown in Fig. 25.7. The konjac flour, Rheolex RS, was characterised by a very fine mesh size (80 mesh sieve) and the measurements were carried out at 25ºC using a viscometer. The data was kindly provided by the Shimizu Chemical Co. Ltd. in Hiroshima Prefecture, Japan. The viscosity of KM aqueous solution increases with stirring time and reaches a constant value after two hours. The viscosity of KM aqueous solution increases gradually with increasing concentration until 1% and then increases remarkably. As seen in Fig. 25.7, the viscosity of a 2% aqueous solution is more than 12 times higher than that of 1% solution. The viscosity of KM aqueous solution is not affected by salt concentration, but is

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Table 25.2

Analytical results of components in commercial konjac flours Japanese konjac tuber

Viscosity (mPas)+ Whiteness Water* Protein* Lipid* Carbohydrate* Fibre* Ash* Sulphur dioxide Arsenic* Lead* Trimethyl amine** Number of germ Coliform bacteria

Chinese konjac tuber (bonded)

Ordinary flour

Purified flour

Ordinary flour

Purified flour

15.0–15.2 66–68 6.5 2.1 1.3 84.6 0.5 5.0 0.65 g/kg Not detected Not detected 490ppm Less than 300/g negative

17.0–18.0 73 6.6 1.1 0.3 89.2 0.6 2.2 0.17 g/kg Not detected Not detected 85ppm Less than 300/g negative

13.5–13.6 69.9 8.4 3.0 0.9 82.2 0.6 4.9 2.1 g/kg Not detected Not detected 760ppm 420/g negative

18.0 68.9 5.3 1.5 0.3 90.0 0.8 2.1 0.64 g/kg Not detected Not detected 170ppm Less than 300/g negative

+ 1% konjac aqueous solution at 35ºC after 4 h stirring at 90 rpm. * g/100 g of konjac flour. ** nitrogen-containing substances.

Fig. 25.7

Relationships between viscosity of purified commercial konjac flour, Rheolex RS, and stirring time: (❍) 1%, (●) 2%.

affected by pH of the solution. The effect of pH on viscosity change for 1% and 2% KM solutions is listed in Table 25.3. The viscosity of KM solution decreases with decreasing pH value. At a high pH, KM solution changes to gel. Konjac mannan interacts synergistically with other polysaccharides and forms thermoreversible gels. The viscosity of the mixtures and the gel strength are listed in

Konjac mannan Table 25.3

421

Effect of pH on viscosity change for 1% and 2% KM solutions Viscosity (cps)

KM* concentration (%)

Water (no pH adjust.)

pH 4

pH 3

pH 2.5

1

31,600

31,800

29,900

18,600

2

341,000

340,000

301,000

251,000

* – Rheolex RS.

Table 25.4 Viscosity of mixtures of KM and other gums with various composition. Total concentration of the mixtures is 1% KM* concentration (%)

Other gums concentration (%)

0.0 0.2 0.4 0.6 0.8 1.0

1.0 0.8 0.6 0.4 0.2 0.0

Viscosity (cps) Xan

LBG

Gel

Pec

Car

Aga

8,250 225 0 0 300 0 8,800 650 125 75 12,750 60 12,000 2,700 1,525 600 17,500 725 13,250 7,500 5,860 3,750 51,000 3,740 161,000 15,750 14,700 11,640 113,600 12,500 29,500 29,500 29,500 29,500 29,500 29,500

GG

CMC

4,250 75 6,800 225 10,000 1,065 14,750 4,075 20,750 12,200 29,500 29,500

* – Rheolex RS; Xan – xanthan gum; LBG – Locust bean gum; Gel – Gelatin; Pec – Pectin; Car – -carrageenan; Aga – Agar; GG – Guar gum; CMC – Carboxymethyl cellulose.

Table 25.5 Gel strength of mixtures of KM and other gums with various compositions. Total concentration of the mixtures is 1% KM* concentration (%)

Other gums concentration (%)

Xan

LBG

Gel

Pec

Car

Aga

GG

CMC

0.0 0.2 0.4 0.6 0.8 1.0

1.0 0.8 0.6 0.4 0.2 0.0

– 7.8 161.7 84.3 34.7 –

– – – – – –

– – – – – –

– – – – – –

24.1 118.7 185.3 129.0 – –

21.4 25.7 20.3 11.7 4.0 –

– – – – – –

– – – – – —

Gel strength (g)

* – Rheolex RS; Xan – xanthan gum; LBG – Locust bean gum; Gel – Gelatin; Pec – Pectin; Car – -carrageenan; Aga – Agar; GG – Guar gum; CMC – Carboxymethyl cellulose.

Tables 25.4 and 25.5, respectively. The synergism is observed for the combination of KM and xanthan gum, KM and carrageenan, and KM and agar. Table 25.6 shows the effect of sugar concentration on the gel strength for 1% mixed gel with various ratios of KM to -carrageenan. The addition of sugar enhances the gel strength slightly for the gel with higher composition of KM but reduces the strength for the gel with lower composition of KM. Table 25.7 shows the influence of the addition of salt on the gel formation for a 1% of mixture of KM and -carrageenan. The synergistic gel formation is inhibited by addition of salt.

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Table 25.6 Effect of sugar concentration on gel strength of mixed gel of KM and -carrageenan with various compositions. Total concentration of the mixtures is 1% Gel strength (g) *

KM /Car ratio 8:2 7:3 6:4 5:5 4:6 3:7 2:8

Sugar concentration (%) 0

5

10

15

– – 121.5 331.0 299.7 216.8 137.5

– 141.6 205.4 275.3 285.2 213.8 100.1

– 134.9 195.8 299.1 297.4 187.8 100.3

– 145.3 220.7 260.9 222.4 101.1 109.7

* – Rheolex RS; Car – -carrageenan. Table 25.7 Effect of salt concentration on gel strength of mixed gel of KM and -carrageenan with various compositions. Total concentration of the mixtures is 1% Gel strength (g) *

KM /Car ratio 8:2 7:3 6:4 5:5 4:6 3:7 2:8

Salt concentration (%) 0

1

3

5

– – 121.5 331.0 299.7 216.8 137.5

41.0 72.0 120.7 247.7 342.2 529.0 265.4

– – – – 27.9 87.5 126.5

– – – – – – –

* – Rheolex RS; Car – -carrageenan.

25.5

Uses and applications

Konjac flour has been used as an important food ingredient for more than a thousand years. With the addition of a mild alkali such as calcium hydroxide, konjac flour aqueous solution (ca. 3% of concentration) changes to a strong, elastic and irreversible gel. The alkali treated konjac gel is quite a popular traditional Japanese food and is called Kon-nyaku in Japanese. Recently, synergistic gels prepared by mixing of other hydrocolloids are major products in the food industry as new types of healthy jellies. Clinical studies indicate that konjac mannan solution has the ability to reduce serum cholesterol and serum triglyceride. Konjac mannan also has an influence on glucose tolerance and glucose absorption. However, the alkali treated gel food does not have such effects. Konjac flour is suitable for thickening, gelling, texturing, and water binding. It may be used to provide fat replacement properties in fat-free and low-fat meat products. Applications and functional uses of konjac mannan are listed in Table 25.8.

Konjac mannan Table 25.8

Applications and functional uses of konjac mannan

Application

Function

Confectionery Jelly Yoghurt Pudding Pasta Beverage Meat Edible film

Viscosity, texture improver, moisture enhancer Gel strength, texture improver Fruit suspension, viscosity, gelation Thickening, mouthfeel Water-holding capacity Fibre content, mouthfeel Bulking, fat replacer, moisture enhancer Water soluble, water insoluble

25.6

423

Regulatory status

In Japan, konjac flour is accepted as a food ingredient and a food additive for thickening and as a stabiliser according to the provisions of the Food Sanitation Act. For regulatory purposes, a distinction must be drawn between konjac flour and konjac mannan, the separated polysaccharide. The Food Chemical Codex lists the current uses of konjac flour in the United States as gelling agent, thickener, film former, emulsifier, and stabiliser. Konjac flour is also used as a binder in meat and poultry products. Konjac mannan has been recognised as GRAS (generally recognised as safe) by the Food and Drug Administration (FDA) since 1994 and the US Department of Agriculture (USDA) accepted the use of konjac flour as a binder in meat and poultry products in 1996. In Sweden, it was recognised that konjac mannan has the ability to reduce serum cholesterol and indication of the effect was officially accepted enabling claims to be made for its use as a functional food. Konjac flour imported into Europe for diet food and pet food is rarely of consistent quality and does not meet EU standards. However, konjac mannan received a provisional European classification number as a food additive (E425) in 1998. Konjac mannan can thus be imported into Europe because it has achieved an E number.

25.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

References

S. OKIMASU (ed.), Science of Konjac, Keisuisha, Hiroshima (1984). S. TAKIGAMI and G. O. PHILLIPS Gums and Stabilisers for the Food

Industry, 8, 391 (1996), (eds G. O. Phillips, P. A. Williams and D. J. Wedlock), IRL Press, Oxford, UK. S. TAKIGAMI, T. TAKIGUCHI and G. O. PHILLIPS Food Hydrocolloids, 11, 479 (1997). K. MAEKAJI Agric. Biol. Chem., 38, 315 (1974). S. OKIMASU (ed.) (1984) Science of Konjac, Keisuisha, Hiroshima, Japan. P. A. WILLIAMS, S. M. CLEGG, M. J. LANGDON, K. NISHINARI and G. O. PHILLIPS Gums and Stabilisers for the Food Industry, 6, 209 (1992), (Eds. G. O. Phillips, P. A. Williams and D. J. Wedlock), IRL Press, Oxford, UK. G. J. BROWNSEY, P. CAIRNS, M. J. MILES and V. J. MORRIS Carbohydr. Research, 176, 329 (1988). P. A. WILLIAMS, D. H. DAY, K. NISHINARI and G. O. PHILLIPS Food Hydrocolloids, 4, 489 (1991). T. KASAI and Y. KOBATA Proceeding of Hokkaido University, 5, 145 (1965). N. KIMURA, K. MOTOKI, T. TAKIGUCHI and Y. SATOU Annual Report of Gunmaken Industrial Research Laboratory (1994), p. 147, Gunma, Japan. K. KATO and K. MATSUDA Agric. Biol. Chem., 33, 1446 (1969). H. SHIMAHARA, H. SUZUKI, N. SUGIYAMA and K. NISHIDA Agric. Biol. Chem., 39, 301 (1975). M. MAEDA, H. SHIMAHARA and N. SUGIYAMA Agric. Biol. Chem., 44, 245 (1980). F. SMITH and C. SRIVASTA J. Am. Chem. Soc., 81, 1715 (1959). T. SATO, A. MORIYA, J. MIZUKUCHI and S. SUZUKI Nippon Kagaku Zasshi, 91, 1071 (1970).

424 16. 17. 18. 19. 20. 21. 22.

Handbook of hydrocolloids and T. SUZUKI Agric. Biol. Chem., 48, 2943 (1984). T. NAKAJIMA and K. MAEKAWA Matsuyama Shinonome Gakuen Kenkyuronshu, 2, 55 (1966); 3, 117 (1967). S. OKIMASU and N. KISHIDA Hiroshima Joshi Daigaku, Kaseigakubu Kiyo, 13, 1 (1982). K. OGAWA, T. YUI and T. MIZUNO Agric. Biol. Chem., 55, 2105 (1991). N. KISHIDA, S. OKIMASU and T. KAMATA Agric. Biol. Chem., 42, 1645 (1978). Unpublished data. T. TAKIGUCHI, T. NARITA, K. SEKIGUCHI, I. YOSHINO and I. KAWANO Annual Report of Gunmaken Industrial Research Laboratory (1990), p. 168, Gunma, Japan. R. TAKAHASHI, I. KUSUKABE, S. KUSANO, Y. SAKURAI, K. MURAKAMI, A. MAEKAWA

26 Philippine Natural Grade or semi-refined carrageenan* H. J. Bixler and K. D. Johndro, Ingredients Solutions Inc., Searsport

26.1

History of product development

This form of carrageenan evolved into a food ingredient from a past that began as a way of treating raw Eucheuma seaweeds in the Philippines prior to export. In the 1960s carrageenan refiners in industrialised countries began to encounter environmental problems with their waste. This waste was high in BOD and colour, and the high level of salt in raw seaweed interfered with conventional waste pre-treatment processes. To counteract the growing cost of treating this waste there developed in the Philippines a pre-treatment process for the raw seaweed which yielded the item of commerce now called ‘alkali treated cottonii chips’. These chips gave substantially higher carrageenan yields in the refineries when compared with yields from dry, raw seaweed and generated much less BOD in waste streams. It was a relatively short step from this chip development to the development in the early 1970s of a powder made from these chips (‘alkali modified flour’) for use by manufacturers of canned pet food. Refined carrageenan had been used for years as a binder, stabiliser, gelling agent in these canned pet foods made from meat byproduct. By optimising the alkali treatment process, the performance properties of the alkali modified flour approached those of refined carrageenan, but at substantial cost savings to the pet food producers. Since sun or open bin drying was employed in the process, bacteria loadings in the flour were quite high. However, all canned pet food is retorted at high temperature, so the finished products containing the alkali modified flour are rendered sterile. By the early 1980s the quality of the alkali modified flour had been improved to the point where at least one major carrageenan processor was offering it in the US to dairy powder blenders for use in chocolate milk for human consumption. Bacterial reduction had rendered the improved product pathogen free, and aerobic plate counts were less than 5000 cfu/g. In addition, improvements had been made in colour, odour and taste. To distinguish the product for human consumption from the alkali modified flour, the names * Processed Eucheuma Seaweed; E407a.

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Philippine Natural Grade (PNG) carrageenan, and semi-refined carrageenan (SRC) were adopted by the producers. Most of the rest of the 1980s and early 1990s saw PNG tied up in various food regulatory jurisdictions while sorting out its food safety, its purity criteria or specifications and how it should be named (or numbered) on ingredients labels. This process was slowed appreciably by the sometimes bitter struggle between Philippine carrageenan producers and international carrageenan refiners. The disagreements were over whether PNG should be permitted as a food ingredient and how rigidly it should be distinguished from the carrageenan in existing food regulations. While sales of PNG for food use grew substantially during the 1990s, it was not until 1999 that the last major regulatory barrier to PNG was removed and its proximity to carrageenan in chemical, biological and ecological properties was confirmed.1 Because the product must be officially labeled ‘carrageenan’ in some countries (e.g. the US and Canada) and ‘PES’ or ‘E407a’ in others (e.g. European Union), the more neutral, but unofficial term PNG will be used in the remainder of this chapter.

26.2

Processing of PNG

The process for making PNG as it has finally evolved is shown schematically in Fig. 26.1. Raw seaweed in wire mesh baskets is contacted with hot KOH solution for a few hours. Impurities such as protein, DNA, lipids, sea salts and colour bodies are extracted by the alkali. In addition, most of the 6-sulfate groups on the 1,4 galactose units are converted to 3,6-anhydrogalactose or 3,6-AG (see Fig. 26.2), thus enhancing performance properties such as water gel strength and milk protein reactivity of the kappa and iota carrageenans found in PNG.2 In the carrageenan industry this enhancement is usually called ‘modification’. It should be emphasised that only kappa and iota carrageenan-bearing seaweeds (Eucheuma cottonii and Eucheuma spinosum, respectively)* are currently approved as sources of PNG. Since these are both gelling type carrageenans, it should not be surprising that optimising 3,6-AG conversion, and thereby gel strength enhancement, is an important processing goal. Extensive work with E. cottonii and its extracts have shown that the carrageenan polysaccharide in the untreated seaweed contains about 37% 3,6AG, whereas in both PNG and refined carrageenan it ranges from 44% to 46%.3 As would be expected from the chemistry described in Fig. 26.2, this increase in 3,6-AG is accompanied by a reduction in sulfate from about 22% in the raw seaweed to 17% to 20% in PNG and refined carrageenan, respectively. After the extraction step the seaweed is washed and chopped and then subjected to additional colour removal, dried in an apron or fluid bed dryer, milled to a powder and blended for standardisation. If necessary, a separate bacteria reduction step may be performed on the powder. Colour removal is usually accomplished with a dilute calcium or sodium hypochlorite wash and bacterial reduction by exposure of PNG to ethanol or iso-propanol vapour or heat. The PNG process parallels the refined carrageenan process, except for the extraction step. In making refined carrageenan, the polysaccharide is extracted or dissolved from the * The taxonomy of seaweeds periodically undergoes change. Scientists currently refer to these two seaweeds as Kappaphycus alverezeii and Eucheuma denticulatum, respectively. However, food additive regulations still include these older names.

Philippine Natural Grade or semi-refined carrageenan

Fig. 26.1

Block diagram of PNG process.

Fig. 26.2

Alkaline modification of PNG.

427

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seaweed to yield a carrageenan solution at a concentration of about two weight percent (which is subsequently reduced to one weight percent to facilitate filtration). PNG uses a reverse extraction process4 where impurities are extracted from the seaweed, and the polysaccharide is left in a gel state containing about 45 weight percent carrageenan and algal cellulose. It is this marked concentration difference during extraction or filtration that greatly reduces energy usage in making PNG and is responsible for most of its lower processing cost. For refined carrageenan, 99kg of water must be removed to recover 1kg of dry carrageenan. For PNG the ratio is reduced to 1.2kg of water per 1kg of dry carrageenan.

26.3

Chemical analysis of PNG

Analysis of numerous commercial samples of PNG from various producers has yielded the average composition by proximate analysis shown in Table 26.1. The most significant difference between different types of PNG and their refined carrageenan counterparts is the high acid insoluble matter (AIM) content of the former relative to the latter. Refined carrageenan generally contains 0.1% or less AIM compared with the six to eight per cent in iota PNG and 10% to 15% in kappa PNG. AIM is the residue of a hydrocolloid left after hydrolysis in 1%v/v H2SO4 at 100ºC. AIM, though widely used to assay crude hydrocolloids, has not been very well characterised in the scientific literature as to chemical composition, and it would be expected to vary with the type of hydrocolloid. AIM in PNG, however, has been analysed by various chemical and physical chemical methods and previously reported.5 The results of these chemical analyses are given in Table 26.2. Most commercially refined kappa and iota carrageenans contain substantially more salt than their PNG counterparts. The net effect is that both contain about the same weight percent of carrageenan polysaccharide. Of course, it is possible to prepare refined Table 26.1

Average composition of PNG1

Seaweed source

Carrageenan Carrageenan type

Eucheuma cottonii Eucheuma spinosum 1 2 3

kappa iota

AIM2

Protein

85% 4%

8% 2% 0.9% 2% 0.2% 0.1% 6% 1%

Composition of AIM

Component

Percent by weight

Cellulose Xylan Carrageenan Protein Fat Salts

90.0 41 2.1 1 2.0 2 4.4 1 0.3 0.2 1.2 0.3

1

95% confidence limits.

Salts

79% 4%3 12% 3% 1.0% 2% 0.2% 0.1% 8% 2%

Dry basis; % by weight. Acid insoluble matter. 95% confidence limits.

Table 26.2

Lipid

Philippine Natural Grade or semi-refined carrageenan

429

carrageenans with very little salt, but these are laboratory chemicals and not the items of commerce used as food ingredients. From the standpoints of major components, therefore, PNG differs from refined carrageenan primarily in its relatively high algal cellulose content and lower salt content. The other components of AIM are also found in commercially refined carrageenans at about the same overall levels as found in commercial PNG. The morphology of the cellulose (and by inference, the morphology of carrageenan) in PNG has been of interest in analysing the performance properties of PNG. 13C-NMR has been used by two laboratories to confirm that the insoluble portion of PNG is cellulose.2, 5 Using X-ray diffraction and 13C-NMR, one of these laboratories was able to conclude that the crystalline fraction of PNG cellulose is a type I polymorph similar to that found in food grade powdered or microcrystalline cellulose derived from wood pulp. In addition, this group estimated that the crystallinity of this cellulose in both kappa PNG from E. cottonii and iota PNG from E. spinosum PNG is 42% to 45%. Electron microscopy7 and atomic force microscopy8 have shed light on the organisation of the cellulose and carrageenan in PNG. Electron microscopy has shown that the cell wall organisation in untreated seaweed, where the carrageenan is known to reside, is at least partially carried over into the PNG product. As will be seen in Section 26.5, this organisation in PNG suppresses the swelling of powder particles in water and in aqueous curing brine, which enhances its use in certain processed meats. Atomic force microscopy of iota PNG has been carried out after dissolving the carrageenan and cooling the solution. The results suggest that the organised carrageenan chains freely interpenetrate the cellulose micro fibres which themselves have become slightly disorganised by the PNG extraction process. These observations support the additional findings that once the carrageenan in PNG has dissolved (heated to at least 85ºC in an aqueous medium), and it then gels on cooling, the cellulose micro fibres play essentially no role in the tensile properties of the gel. The only property of interest to food processors that is imparted by the cellulose is to make the gels cloudy or turbid. Cellulose does appear to elevate slightly the viscosity of hot PNG solutions due to the relatively high excluded volume of the insoluble cellulose micro fibres. The allowable content of heavy metals in food hydrocolloids is currently undergoing review by regulatory bodies. For instance, the Joint Expert Committee on Food Additives (JECFA) of the FAO/WHO Codex Alimentarius has recently proposed removing total heavy metals from their specifications for PNG and carrageenan and to reduce lead from 5mg/kg to 2mg/kg.9 In addition, JECFA proposed adding cadmium and mercury specifications of 1mg/kg while maintaining arsenic at 0.3 mg/kg. The European Communities has tentatively adopted similar changes, except that the lead limit remains at 5mg/kg, and total heavy metals has been reduced from 40mg/kg to 20mg/kg.1 None of these changes have been fully adopted, and various hydrocolloid trade groups are proposing that the reductions be taken in two steps. The industry needs time to develop reproducible test procedures specific to carrageenans and to make any process changes that may be necessary to meet the tighter standard. The timing seems not to be critical, since present heavy metals specifications at allowable daily intakes (ADI) for hydrocolloids as a group do not pose a health hazard. Atomic absorption spectroscopy (AAS) has been used by the authors’ laboratory to determine individual heavy metals in commercial samples of PNG and refined carrageenan.10 The results of this testing are shown in Table 26.3. If the heavy metals in carrageenans come from the seaweed raw material, the lesser processing received by PNG would explain its heavy metals content being on average somewhat higher than for

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Table 26.3 AAS analysis of commercial samples of PNG and refined carrageenan for regulated heavy metals Concentration (mg/kg) Refined cgn (39 samples) Metal As Cd Hg Pb

PNG (30 samples)

Average

Std. dev.

Average

Std. dev.

0.066 0.107 0.025 0.203

0.046 0.091 0.024 0.121

0.150 0.263 0.141 0.561

0.119 0.279 0.193 0.391

refined carrageenan, and the scatter in the data as indicated by standard deviation somewhat greater. However, other sources of the differences cannot be ruled out. The source of the heavy metals in PNG is currently under investigation by the author in collaboration with the Philippine industry and the Philippine government. However, even without the additional research, there appears to be no problem for PNG to meet the heavy metals specifications, including the proposed new level for lead of 2mg/kg, or an even tighter standard of 1mg/kg that has been discussed. The only other chemical difference between PNG and refined carrageenan that has received attention is molecular weight and molecular weight distribution. These physical chemical properties of food grade carrageenan have long been a regulatory issue. In the 1960s a highly depolymerised form of the polysaccharide was proposed for use as an ulcer therapeutic, but was found to cause ulcerative colitis in certain test animals.11 It was determined at that time that conventionally processed refined carrageenan was high enough in molecular weight and had a narrow enough molecular weight distribution so as not to overlap with the ulcer therapeutic (‘polygeenan’). That is, food grade carrageenan contains an undetectable amount of the low molecular weight polysaccharide ( 10,000 daltons) implicated in the ulcerative colitis syndrome. Representative commercial samples of kappa PNG have been subjected to gel permeation chromatography for molecular weight distribution determination. 12 A comparison was made with two typical refined carrageenans and one PNG. The chromatograms are shown in Fig. 26.3, where it can be seen that PNG has a slightly higher peak molecular weight and a somewhat narrower molecular weight distribution than the refined carrageenans. From this standpoint, therefore, neither PNG nor refined carrageenan are toxicologically active.

26.4

The regulatory status of PNG as a food ingredient

An exhaustive discussion of this subject would subject the reader to a lot of extraneous interplay between industry and regulators wherein food safety questions were raised to slow the approval of PNG, so refined carrageenan producers could adjust to the economic impact of lower cost competitors. In the end, science and reason prevailed, and this chapter will simply review the regulatory status of PNG in various jurisdictions as it existed at the beginning of 2000. Table 26.4 summarises the specifications for purity criteria for PNG as most recently published by Codex Alimentarius, the European Commission and the Food Chemical Codex.9, 1, 13

Philippine Natural Grade or semi-refined carrageenan

431

Fig. 26.3 Mw distribution curve of two commercial, refined (Sherex 610 and Genulacta L100) and PNG (Sherex IC 109) (kappa)-carrageenan stabilisers measured using HPSEC-MALLS and eluted in 0.1M LiCl at 60ºC.12

Table 26.4

Purity criteria for PNG

Criterion

European Commission1

Codex Alimentarius9

Food Chemical Codex13

Residual alcohols pH Viscosity of a 1.5% sln @ 75ºC Loss on drying after 4 h at 105ºC Sulfate (as SO4) Total ash (550ºC) Acid insoluble ash (in 10% HCl) Acid insoluble matter (in 1% v/vH2SO4) Arsenic Lead Mercury Cadmium Heavy metals (as Pb)

0.1% total – 5 mPa/s 12% 15% 40% 1% 40% 1% 8% 15% 3 mg/kg 5 mg/kg 1 mg/kg 1 mg/kg 20 mg/kg

0.1% total 8 to 11 5 mPa/s 12% 15% 40% 15% 30% 1% 8% 15% 3 mg/kg 2 mg/kg6 1 mg/kg6 1 mg/kg6 –6

Total plate count Yeast and mould E. coli Salmonella spp.

5000 cfu/g 300 cfu/g negative in 5g negative in 10g

5000 cfu/g – negative in 1g negative to test

– – 5 mPa/s 12% 18% 40%4 35% 1% –5 3 mg/kg (ppm) 10 mg/kg – – 40 mg/kg (0.004%) – – negative to test negative to test

1. See Ref. 1. 2. See Ref. 9. 3. See Ref. 13. 4. PNG as a food ingredient in the US is defined by the Code of Federal Regulations (21 CFR 172.620) for carrageenan after a ruling in 1990 stated that PNG met the definition of carrageenan. The FCC purity criteria are in agreement with the CFR except for sulfate which in the CFR is 20% 40%. 5. The Second Supplement to the Third Edition of FCC contains an AIM limit of 2% for carrageenan, but this has never been included in the CFR definition. The NAS committee that developed the Fourth Edition of the FCC could not resolve this matter and as a result, there is no carrageenan monograph in the Fourth Edition. 6. Proposed; currently under review (2000).

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PNG was confirmed in 1990 to meet the existing US specifications for carrageenan contained in 21 CFR 172.62014 and the Food Chemical Codex.13 This permitted PNG to be labelled simply as carrageenan on processed food ingredients labels in the US so long as it meets the specifications spelled out in the referenced documents. The European Commission (EC) in 1998 issued a directive in which PNG carrageenan was given the label name of PES or Processed Eucheuma Seaweed with an E-number of E407a (refined carrageenan has an E-number of E407). This was accomplished after a thorough review by the EC-Scientific Committee for Food (EC-SCF) of a dossier on PES prepared by the Seaweed Industry Association of the Philippines (SIAP),1 which included a 90-day rat feeding study and an Ames test for mutagenicity performed by the Bureau of Food and Drug (Philippine BFAD) and the University of the Philippines (UP), respectively. The FAO/WHO Codex Alimentarius under its Joint Expert Committee on Food Additives (JECFA) concluded in 1995 that it was not satisfied with the BFAD and UP toxicology studies and requested the Philippine industry to upgrade the level of the studies by choosing an internationally recognised organisation to perform the work. The new work was performed by BIBRA in the UK, and the results were submitted to JECFA for their forty-first meeting in 1998. In 1999, JECFA published its conclusions that PES extracted from E. cottonii and E. spinosum lacked toxicity.15 Further, it recommended to the Codex Committee on Food Additives and Contaminants (CCFAC) that PES be included with refined carrageenan having a temporary group ADI ‘not specified’ to be reviewed in 2001 for removal of the temporary limitation. The term ‘not specified’ means there is no limit on the daily intake of PES by humans so long as the use is technically justified and the food processor employs Good Manufacturing Practice (GMP). Most nations of the world regulate the use of food ingredients through the practices of one of the regulatory or advisory bodies noted above or some combination of them. Some lack of harmonisation of the ingredients label name still exists. In the US (and Canada) PNG is carrageenan and in Europe and elsewhere PNG is PES (or E407a).

26.5

Applications of PNG in foods

26.5.1 Introduction In many food applications, iota and kappa carrageenans, whether PNG or refined, are interchangeable. Therefore, it may be instructive to compare similarities and differences of these forms of carrageenan before launching into specific applications. First of all, only gelling type PNGs are approved for food use. Therefore, there is no PNG that could be used in a cold soluble dairy application, i.e. a lambda carrageenan in a chocolate dairy powder to be dispersed in cold low-fat milk to make a cold instant chocolate drink where the carrageenan provides ‘mouthfeel’. While kappa carrageenan is widely used in Asia to make water dessert jellies, PNG kappa has not found use in this application. Asians like their jellies to be sparkling clear, and the cellulose or AIM in PNG make the jellies cloudy or opaque. Except for these two applications, PNG has found its way into most other food applications for cost savings as well as performance advantages. In general, PNG is about two thirds the cost of an undiluted refined carrageenan. Performance advantages cannot be generalised, but will be covered in specific applications below. Frequently PNG and refined carrageenans are blended to form a product with unique properties taking advantage of the additive, or sometimes synergistic, attributes of PNG and refined

Philippine Natural Grade or semi-refined carrageenan

433

carrageenans. Therefore, in the industrial marketplace the price distinction made above becomes fuzzy and the market driven commercial balance of cost and performance takes over. Occasionally an application for PNG arises that is neither a processed meat nor dairy product, i.e. an unusual salad dressing or canned soup, but well over 90% of the use of PNG is either in a meat or a dairy product. The remainder of this chapter will consider the technology of meat and dairy applications for PNG separately. It should be pointed out that the following information on applications for PNG has been taken primarily from the authors’ experience in the US market. PNG has penetrated more or less uniformly meat and dairy applications in the US. This is not the case in Western Europe where there has been a resistance to change from a long history of using refined carrageenan, and cost pressure on ingredients, until recently, has been less of a factor than in the US. On the other hand, PNG has penetrated more heavily meat applications in Central and South America and in Eastern Europe than it has in the US. This trend has not carried over to dairy applications which are either less highly developed in this region, or they are patterned after Western European products and carrageenan usage.

26.5.2 Meat applications Starting in the 1980s, refined carrageenan has been employed by meat processors as a texturising and water binding agent. This was particularly beneficial in precooked and vacuum packaged ham and turkey breast, although more recently the technology and benefits have been extended to chicken, bacon, roast beef and fish. Whole muscle products as well as restructured, comminuted and emulsion products are involved. Initially, carrageenan was used to retain the natural juices of meats that were lost in cooking and smoking or showed up as purge in vacuum packages. The advantages to the meat processor were a juicier product in a more attractive package and a higher product yield from slaughtered animals. The technology has since been extended to pumped products where additional water in the form of curing brines is added to the meat through injection and/or massaging. In the United States, pumping is usually limited to 100% added ingredients (including water), but in countries where meat is sold for a higher retail price, relative to other grocery products, extensions by pumping may reach 200% added ingredients. Labelling of extended meat products is controlled in the US by the Department of Agriculture (USDA). For instance, in the case of ham, the label categories shown in Table 26.516 are based on the amount of extension as determined by the concentration of original meat protein in the finished product. For other meat products, where Standards of Identity may control familiar label names, fanciful names for products containing carrageenan have been approved by USDA, e.g. beef roll for a processed roast beef containing carrageenan. The way in which carrageenan functions in these meat applications is still subject to debate and displays the need for more in-depth experimentation. At the time of writing, it is generally agreed that the carrageenan and brine juices in the meat form a water gel that penetrates the interstices of the muscle fibre to add strength and syneresis control. However, there is also some suggestion that the carrageenan interacts with the myosin protein solubilised during tumbling and massaging to form a carrageenan/protein gel. Otherwise, how would the kappa carrageenan completely dissolve to form a strong gel on cooling when the cooking temperatures (65–75ºC) are below the melting/dissolution

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Table 26.5

Labelling of cooked ham products as required by the US Department of Agriculture

Label designation Ham Ham with natural juices Ham, water added Ham and water product – X% of weight is added ingredients

Protein fat-free (minimum %) 20.5 18.5 17.0 < 17.0

temperature of the carrageenan? Another model has the carrageenan particles simply swelling after being cooked and forming on cooling a swollen carrageenan particle, coagulated myosin gel, which provides the texture and water-binding attributes. It is not the purpose of this chapter to present a primer in meat processing, and the reader is referred to standard references for more in-depth discussion.17, 18 Instead, the introduction of PNG into this application and its performance will be considered. By the early 1990s kappa PNG began to make inroads into these meat applications, initially for cost savings, but later on for some distinct performance advantages over refined carrageenan. Carrageenans with relatively strong break forces or gel strengths are needed to enhance the texture of extended products. Therefore, iota PNG has found little use in meats because of the weak, rubbery texture of its gels. The performance advantages of PNG are: • less particle swelling in cold brine, resulting in less injector needle clogging and better meat tissue penetration • less syneresis or purge from a kappa PNG when adjusted in gel strength to meet texture requirements • better hiding of the inevitable ‘gel pockets’ or ‘tiger striping’ in highly extended meat products by opacifying the gel. While proof of these advantages will be presented later in this section, the selection of PNG versus refined carrageenan by a meat or poultry processor is still largely subjective . . . some swear by refined carrageenan and others by PNG with conflicting technical justifications for either decision. However, it is safe to say that PNG is well respected and widely used in the meat and poultry industries and, in the course of a few years’ use, now stands side by side in performance and reliability with refined carrageenan. Not all refined kappa carrageenans are equal, and the type recovered by KCl precipitation and gel pressing is preferred by meat and poultry processors over the type recovered by alcohol precipitation. The lower cost of the former, is the overriding consideration since syneresis and purge are generally lower for the latter when they are compared at equal gel strength. The very strong gel strength of the KCl precipitated carrageenan also gives it a slight performance edge in very thinly sliced or ‘slivered’ extended meats. Some carrageenan products for meat and poultry applications are blends of PNG and refined. In this way, some of the advantages of each type can be preserved. KCl is frequently added to these blends to reduce their cost while taking advantage of the strong effect of K+ ions on the gel strength of both the refined and PNG kappas, and making use of PNG’s superior syneresis control. Figure 26.4 is a bench-top syneresis experiment. Gels made with 1% carrageenan and a 2% NaCl brine were heated to the specified temperature, held at that temperature for 10 min., and then refrigerated over night at 4ºC, after which the liquid lost from the gel was

Philippine Natural Grade or semi-refined carrageenan

Fig. 26.4

435

Syneresis/purge in carrageenan gels made in NaCl brine.

measured (syneresis or purge). The gelling agents are a KCl precipitated, gel pressed refined carrageenan, a PNG and a 50:50 blend of the two. There is little difference in the relative syneresis of the three carrageenans if they are heated to 68ºC or higher, and the relative values are quite low. However, if the gels are heated to less than 68ºC, the carrageenan particles do not swell sufficiently to give good water binding. The stronger gelling K+-rich gel pressed carrageenan gives more syneresis than the PNG or to a blend of the two. Figure 26.5 examines the swelling of carrageenan particles in 4ºC brine as an indication of how prone the carrageenan types would be to clogging injector needles or interfering with particle dispersions in the meat muscle fibre interstices. Again, three types of carrageenan (refined, PNG and a 50:50 blend of the two) are being considered. The cumulative distribution plot in Fig. 26.5 clearly shows that PNG swells less than the

Fig. 26.5

Carrageenan particle swelling at 4ºC in 2% NaCl brine.

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Handbook of hydrocolloids

Fig. 26.6

Hydration profile for a sodium PNG and a potassium PNG (1.5% gum in 2% NaCl brine).

blend, and both swell less than the refined carrageenan. For instance, only about 50% of PNG in the swollen state are larger than 200 mesh ( 74m), whereas about 75% of the particles of the blend are larger than 200 mesh and about 90% of the refined carrageenan exceed this size. Not all PNGs are equal. Figure 26.6 shows the hydration of two PNGs in 2% NaCl brine as a function of temperature from 0–80ºC. Relative hydration was determined by Brookfield viscosity of the brine slurry of PNG powders. A typical temperature sweep of this type will show a rise in viscosity as the excluded volume of swollen particles increase (rise in Einstein viscosity) with increasing temperature. As the swollen particles reach their melting point, the viscosity will begin to fall with temperature as the carrageenan goes into solution. For the potassium treated PNG, hydration begins at about 45ºC, but only rises slightly and levels off after about 50ºC. It begins to go into solution at about 70ºC. For the sodium-treated PNG, swelling begins at about 25ºC and rises rapidly above 40ºC to a maximum at about 65ºC and stays at this degree of swelling until it reaches a melting point of about 80ºC. These observations are a manifestation of the well-known effect of K+ and Na+ on the double helix formation and helix aggregation and their effect on the gel strength of kappa carrageenan.19 With respect to meat applications, a range of performance can be achieved by blends of these two types of PNG. For a tumbled or massaged product without injection, a relatively high percentage of the high swelling sodium treated PNG is desired. For injected and tumbled product, a higher percentage of the potassium treated PNG with less particle hydration at lower temperatures is desired. Finally, consider some of the cost/performance tradeoffs available to the meat processors when using carrageenan. Figure 26.7 is a plot of a ratio of syneresis to gel strength against the amount of KCl or PNG added to KCl gel pressed refined carrageenan to reduce cost. In general, syneresis, and thereby package purge, increases as gel strength increases, so the aim in making the blends shown here is to extend the flat portion of the curve to the highest concentration of cost reducing additive. While KCl is the cheaper

Philippine Natural Grade or semi-refined carrageenan

Fig. 26.7

437

Extending KCl gel pressed carrageenan with added KCl or PNG.

additive, the overall performance of the system using PNG is much better, especially when compared at approximately equivalent cost (10% additive when KCl is used versus 20% additive when PNG is used). Part of the superior performance of the PNG/gel pressed refined carrageenan blend is its higher gum solids. Even though KCl can greatly increase the gel strength of kappa carrageenan, gum solids are needed to reduce syneresis. Higher gum solids also contribute to texture enhancement not captured in a simple gel strength measurement.

26.5.3 Dairy applications The principal uses of PNG carrageenan by US dairies are in chocolate milk and ice cream, both hard pack and soft serve. Minor uses include puddings and dairy-based dressings such as cottage cheese dressing, but only the major uses will be considered in this chapter. Very few dairies in the US make their own stabiliser formulations for these applications. Instead, a whole industry has been built around formulating or blending technology. These dairy blenders are the customers for producers of dairy type carrageenans. This is different from current dairy practice in Europe, where many dairies still do their own stabiliser formulation. Furthermore, flans and ready-to-eat dairy products are big supermarket items in Europe, but not in the US. With the slow introduction of PNG in Europe, its potential in these dairy products is virtually unknown. Carrageenan is added to chocolate dairy powders to suspend the cocoa particles in the finished chocolate milk and to add mouthfeel (viscosity) to skim or low fat (2% butterfat) milk-based chocolate milks. Approximately 200 to 300ppm of PNG in the finished milk is used for these purposes. Since each blender of dairy powders sells to a large number of dairies with quite different processing equipment, the carrageenan stabiliser must be very forgiving. Otherwise, the different levels of shear and holding times to which the chocolate milk is subjected, can adversely affect performance. Besides gross settling of cocoa, poor stabiliser performance might be manifested in several ways from ‘white cap’

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(incipient separation of curds and whey with a thin layer of white whey forming on top of the chocolate milk) to ‘mottling’ (where the curds and whey separation penetrates as a fibrous web of white into the interior or the chocolate milk mass). PNG carrageenan offers no outstanding performance attributes in chocolate milk when compared with comparable refined carrageenans. It comes down to a matter of PNG saving the dairy blender cost at equivalent performance, and since the chocolate milk business is one of thin profit margins, cost savings are very welcome. PNG carrageenan has 70% to 80% of the chocolate dairy powder market in the US at the time of writing. Blends of kappa and iota PNG have been found to give the best chocolate milk performance. The kappa component seems to provide moderate milk viscosity or mouthfeel and good cocoa suspension, while the iota component contributes mostly mouthfeel. If the level of iota is too high, it can actually reduce the carrageenan’s cocoa suspension capabilities. A more complex blend of kappa and iota PNG plus lambda/kappa-2* refined carrageenan offers further performance advantages while still maintaining a cost advantage over an all-refined carrageenan blend. The lambda/kappa-2 component enhances the stability of the PNG components by extending the concentration range between the onset of cocoa settling and the onset of milk gelation (with its undesirable livery texture). The lambda/kappa-2 component also makes the stability of chocolate milks less sensitive to the wide variation in processing shear encountered in dairies across the US. Figure 26.8 is a plot of test results used to characterise carrageenan efficacy in chocolate milk stabilisation. Pasteurised chocolate milk is prepared from cocoa, sugar, low-fat milk, and various levels of carrageenan (as ppm in the finished chocolate milk). After overnight refrigeration at 4ºC, the viscosity of the chocolate milk is measured using a Krimko cup (a cup-type capillary viscometer). The settlement of cocoa, the occurrence of white cap or mottling and the presence of any liveriness (gelation) is also noted from visual observation. The ranges shown by arrows in the figure are referred to as the spread for the carrageenan and mark the onset of cocoa settling (called dusting) at the low end of the spread and the onset of gelation (called ripple) at the high end of the range. The spread is recorded in ppm as the difference in concentration between dusting and ripple. The useful viscosity building or mouthfeel potential of the carrageenan is characterised by the milk viscosity ratio which is simply the slope of the lines in Fig. 26.8 reported in sec/ppm. The carrageenan stabilisers used in constructing Fig. 26.8 are a pure refined carrageenan of the lambda/kappa-2 type and a PNG of both the kappa and iota type, to which small amounts (<10%) of the pure refined carrageenan have been added. As a control, a formulated refined carrageenan blend containing kappa/kappa-2/lambda and dextrose, was used. The pure refined carrageenan displays a high milk viscosity ratio indicating good viscosity or mouthfeel building potential. However, its spread is quite narrow (20ppm). In practical terms, this type of stabiliser would be too unforgiving, and its concentration in dairy powders being used by different dairies with different procedures would require frequent adjustment. Therefore, the industry has demanded products with better spread

* Kappa-2 is the weak gelling copolymer of kappa and iota carrageenan that comes from commercial temperate water red seaweed and accompanies the lambda carrageenan that also comes from the same commercial seaweeds. There is no practical way to separate these fractions, and the best one can do in practice is to control their ratio by seaweed selection and in-process blending.

Philippine Natural Grade or semi-refined carrageenan

Fig. 26.8

439

Chocolate milk stability for various carrageenans as measured by milk viscosity ratio and the milk index spread.

(50ppm) to make one dairy powder formulation fit the end use requirements of more dairies. Both the refined carrageenan blend used as a control and the PNG formulation have the desired spread and reasonably good milk viscosity ratios. However, there is a cost savings advantage to using the PNG formulation, even with the addition of the small amount of refined carrageenan. The refined carrageenan is not added to the PNG to modify either the spread or the milk viscosity ratio. In fact, a straight PNG kappa/iota would be indistinguishable from the PNG/refined formulation shown in Fig. 26.8 if it had been included in the study. Instead, the refined lambda/kappa-2 carrageenan is added to give better shear stability. This is confirmed in the laboratory by subjecting the pasteurised chocolate milk after overnight standing at 4ºC to uniform shear from a laboratory propeller stirrer. The sheared chocolate milk is allowed to stand at room temperature for four hours, after which it is observed for cocoa settling, white cap and/or mottling. The small addition of the refined carrageenan to the PNG noticeably improved the stability of the chocolate milk to shear. Figure 26.9 shows the results of a milk test designed to characterise carrageenans for stabilising hard pack ice cream. Carrageenan is used in the ice creams for two purposes: 1. 2.

to prevent whey-off (severe curds and whey separation) in ice cream mixes before they are frozen to retard ice crystal growth.

Only (1) is normally tested for during development of carrageenan stabilisers, since all carrageenans behave more or less the same with respect to (2). Liquid ice cream mixes generally contain relatively high concentrations of hydrocolloids (e.g. locust bean gum, carboxymethyl cellulose) that are notorious for promoting whey-off. Carrageenan is added to retard this whey-off, although with shorter and shorter liquid mix holding times in dairies these days, this application for carrageenan is less critical than it once was. In Fig. 26.9 a refined kappa/kappa-2/lambda carrageenan is compared with a kappa/iota PNG. Over the entire range of carrageenan concentrations studied, the PNG outperformed the more complex refined product. It is suspected that the high milk protein reactivity of

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Fig. 26.9

Comparing two carregeenans for hard pack ice cream mix stability.

kappa-2 carrageenan is responsible for the poorer whey-off protection offered by the refined carrageenan blend. The final dairy application that will be considered here is soft serve ice cream, which falls more or less between chocolate milk and hard pack ice cream in its demands on carrageenan. Chocolate milk puts the greatest demand on carrageenan for both stability and viscosity building. Hard pack ice creams with their relatively high fat and high solids content and their need only for whey-off protection, place the least demands on the carrageenan. Soft serve ice cream, which is generally low in fat and intermediate in solids, needs good viscosity build and viscosity control from the carrageenan plus some whey-off protection and ice crystal growth retardation. PNG for the soft serve ice cream application needs a higher iota to kappa ratio than for hard pack ice cream or chocolate milk. Figure 26.10 shows the results of experiments to optimise the stabiliser composition. Higher iota to kappa ratio helps to reduce whey-off, but less kappa in the blend means less viscosity build. A satisfactorily low level of wheyoff has only been achieved by adding a refined kappa2/lambda carrageenan to the blend. To date the required balance of whey-off and viscosity has only been achieved with a blend of PNG with a high iota to kappa ratio (70/30) and a relatively high percentage (30+%) of refined kappa-2/lambda carrageenan (‘G. radula extract’). This optimum range of blend composition is highlighted in Fig. 26.10.

26.6

Concluding remarks

This chapter updates and extends the scope of an earlier article on PNG;20 in neither have the authors intended to imply that this newest member of the carrageenan family of hydrocolloids has revolutionised the industry. However, it has offered food processors a lower cost alternative to all refined products and, in some situations, superior performance. Long resisted by makers of refined carrageenan, PNG has now been embraced by most of them at the time of writing in early 2000. Either through joint

Philippine Natural Grade or semi-refined carrageenan

441

Fig. 26.10 Optimising the composition of PNG-based stabiliser for soft serve ice cream.

ventures or through their own factories, the refiners can now offer PNG and PNG/refined carrageenan blends for most applications. After a somewhat rocky start, the independent PNG producers have proven themselves capable of serving the world’s markets with high quality and reliable products. They have proven themselves at not only being adept at manufacturing, but also at applications development and marketing. For more information on PNG and how it might fit into your application, contact the marketing department of one of the following: Shemberg Marketing Corp. Osmen˜a Blvd. Cor. Lapu-Lapu St. Cebu City, Philippines Fax: +63-32-3461-892 Marcel Carrageenan Corp. 5th Floor, First Marcel Tower 926 Araneta Ave. Quezon City 1104, Philippines Fax: +63-2-712-1989 Quest International (Philippines), Inc. GF/SFB#1, Mactan Export Processing Zone Lapu-Lapu City 6015 Cebu, Philippines Fax: +63-32-340-0328 This is not an exhaustive list of producers, but lists those who have been in the business for the longest and, therefore, have the most experience. In the US the distributor/blender with the greatest concentration of effort on developing high performance carrageenan blends based on PNG is:

442

Handbook of hydrocolloids Ingredients Solutions, Inc. 33 Mt. Ephraim Road Searsport, ME 04974 USA Fax: +1-207-548-2921

26.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

References Official Journal of the European Communities L334 (1998), Commission Directive 98/86/EC. HOFFMANN, R. A., GIDLEY, M. J., COOKE, D. and FRITH, W. J. (1995), Food Hydrocolloids 9 (4), 281–9. TYE, R. J. (1994) Gums and Stabilisers for the Food Industry 7, 125 ff., (eds) G. O. Phillips, P. A. Williams and D. J. Wedlock, Oxford University Press, Oxford. Report to Congressional Requesters GAO/HEHS-94-11, (1994), United States General Accounting Office, Washington. BIXLER, H. J. (1995) Hydrobiologia 326/327, 35–57. FURNEAUX, R. (1995) New Zealand Institute for Industrial Research and Development, Personal Communication. SCOTT, J. (1998) William and Mary College, USA, Personal Communication. MORRIS, V. (1998) Gums and Stabilisers for the Food Industry 9, 361 ff., (eds) P. A. Williams and G. O. Phillips, The Royal Society for Chemistry, Cambridge. Food and Nutrition Paper 52, Addendum 6, (1998), FAO, Rome. COOK W. (1999) Soils Laboratory, University of Maine, USA, Work performed under contract to the authors. WATT, J. and MARCUS, R. (1969) J. Pharm. Pharmacol. 21, 1875–85. HOFFMANN, R. A., RUSSELL, A. L. and GIDLEY, M. J. (1996) Gums and Stabilisers for the Food Industry 8, 137 ff., (eds) G. O. Phillips, P. A. Williams and D. J. Wedlock, Oxford University Press, Oxford. Carrageenan Monograph, Food Chemical Codex, Third Edition, (1981), National Academy Press, Washington DC, 74–5. Code of Federal Regulations (1990), 21 CFR 172.620, US Government Printing Office. GREIG, J. B. (1999), Safety evaluation of certain food additives, WHO Food Additives Series 42, IPCS, WHO, Geneva. Code of Federal Regulations (1995), 9 CFR 319.104, US Government Printing Office. OCKERMAN, H. W. (1989) Sausage and Processed Meat Formulations, Van Nostrand Reinhold, New York. Handbook for the Meat Processing Industry (1990) Copenhagen Pectin A/S, Denmark. REES, D. A. (1972) Biochem. J. 126, 257–73. PHILLIPS, G. O. (1996) Gums and Stabilisers for the Food Industry 8, 403 ff., (eds) G. O. Phillips, P. A. Williams and D. J. Wedlock, Oxford University Press, Oxford.

Index

Acetobacter 321 acid hydrolysis 33, 48, 332 acid insoluble matter (AIM) 428–9 acid milk drinks 183, 184, 228–9, 315–16 acid stability alginates 386 carrageenan 91–2 karaya 242 MCC 340 SSPS 312 starch selection and 54–5 tragacanth 235 TSX 258 xanthan gum 109–10 acid treatment 69 acidic conditions 315–16 acrylic acid 375 acylation 371–2 adhesive properties 242, 313 adhesives 134, 347–66 adhesion tests 357 bioadhesives 356–7 food uses/applications 355–6 future prospects 361–2 non-food uses/applications 354–5 wet glues 357–61 adsorption 335 agar 14, 21–40, 421 applications 33–8 chemical structure 26–8 gelation 28–33 manufacture 23–6 new developments 38–9 agarobioses 26–8 agaropectins 28 agarophyte seaweed 22–3 alcohol 110, 255, 257, 341 aldehydes 372–3

aldimine cross-links 71–2 aleurone cells 137–8 alginates 14, 347–8, 379–95 alginate-chitosan complexes 374–5 gels and gelling 387–93 limitations 384–7 liquid phase 387 manufacture 380–1 properties 381–4 regulatory status 393 structure 381–2 alginic acid gels 384–5, 392–3 alkali hydrolysis 33 alkali modified flour 425 alkaline treatment 69 -lactalbumin 190–2, 200 amidated pectins 173 Amorphophallus 413, 415 amyloids 259 amylopectin 42, 44, 45 amylose 42, 44, 45 antifungal preservative 375 anti-oxidative properties 314 anti-sticking effect 318 arabic, gum see gum arabic attrition 332 bacterial cellulose 321–30 manufacture 321–2 regulatory status 330 structure 322–3 technical data 324–6 uses/applications 326–30 baked goods 57, 112–13 bakery fillings 112–13, 223–4, 237 bakery products 244 cellulosics 228 milk proteins 205, 206

444

Index

bakery products (continued) pectins 179–81 bark and foreign matter (BFM) content 239–40 barley 288–9 Bassorin 233 batters 57–8, 112 beef patty 304 -glucans 287–307 manufacture 288–91 regulatory status 305 structure 291–7 molecular structure 291–3 tertiary structure 293–5 technical data 297–300 uses/applications 301–5 -lactoglobulin 190–2, 200 beverages see drinks bioadhesives 356–7 birefringence 46 blood substitutes 83 Bloom strength 76–7 blueberry muffin 302–3 bonding strength 357, 359–61 Brabender viscoamylograph profiles 50, 51 breadings 57–8 cakes 402 calcium fortified milk 342–4 capsules 81–3 carboxymethyl cellulose (cmc) 11, 219, 222, 225–9, 350 carob bean gum see locust bean gum carrageenan 4, 14, 87–102, 139–40 coatings 349, 350 food applications 95–100 manufacture 87–9 physical properties 91–5 PNG/PES see Philippine Natural Grade regulatory status 101 structure 89–90 synergism with konjac mannan 94–5, 98, 421, 422 caseinates 193–5 caseins 189–93, 196 carrageenan-casein interaction 95 CMC-casein complex 222, 223 manufacture 193–5, 201 see also milk proteins cellulose 145, 247, 248, 349–50, 429 bacterial see bacterial cellulose MCC see microcrystalline cellulose cellulosics 219–30 applications 222–9 manufacture 219–20 properties 220–2 regulatory status 229–30 structure 220, 221 chain length 221, 333, 400 chaotropic agents 32 cheeses 100, 206–7 chemical gels 28 chemically modified gelatin 85 chitin 367–8

chitosan 348–9, 367–78 chemical structure and molecular characterisation 368–9 derivatives 369–70 production 369 properties 370–5 chocolate milk 437–9 cholesterol 287, 295–6 coagulation 202–3 coatings 213, 347–66 edible protective films 347–50 film-application techniques and stages 353 future prospects 361–2 methods for testing 353–4 parameters to consider 351 non-food coatings 352–3 Codex Alimentarius 5 Codex Alimentarius Commission (CAC) 5 Codex Committee for Food Additives and Contaminants (CCFAC) 5, 432 coffee creamers 206 cold water soluble gelatins 84 collagen 67, 70–3 colloidal type MCC 332–3, 334, 336–45 see also microcrystalline cellulose compatibility 351 karaya 243 tragacanth 235 TSX 259 xanthan gum 110 compressibility 335 confectionery 80 gellan gum 129–31, 132 gum arabic 166–7 milk proteins 209–10 pectins 184–6, 187–8 starch 58–9 tragacanth 237 conformations -glucans 295 curdlan 271–4 molecular conformations 277–8 xyloglucan 253–4 convenience foods 212 conversions 48–9 cooked rice/noodles 318 co-precipitates 201 cosmetics 237–8, 244, 354 CRC Biopolymer 405–12 composition 410–11 CRC process 406, 407 emulsion stability 409–10 patent status 411 product specifications 406–8 properties and efficacy 408–9 cream paste type MCC 332–3, 334 see also microcrystalline cellulose critical polymer concentration (C*) 7–8, 10 Cross equation 8–9 cross-linking alginates and ionic 388–92 in collagen 70–3 starch 47

Index crystallinity 335 curdlan 269–86 applications 283–4 chemical structure 270–1 functional properties 271–83 native curdlan 271 production 370 regulatory status 285 dairy products 81, 113, 243–4, 403 carrageenan 99–100 gellan gum 128–9 milk proteins 205–7 pectins 183–4 PNG 437–40 starch 59–60 see also milk products dairy spreads 401 degree of polymerisation (DP) 221, 333, 400 degree of substitution (DS) 48, 220, 222 dental moulds 37–8 depolymerisation 369 dessert products 36 frozen desserts 328–9 jellies 125–6 milk proteins 208–9 pectins 183–4, 185–6 dextran sulfate 374 dextrinisation 47, 48–9 dextrins 48–9, 290 dextrose equivalent (DE) 49 diafiltration (DF) 199 dietary fibre 16–18, 312 dietary preparations 210–12 diffusion setting 389, 390–1 dilatant viscosity 324–5 dispersion colloidal-type MCC 339–41 gellan gum 118 stabilisation and SSPS 314–16 stability of bacterial cellulose 325 xanthum gum 105–7 drinks 58, 81 bacterial cellulose 327–8 cellulosics 225–6, 227–8 gum arabic 167 milk proteins 208 soft drinks 227–8, 237 droplet size 408–9, 409–10 dry mixes 61, 114 E numbers 7, 8, 229–30 edible protective films 347–50 emulsification/emulsions 58 CRC Biopolymer 408–10 gum arabic 165–6 milk proteins 204–5 SSPS 316–17 tragacanth 235, 237 TSX 260 encapsulation, flavour 58, 167–8 end use 57 endosperm 137–8

445

enzyme hydrolysis 49 enzymes 110 gelation of chitosan 372 tailoring of TSX properties 263–5 European regulation 5–6, 160, 229–30, 423 label declarations 64–5 fat replacement 53, 261, 330 -glucans 301–5 fat spreads 401 fats 56 fermentation 17, 321–2 fibre, dietary 16–18, 312 fill-viscosity starches 61 fillings 402 bakery fillings 112–13, 223–4, 237 pie fillings 112–13, 181, 182 films 134, 224, 242, 313 application techniqes and stages 353 edible protective films 347–50 milk proteins 213 see also adhesives; coatings flavour emulsions 316–17 flavour encapsulation 58, 167–8 flies 37 fluid gels 126–8 fluorescent lights 352 foams 204–5, 225 stabilising 317–18 food applications adhesives 355–6 agar 33–6 bacterial cellulose 326–30 -glucans 301–5 carrageenan 95–100 cellulosics 222–9 curdlan 283–4 FrutafitÕ-inulin 401–3 galactomannans 153 gelatin 80–1 gellan gum 124–34 gum arabic 166–8 karaya 243–4 konjac mannan 422–3 MCC 341–5 milk proteins 205–13 pectins 176–88 PNG 97–8, 432–40 SSPS 314–19 starch 57–63 tragacanth 236–7 TSX 259–61 xanthan gum 112–15 Food Chemical Codex 6–7, 160–1 fractionation of milk proteins 196, 200–1 freezing-thawing method 24–5 frozen products 80, 114 bacterial cellulose and frozen desserts 328–9 cellulosics 226, 227 ice cream see ice cream karaya 243 tragacanth 237 fruit flesh, jellies with 329

446

Index

fruit preparations gellan gum 131–4 pectins 179, 181–3 starch 60 FrutafitÕ-inulin 397–403 applications 401–3 gel characteristics 399–401 gelling agent 399 and hydrocolloids 297–8, 403 properties 398 synergism 398–9 Fry Shield 348 furcellaran 87, 90, 101 furniture glues 355 galactomannans 11, 137–54 manufacture 140–4 regulatory status 153 structure 144–6 synergism with xanthan gum 110–12 technical data 146–53 uses/applications 153 see also guar gum; locust bean gum galactose 137, 144–6 removal from TSX 263–5 galacturonic acid 310–11 G-blocks 381, 382, 390, 391 gel strength 420–2, 436–7 Gelidium seaweed 22–3, 24 gelatin 4, 14, 67–86, 125–6 manufacture 67–70 new derivatives 84–5 regulatory status 85 structure 70–6 technical data 76–9 uses 79–84 gelatin hydrolysates 84–5 gelation 12–15 agar 28–33 alginates 387–93 chitosan 371–3 curdlan 274–80 gellan gum 120–2 milk proteins 202–3 pectins 173–4 xyloglucan 255–8 gellan gum 4, 14, 117–35 manufacture 117 regulatory status 134 structure 117–18 technical data 118–24 uses/applications 124–34 gelling agents FrutafitÕ-inulin 399–401 xyloglucan 260, 261 gelling hysteresis 29 gelling memory 31 gels agar 31–3 alginates 387–93 -glucans 293–5 carrageenan 92–4 curdlan 275–7

FrutafitÕ-inulin 399–401 gelatin 73–6 konjac mannan 420–2 milk gels 99, 100, 128–9, 183–4 multilayered 355–6 glazing jelly 181 Glucagel 288, 290–1, 294–5, 296, 297–8, 301 see also -glucans glucomannans 110–12 glutaraldehyde 372–3 glycaemic response 296 glycomacropeptide (GMP) 201 Gracilaria seaweed 22 granulation 335 gravies 60–2, 115 ground green tea milk 341–2, 343 guar gum 137–54 passim manufacture 142–4 synergism with xanthan gum 111 see also galactomannans guluronic acid 381–2 gum arabic 4, 155–68 applications 166–8 manufacture 157–8 properties 164–6 regulatory status 158–61 structure 161–4 supply and market trends 155–7 gum karaya see karaya gum gum tragacanth see tracaganth gum hard pack ice cream 439–40 hashab 155 see also gum arabic hazelnut spread 402–3 health benefits 17–18 -glucans 287, 295–7 MCC 339 health foods 244 health and safety 176 heat stability 235, 242, 258, 312–13 heavy metals 429–30 high acyl (HA) gellan gum 117, 118, 120, 122, 123–4, 125, 130–1 see also gellan gum high methoxyl (HM) pectins 14, 173–4, 175, 315–16 see also pectins homogalacturonan 310–11 hydration 242, 338–9 gellan gum 118–20 milk proteins 203 PNG 436 xanthan gum 105–7 xyloglucan 261 hydrocolloids 1–19 fibres 16–18 future prospects 18–19 prices 3 regulation 4–7 sources 1 synergistic combinations 15–16 thickening characteristics 7–11

Index viscoelasticity and gelation 12–15 hydrogen bonding 46 hydrolysates, gelatin 84–5 hydrolysis acid 33, 48, 332 partial 262–3 hydroxyproline 73 hydroxypropyl cellulose (hpc) 219, 221, 224–5, 349–50 hydroxypropyl methyl cellulose (hpmc) 11, 14, 219, 221, 222–4, 349–50 hypocholesterolemic properties 295–6 ice cream 99–100, 237, 243 cellulosics 226, 227 PNG 439–40, 441 ice crystal stabilisation 260–1 icings 36, 237 immunoglobulins (Ig) 201 incipient gel temperature (IGT) 221 industrial applications adhesives 354–5 agar 37–8 curdlan 284 karaya 244–5 tragacanth 238 infant formulae 210–11 ingredient declarations 64–5, 174–6, 230 insect culture 36–7 instant products 225–6 internal setting 389, 391–2 international numbering system (INS) 7, 8 international regulation 4–5 inulin concentration 400 see also FrutafitÕ-inulin iodine color reaction 259 ion binding 384 ionic crosslinking 388–92 iota carrageenan 14, 89, 90, 91, 92–3, 96, 98 see also carrageenan Italian dressing 304–5 jams 133, 177–9, 179–80 jellies 125–6, 178–9, 344–5 with fruit flesh 329 glazing jelly 181 jelly sweets 131, 132, 166, 184–6, 187–8 Joint Expert Committee on Food Additives (JECFA) 4–5, 429, 432 gum arabic 158–60 kappa carrageenan 14, 87, 89, 90, 91, 92–3, 96 interaction with kappa casein 95 see also carrageenan karaya gum 238–45 manufacture 239–40 regulatory status 245 structure 240 technical data 241–3 uses/applications 243–5 keto-imine crosslinks 71–2 kibbling 141, 157–8 konjac flour 414–15, 415–17

447

purification 416–17 konjac mannan (KM) 14, 18, 413–24 manufacture 415–17 regulatory status 423 structure 418–19 synergism with carrageenan 94–5, 98, 421, 422 technical data 419–22 uses/applications 422–3 labelling 64–5, 174–6, 230, 433, 434 lactalbumin 200 lactoperoxidase (LPO) 200 lactotransferrin (Lf) 200–1 lambda carrageenan 90, 91, 96 see also carrageenan lap-shear tests 357, 359–61 leather 355 lichenase 291–3 lipophilic substitution 47, 49 liquid phase 387 loaf volume depression 205 locust bean gum (LBG)/carob bean gum 14, 137–54 passim manufacture 140–1 synergism with agar 31–2, 33–4 synergism with carrageenan 94, 98 synergism with xanthan gum 111–12 see also galactomannans low acyl (LA) gellan gum 117, 118–20, 120–2, 123–4, 125, 129–30 see also gellan gum low-fat products 402–3 spreads 80, 402–3 low methoxyl (LM) pectins 14, 173, 174, 175, 348 see also pectins maltodextrins 299–300 mannose 137, 144–6 mannuronic acid 381–2 marmalade 177, 178 marshmallows 166–7 mayonnaise 62–3, 330, 402 see also salad dressings meat products 244 carrageenan 97–8 gelatin 80–1 milk proteins 210, 211 PNG 433–7 starch 63 medical applications 354 gelatin 81–3 milk proteins 210–12 tragacanth 237–8 melting temperature 104–5, 110 methyl cellulose (mc) 11, 14, 219, 221, 222–4, 349–50 methyl ethyl cellulose (mec) 219, 222, 225 micelles, casein 190, 192 microbial cultivation 37 microcapsules 374 microcrystalline cellulose (MCC) 331–46 food applications 341–5

448

Index

microcrystalline cellulose (continued) manufacture 331–3 regulatory status 345–6 structure 333–5 technical data 335–41 milk desserts 183–4, 185 milk gels 99, 100 gellan gum 128–9 pectins 183–4 milk products acid milk drinks 183, 184, 228–9, 315–16 MCC 341–4 milk protein concentrates (MPC) 201 milk proteins 95, 189–217 food uses 205–13 functional properties 202–5 future developments 213–14 manufacture 193–201 milk protein system 189–93 Miscellaneous Additives Directive (MAD) 5–6 mitomycin C 265 modified starch 42, 43, 46–50 modified whey powders 196–9 molecular mass/weight 9–10 alginates 383–4 chitosans 368–9 molecular mass/weight distribution galactomannans 139–40 gelatin 78–9 gum arabic 161–2, 163 PNG 430, 431 molecular structure 10 see also under individual hydrocolloids morphological analysis 280–3 mousses 184, 186 multilayered foods 355–6 native curdlan 271 see also curdlan native starches 42, 50–2, 62 see also starch Newtonian viscosity 8, 253–4, 325 noodles 318 Nu-trim 288, 290, 301 see also -glucans Oatrim 288, 289, 290, 296–7, 298–300, 301 see also -glucans oats 288–9 oils 56 okara 309, 310 oxidation 48 oxychitin chitosan complexes 375 paper coatings 352 paper industry 244, 354–5 partial hydrolysis 262–3 pasta products 209 pastilles 166 see also jelly sweets patent status 411 pectins 14, 169–88, 315–16, 348 chemical nature 171–2

properties, modification and function 172–4 manufacture 169–71 nutrition and safety 174–6 regulatory status 188 uses/applications 176–88 peel testing 354, 357, 359–61 peeling processes 141 pH stability see acid stability pharmaceuticals 237–8, 244 phenolic compounds 386 Philippine Natural Grade (PNG)/processed Euphemia seaweed (PES) 4, 89, 90, 93, 425–42 chemical analysis 428–30 food applications 97–8, 432–40 history of product development 425–6 processing 426–8 regulatory status 101, 430–2 phosphor 352 photography 83–4 phycocolloids 21 see also agar; alginates; carrageenan physical gels 28 phytoalexin 265–6 pie crust 301–2 pie fillings 112–13, 181, 182 plant defence system 265–6 polyacrylic acid (PAA) 352 polydispersity index (PI) 383 polyelectrolyte complex formation 373–5 poly-L-lysine 374 poly-L-proline II helix 73, 74 potassium chloride 436–7 potassium PNG 436 powder type MCC 332, 334, 335–6 see also microcrystalline cellulose pregelatinisation 49–50 preservatives 236, 243, 375 preserves 177–8 price 3, 156–7 process-tolerant starches 52–3 processed cheese 100 processed Euchema seaweed (PES) see Philippine Natural Grade processing equipment and conditions 56 proline 73 propylene glycol alginate (PGA) 380 proteins 222, 223 milk proteins see milk proteins SSPS and dispersion stabilisation effects on protein particles 314–16 pseudoplastic viscosity 235, 324–5 pumping 433 purification 416–17 purity 78 regulation 4–7 see also under individual hydrocolloids resistant starch (RS) 53–4 retorted products 114 pudding 329 rhamnogalacturonan 310–11 rheological properties 7–11

Index MCC 337–8 tragacanth 235 xanthan gum 107–10 xyloglucan 253–4 see also viscosity rice 318 salad dressings 98 bacterial cellulose 329, 330 -glucans 304–5 cellulosics 226–7 FrutafitÕ-inulin 402 karaya 243 starch 62–3 tragacanth 236–7 xanthan gum 113–14 salt stability 109, 312–13, 421, 422 sauces 115 bacterial cellulose 329–30 cellulosics 226–7 starch 60–2 tragacanth 236–7 savoury snacks 63 seaweed, agarophyte 22–3 seed coatings 352 seeding process 401 semi-refined carrageenan (SRC) see Philippine Natural Grade Semperfresh 350 sequestrants 118–19, 174 shape retention 223 shear rate 8–10 shear stability 439 shear treatment 401 sheared gels 18 shelf stability 56 short-chain fatty acids (SCFA) 17–18 silkworms 36–7 small-angle X-ray scattering (SAXS) 250–1 snacks 63, 213 sodium PNG 436 soft drinks 227–8, 237 soft-mix 328–9 soft serve ice cream 440, 441 solubility alginates 384–5 chitosan 370–1 karaya 241 milk proteins 202 SSPS 312 tragacanth 234 soluble lacto-protein (SLP) 201 soluble soybean polysaccharide (SSPS) 309–20 functions and applications 314–19 manufacture 309–10 properties and characteristics 312–14 regulatory status 319 structure 310–11 solutions bacterial cellulose 324 -glucans 293–5 carrageenan 91 curdlan 271–4

449

dissolving konjac mannan 419–20 dissolving pectin 176 gelatin 70–3 tragacanth 236 xanthan gum 105–10 xyloglucan 259 soups 60–2 source dependence 382–3 soybean polysaccharide see soluble soybean polysaccharide sponges, gelatin 83 stabilisation/stabilisers PNG 437–40, 441 SSPS and dispersion stabilisation effects on protein particles 314–16 starch 47–8 xyloglucan 259–61 stability acid/pH see acid stability alginates 385–7 emulsion 316–17, 409–10 heat 235, 242, 258, 312 salt 109, 312–13, 421, 422 SSPS 312–13 suspension 316–17, 325, 337 xyloglucan 258 starch 41–65, 330, 349 applications 57–63 FrutafitÕ-inulin and 399, 403 manufacture 41–2 modification by xyloglucan 260, 261 modifications 42, 43, 46–50 regulatory status 64–5 starch selection 54–7 structure 42–6 technical data 50–4 viscosity-troubleshooting 63–4 starch granules 46 sterilisation 386–7 stirring time 419–20 sugar icings 36 sugars 421, 422 agar reactivity with 32 and gellan gum 129–31 and starch 55 xyloglucan synergism with 254, 256 surface activity 204, 235 surface roughness 351, 354 surface tension 351, 354 suspension stability 316–18, 325, 337 suspensions curdlan 274, 275 gellan gum 126–8 sweet potato sweet 36 swelling 435–6 bacterial cellulose 326, 327 syneresis agar 25–6, 30–1 PNG 434–5, 436–7 synergistic combinations 15–16 agar gels 31–3 carrageenan 94–5 konjac mannan 414, 420–2

450

Index

synergistic combinations (continued) xanthan gum 110–12 xyloglucan 254, 256 syrups 115 tailoring of properties 263–5 Tal Pro-long 350 talha 155, 157 see also gum arabic tamarind gum 249 tamarind seed xyloglucan (TSX) 247, 249 modification 262–6 uses/applications 259–61 see also xyloglucan tannic acid (TA) 32 tara gum 137–8, 145, 148 see also galactomannans temperature and curdlan gel 275–6, 277, 280 and FrutafitÕ-inulin gel 400 gel temperatures of agars 29–30 IGT 221 and viscosity 325–6, 341 xanthan gum and melting temperature 104–5, 110 see also heat stability tensile-bond tests 357, 358, 359–61 testing adhesives 357, 358, 359–61 coatings 353–4 textiles 238, 244 texture gellan gum 122–4 MCC 338 texture profile analysis (TPA) 122–4 textured products 213 thermal analysis 280–3 thermal treatment 50 thickening agents 139, 398 inulin and 403 TSX 259–60 thickening characteristics 7–11 thixotropy 337–8 toffees 166–7 tomato ketchup 227 toppings 115, 224–5 total milk protein (TMP) 201 tragacanth gum 231–8 manufacture 232 regulatory status 238 structure 233–4 technical data 234–6 uses/applications 236–8 tragacanthic acid 233 tragacanthin 233 trimethylamine 417 ‘Turkish delight’ 187–8 ultrafiltration (UF) 199 United States Food Chemical Codex 6–7, 160–1

Pharmacopia and the National Formulary 161 urea 276 vegetable tissue culture 37 viscoelasticity 12–15 viscosity 7–11 bacterial cellulose 324–6 cmc 225 curdlan 272–4 effect of inulin 398–9 galactomannans 151–3 gelatin 78 gum arabic 164–5 karaya 241–2 konjac mannan 419–20, 421 MCC 337, 340–1 milk proteins 203–4 SSPS 312–13 tragacanth 234–5 troubleshooting and starch 63–4 xanthan gum 107–10 xyloglucan 253–4, 255 see also rheology wafers 402 water gels 96–9 water holding see hydration wattle blossom-type structure 162, 164 welan gum 18 wet glues 357–61 whey-off 439–40 whey powders 196–9 whey protein concentrates (WPCs) 199, 208, 209, 210 whey protein isolate (WPI) 199 whey proteins 189–93, 196–9 see also milk proteins whipped toppings 115, 224–5 wine gums 166 wound dressings 375 xanthan gum 3–4, 11, 14, 103–15, 139–40, 232, 421 manufacture 103 regulatory status 115 structure 103–5 technical data 105–12 uses/applications 112–15 xyloglucan 247–67 modification of TSX 262–6 origin and manufacture 247–9 regulatory status 261–2 structure 250–2 technical data 253–9 uses/applications 259–61 yield value 325 yogfruit 133 yoghurt 129, 206–7, 402 Yokan 35