Handbook of hydrocolloids Second edition Edited by G. O. Phillips and P. A. Williams
Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi ± 110002, India Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2009, Woodhead Publishing Limited and CRC Press LLC ß 2009, Woodhead Publishing Limited The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing Limited ISBN 978-1-84569-414-2 (book) Woodhead Publishing Limited ISBN 978-1-84569-587-3 (e-book) CRC Press ISBN 978-1-4398-0820-7 CRC Press order number: N10074 The publishers' policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elemental chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Godiva Publishing Services Limited, Coventry, West Midlands, UK Printed by TJ International Limited, Padstow, Cornwall, UK
Preface
The first edition of the Handbook of Hydrocolloids was published in 2000 and was exceptionally well received. It has been used as the substantive reference book in the subject. It was not meant to be a textbook, but a convenient reference to provide the relevant information readily and at the same time authoritatively. The chapters were all written by top specialists in their fields. Since the year 2000, the subject has moved on with remarkable speed. The dominant industrial influence has been the succession of mergers and acquisitions. The names of very many of the companies which participated over the years have either disappeared or now form part of bigger amalgamations. The specialist single product producer has given way to the global multi-ingredient suppliers. Now it is the global giants who dominate the ever increasing technological industry. While such progress is, I suppose, inevitable, it has taken its toll on individuals who are finding it increasingly difficult to keep pace with changing materials, technologies, loyalties and names. Much corporate memory has been lost in these frantic changes. In this Second Edition we have kept this in mind and now provide a single and reliable reference volume where hydrocolloids structure, functionality, synergistic behaviour, applications and regulatory aspects are brought together. These are the new improvements we have taken to ensure that we fully meet the core and developing hydrocolloid areas: · First, all chapters in the first edition were re-visited and, where necessary, these chapters were up-dated. · Now there is a much greater emphasis on the protein hydrocolloids. New chapters have therefore been included on egg proteins and vegetable proteins (soybean, pea, wheat, and other related protein isolates) and the more recent information about fish gelatin has been added.
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· We have increased the coverage of microbial polysaccharides, with a new chapter on the newly emerging microbial polysaccharides such as pullulan, scleroglucan, elsinan, levan, alternan, etc., which are now moving out of the scientific novelty area into practical use. · The developing role of the exudate gums has been recognized, with a new chapter on gum ghatti included. This old gum is rapidly making a resurgence, particularly as a clean label natural emulsifier and adhesive. New sections have been added also on mesquite gum and larchwood arabinogalactans. · Protein-hydrocolloid complexes are now extensively studied in order to gain synergy from both components. A new chapter is, therefore, included to chart these new developments. · There is a new chapter on the function of hydrocolloids as emulsifiers and the factors which determine the stabilization and long-term stability of the emulsions. · 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 risen exponentially. Obesity, calorific value, fat replacement, glycaemic index and dietary fibre are at the forefront of the medical and political platform. A new chapter has been added to acknowledge this vital new emphasis in using hydrocolloids in foods. Information has also been added about health aspects where relevant to other updated chapters. · There is a complete new chapter on the extraction, structure, analysis, physical chemical properties, technology, applications and health benefits of arabinoxylans. 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 in its new and enlarged form 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 experienced scientist, to understand this rapidly growing, enjoyable yet challenging subject. Glyn O. Phillips and Peter A. Williams Phillips Hydrocolloids Research Ltd and the Centre for Water Soluble Polymers, Glyndwr University
Contributor contact details
(* = main contact)
Chapters 1 and 11 P. A. Williams Glyndwr University Plas Coch Mold Road Wrexham LL11 2AW UK E-mail: [emailprotected] G. O. Phillips Phillips Hydrocolloids Research Ltd 45 Old Bond Street London W15 4AQ E-mail: [emailprotected]
Chapter 2 E. Dickinson Procter Department of Food Science University of Leeds Leeds LS2 9JT UK E-mail: [emailprotected]
Chapter 3 C. A. Edwards* and A. L. Garcia Human Nutrition Section Division of Developmental Medicine University of Glasgow Yorkhill Hospitals Glasgow G3 8SJ UK E-mail: [emailprotected]
Chapter 4 R. ArmiseÂn* Hispanagar S. A. R & D Director (Retired) Independent Chemistry Consultant Calle Sierra Ovejero 8 Pozuelo de AlarcoÂn 28224 Madrid Spain E-mail: armisen.consulting@ armisen.e.telefonica.net
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Contributors
F. Galatas Hispanagar S.A. Calle Pedro de Valdivia 34 28006 Madrid Spain
Chapter 5 P. Taggart National Starch Food Innovation Prestbury Court Greencourts Business Park 333 Styal Road Manchester M22 5LW UK E-mail: [emailprotected] J.R. Mitchell* Division of Food Sciences Sutton Bonnington Campus University of Nottingham Loughborough LE12 5RD UK E-mail: [emailprotected]
Chapter 7 Alan Imeson FMC Biopolymer 12 Langley Close Epsom Surrey KT18 6HG UK E-mail: [emailprotected]
Chapters 8 and 9 Graham Sworn Danisco France SAS 20 rue Brunel 75017 Paris France E-mail: [emailprotected]
Chapter 10 W. Wielinga Hinterdorfstrasse 41 8274-TaÈgerwilen Switzerland E-mail: [emailprotected]
Chapter 6 I. J. Haug* and K. I. Draget Norwegian University of Science and Technology (NTNU) N-7491 Trondheim Norway E-mail: [emailprotected] [emailprotected]
Chapter 12 Hans-Ulrich Endreû* Herbstreith & Fox KG Pektin-Fabrik NeuenbuÈrg 75305 NeuenbuÈrg Germany E-mail: [emailprotected] Steen Hoejgaard Christensen CP Kelco ApS 4621 Lille Skensved Germany E-mail: [emailprotected]
Contributors
Chapter 13 J. O'Regan, M. P. Ennis and D. M. Mulvihill* Department of Food & Nutritional Sciences National University of Ireland Cork University College Cork Cork Ireland E-mail: [emailprotected]
Chapter 14 M. Anton* INRA Nantes Unite 1268 BiopolymeÁres Interactions Assemblages BP 71627 44316 Nantes cedex 3 France E-mail: [emailprotected] F. Nau and V. Lechevalier DeÂpartement AgroAlimentaire ± Agrocampus Ouest UMR INRA Science et Technologie du Lait et de l'Oeuf 65 rue de Saint Brieuc CS 84215 35042 Rennes cedex France E-mail: [emailprotected] [emailprotected]
Chapter 15 S. GonzaÂlez-PeÂrez* and J. B. Arellano Institute of Natural Resources and Agrobiology Consejo Superior de Investigaciones CientõÂficas (IRNASA-CSIC) Cordel de Merinas 40-52 37008 Salamanca Spain
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E-mail: [emailprotected] [emailprotected]
Chapter 16 C. Schmitt* Department of Food Science and Technology Nestle Research Center Vers-chez-les-Blanc CH-1000 Lausanne 26 Switzerland E-mail: [emailprotected] L. Aberkane Laboratoire d'IngeÂnierie des BiomoleÂcules INPL-ENSAIA 2 Avenue de la ForeÃt de Haye F-54505 Vandoeuvre-leÁs-Nancy cedex 5 France C. Sanchez Laboratoire Biocatalyse BioproceÂdeÂs INPL-ENSAIA 2 Avenue de la ForeÃt de Haye F-54505 Vandoeuvre-leÁs-Nancy cedex 5 France
Chapter 17 Saphwan Al-Assaf,* G.O. Phillips and V. Amar Phillips Hydrocolloids Research Centre Glyndwr University, Wrexham Mold Road Wrexham LL11 2AW UK E-mail: [emailprotected] [emailprotected]
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Contributors
Chapter 18
Chapter 19
Y. LoÂpez-Franco Laboratory of Biopolymers Centro de InvestigacioÂn en AlimentacioÂn y Desarrollo A.C. P.O. Box 1735 Hermosillo Sonora 83000 Mexico
K. Nishinari* and M. Takemasa Department of Food & Nutrition Faculty of Human Life Science Osaka City University Sumiyoshi Osaka 558-0022 Japan E-mail: [emailprotected]
I. Higuera-Ciapara Laboratory of Natural Polymers Centro de InvestigacioÂn en AlimentacioÂn y Desarrollo A.C. P.O. Box 284 Guaymas Sonora 85440 Mexico F. M. Goycoolea* Department of Pharmacy and Pharmaceutical Technology Universidad de Santiago de Compostela Campus Sur s/n A CorunÄa 15782 Spain E-mail: [emailprotected] and Laboratory of Biopolymers Centro de Investigacion en Alimentacion y Desarrollo, A. C. P.O. Box 1735 Hermosillo Sonora 83000 Mexico E-mail: [emailprotected] Weiping Wang Andi-Johnson Konjac Co Ltd. 1-504 Long Spring Gardens 118 Yang Bridge West Road Fuzhou China 350002 E-mail: [emailprotected]
and Phillips Hydrocolloid Research Centre/Glyndwr University Plas Coch Mold Road Wrexham LL11 2AW UK and Department of Polymer Science & Engineering Shanghai Jiao Tong University Shanghai 200240 People's Republic of China K. Yamatoya and M. Shirakawa Dainippon-Sumitomo Pharma Co. Ltd 1-5-51 Eble, f*ckushima Osaka 553-0001 Japan
Chapter 20 K. Nishinari* Department of Food & Nutrition Faculty of Human Life Science Osaka City University Sumiyoshi Osaka 558-0022 Japan E-mail: [emailprotected]
Contributors and Phillips Hydrocolloid Research Centre/Glyndwr University Plas Coch Mold Road Wrexham LL11 2AW UK and Department of Polymer Science & Engineering Shanghai Jiao Tong University Shanghai 200240 People's Republic of China H. Zhang Department of Polymer Science & Engineering Shanghai Jiao Tong University Shanghai 200240 People's Republic of China T. Funami Hydrocolloid Laboratory San-Ei Gen F.F.I. Inc. Toyonaka Osaka 561-8588 Japan
T. Khan Department of Pharmaceutical Science COMSATS Institute of Information Technology University Road Post Code 22060 Abbottabad NWFP Pakistan E-mail: [emailprotected]
Chapter 22 D. G. Stevenson* National Starch Bridgewater, NJ 08807 USA E-mail: [emailprotected] G. E. Inglett National Center for Agricultural Utilization Research (NCAUR) Agricultural Research Center USDA Peoria, IL 61604 USA
Chapter 23 Chapter 21 J. K. Park Department of Chemical Engineering Kyungpook National University Sankyuk-dong 1370 Buk-ku Daegu 701-702 Korea E-mail: [emailprotected]
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M. S. Izydorczyk Grain Research Laboratory Canadian Grain Commission 14040303 Main Street Winnipeg, MB Canada R3C 3G8 E-mail: marta.izydorczyk@ grainscanada.gc.ca
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Contributors
Chapter 24 H. Maeda Protein and Food Ingredients Division Soy Protein Processed Foods Company Fuji Oil Co., Ltd 1 Sumiyoshi-cho Izumisano-shi Osaka 598-8540 Japan E-mail: [emailprotected] A. Nakamura Research and Development Headquarters Food Science Research Institute Fuji Oil Co., Ltd. 4-3 Kinunodai Tsukubamirai-shi Ibaraki-Pref. 300-2497 Japan E-mail: [emailprotected]
J. Y. Jung Search & Analysis Team 2 Korea Institute of Patent Information (KIPS) 647-9 Yeoksam-dong Gangnam-gu Seoul Korea 135-980 E-mail: [emailprotected] or [emailprotected] T. Khan Department of Pharmaceutical Science COMSATS Institute of Information Technology University Road Post Code 22060 Abbottabad NWFP Pakistan E.mail: [emailprotected]
Chapter 27 Chapter 25 J. C. F. Murray London UK E-mail: [emailprotected]
Chapter 26 J. K. Park Department of Chemical Engineering Kyungpook National University Sankyuk-dong 1370 Buk-ku Daegu 701-702 Korea E-mail: [emailprotected]
G. R. Krawczyk,* A. Venables and D. Tuason FMC BioPolymer PO Box 8 Princeton, NJ 08543 USA E-mail: [emailprotected] [emailprotected] [emailprotected]
Contributors
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Chapter 28
Chapter 31
A. Nussinovitch The Hebrew University of Jerusalem Institute of Biochemistry Food Science and Nutrition The Robert H. Smith Faculty of Agriculture, Food and Environment P.O. Box 12 Rehovot 76100 Israel E-mail: [emailprotected]
R. A. A. Muzzarelli Muzzarelli Consulting Via Volterra 7 IT-60123 Ancona Italy E-mail: [emailprotected]
Chapter 29 K. I. Draget Department of Biotechnology Norwegian University of Science and Technology (NTNU) N-7491 Trondheim Norway E-mail: [emailprotected]
Chapter 30 D. Meyer Sensus PO Box 1308 4700 BH Roosendaal The Netherlands E-mail: [emailprotected] J.-P. Blaauwhoed Cosun Food Technology Centre PO Box 1308 4700 BH Roosendaal The Netherlands
and R. A. A. Muzzarelli and C. Muzzarelli Institute of Biochemistry University of Ancona Via Ranieri 67 IT-60100 Ancona Italy
Chapter 32 S. Takigami Center for Material Research by Instrumental Analysis Gunma University 1-5-1 Tenjincho Kiryu-shi Gunma 376-8515 Japan E-mail: [emailprotected]
Contents
Contributor contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1 Introduction to food hydrocolloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. A. Williams and G. O. Phillips, Glyndwr University, UK 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Thickening characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Viscoelasticity and gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Synergistic combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Hydrocolloid fibres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
2
Hydrocolloids and emulsion stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Dickinson, University of Leeds, UK 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Principles of emulsion stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Effect of non-adsorbing hydrocolloids on emulsion stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Effect of adsorbing hydrocolloids on emulsion stability . . . . . 2.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 5 8 14 18 19 21 22 23 23 26 35 42 46 47
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Contents 3 The health aspects of hydrocolloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. A. Edwards and A. L. Garcia, University of Glasgow, UK 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Hydrocolloids and non-digestible carbohydrates in food . . . . . 3.3 Effects on metabolism and health . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Clinical nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. ArmiseÂn and F. Galatas, Hispanagar S. A., Spain 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Agar manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Chemical structure of agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Agar gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Agar applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Taggart, National Starch Food Innovation, UK and J.R. Mitchell, Division of Food Sciences, University of Nottingham, UK 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 Regulatory status: European label declarations . . . . . . . . . . . . . . . 5.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. J. Haug and K. I. Draget, Norwegian University of Science and Technology (NTNU), Norway 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Manufacturing gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Regulations, technical data and standard quality test methods 6.4 Chemical composition and physical properties of collagens and gelatins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Gelatin derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Applications of gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.8 References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50 50 51 55 69 70 71 82 83 86 89 91 96 104 105 108
108 109 110 115 121 125 137 140 142
142 143 147 149 157 158 162 162
Contents 7 Carrageenan and furcellaran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. P. Imeson, FMC Biopolymer, UK 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Xanthan gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Sworn, Danisco France SAS, France 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Applications in food products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 8.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Gellan gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Sworn, Danisco France SAS, France 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 9.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Galactomannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. C. Wielinga, retired in 2000 from Meyhall AG, Kreuzlingen, Switzerland, which was subsequently acquired by Danisco A/S 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Raw materials and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii 164 164 165 167 169 170 177 183 183 184 186 186 187 187 188 195 201 201 202 202 204 204 205 205 206 213 225 225 226 226 228
229 229 237 243 249 249
viii
Contents 10.7 10.8
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
250 250
11 Gum arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. A. Williams and G. O. Phillips, Glyndwr University, UK 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Supply and market trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
252
12 Pectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H.-U. Endreû, Herbstreith & Fox KG Pektin-Fabrik NeuenbuÈrg, Germany and S. H. Christensen, CP Kelco ApS, Germany 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 The chemical nature of pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Commercial pectin: properties and function . . . . . . . . . . . . . . . . . . 12.5 Nutritional and health aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.7 Legal status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Milk proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. O. Regan, M. P. Ennis and D. M. Mulvihill, University College Cork, Ireland 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 The milk protein system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Production of milk protein products . . . . . . . . . . . . . . . . . . . . . . . . . 13.4 Functional properties of milk protein products . . . . . . . . . . . . . . . 13.5 Biological activity of milk protein products . . . . . . . . . . . . . . . . . . 13.6 Food uses of milk protein products . . . . . . . . . . . . . . . . . . . . . . . . . . 13.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 13.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Egg proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Anton, INRA Nantes Unite 1268 BiopolymeÁres Interactions Assemblages, France and F. Nau and V. Lechevalier, UMR INRA Science et Technologie du Lait et de l'Oeuf, France 14.1 Introduction: technofunctional uses of egg constituents . . . . . . 14.2 Physico-chemistry and structure of egg constituents . . . . . . . . .
252 254 255 256 260 265 270 273 274
274 275 277 277 281 282 296 297 298
298 299 303 317 324 329 341 342 343 359
359 360
Contents 14.3 14.4 14.5 14.6 14.7
ix
Egg yolk emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Egg white foams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
365 369 373 376 376
15 Vegetable protein isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. GonzaÂlez-PeÂrez and J. B. Arellano, IRNASA-CSIC, Spain 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Chemical composition of vegetable proteins . . . . . . . . . . . . . . . . . 15.3 Protein composition and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5 Technical data: functional properties . . . . . . . . . . . . . . . . . . . . . . . . . 15.6 Functional properties for industrial applications . . . . . . . . . . . . . . 15.7 Chemical and enzymatic modification of protein products . . . 15.8 Vegetable proteins: choosing the best functionality for food application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.9 Applications of vegetable proteins in food products . . . . . . . . . 15.10 Nutritional and health effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.11 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.12 References and sources of further reading . . . . . . . . . . . . . . . . . . .
383
16 Protein±polysaccharide complexes and coacervates . . . . . . . . . . . . . C. Schmitt, Nestle Research Center, Switzerland and L. Aberkane and C. Sanchez, INPL-ENSAIA, France 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 Thermodynamic background, theoretical models and energetics of the formation of protein±polysaccharide complexes and coacervates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Parameters influencing the formation of protein± polysaccharide complexes and coacervates . . . . . . . . . . . . . . . . . . . 16.4 Structure, morphology and coarsening of protein± polysaccharide complexes and coacervates . . . . . . . . . . . . . . . . . . . 16.5 Functional properties of protein±polysaccharide complexes and coacervates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.6 Food applications of protein±polysaccharide complexes and coacervates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.7 Non-food applications of protein±polysaccharide complexes and coacervates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.9 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
383 387 389 394 399 401 404 406 406 412 415 418 420
420
422 425 440 445 452 458 460 462 462
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17 Gum ghatti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Al-Assaf, G. O. Phillips and V. Amar, Glyndwr University, UK 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other exudates: tragancanth, karaya, mesquite gum and larchwood arabinogalactans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y. LoÂpez-Franco and I. Higuera-Ciapara, Centro de InvestigacioÂn en AlimentacioÂn y Desarrollo, Mexico, F. M. Goycoolea, Universidad de Santiago de Compostela, Spain and Centro de InvestigacioÂn en AlimentacioÂn y Desarrollo, Mexico and W. Wang, Andi-Johnson Konjac Co. Ltd., China 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
477 477 480 480 483 487 491 493
18
19 Xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Nishinari and M. Takemasa, Osaka City University, Japan and K. Yamatoya and M. Shirakawa, Dainippon-Sumitomo Pharma Co. Ltd, Japan 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 Origin, distribution and preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 Structure and fundamental properties . . . . . . . . . . . . . . . . . . . . . . . . 19.4 Interactions with tamarind seed xyloglucan . . . . . . . . . . . . . . . . . . 19.5 Applications in the food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6 Regulatory status in the food industry . . . . . . . . . . . . . . . . . . . . . . . 19.7 Physiological effects of xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . . . 19.8 Other aspects and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.9 References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Nishinari, Osaka City University, Japan, and Glyndwr University, UK, and Shanghai Jiao Tong University, China, H. Zhang, Shanghai Jiao Tong University, China and T. Funami, San-Ei Gen F.F.I. Inc, Japan 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
495
495 496 501 508 519 525 527 535
535 535 538 546 553 557 558 561 563 567
567
Contents 20.2 20.3 20.4 20.5 20.6 20.7 20.8
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Native curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other microbial polysaccharides: pullulan, scleroglucan, elsinan, levan, alternan, dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. K. Park, Kyungpook National University, Korea and T. Khan, COMSATS Institute of Information Technology, Pakistan 21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Pullulan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Scleroglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Elsinan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5 Levan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.6 Alternan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.7 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.8 References and sources of further reading . . . . . . . . . . . . . . . . . . .
xi 568 568 568 570 585 588 588
21
22 Cereal -glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. G. Stevenson, National Starch, USA and G. E. Inglett, USDA, USA 22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Botanical distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Structure and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.4 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.5 Extraction and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.6 Health benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.7 Commercial products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.8 Food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.9 Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.10 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.11 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Arabinoxylans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. S. Izydorczyk, Canadian Grain Commission, Canada 23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 Occurrence and content of arabinoxylans . . . . . . . . . . . . . . . . . . . . 23.3 Extraction, isolation and purification of arabinoxylans . . . . . . . 23.4 Molecular structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.5 Analysis and detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6 Physico-chemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
592
592 593 596 600 603 606 609 612 615
615 617 617 621 622 624 628 632 635 637 638 639 653 653 655 658 664 669 671
xii
Contents 23.7 23.8 23.9
Technological functionality and potential applications of arabinoxylans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological effects of arabinoxylans . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24 Soluble soybean polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Maeda, Soy Protein Processed Foods Company, Japan and A. Nakamura, Fuji Oil Co., Ltd., Japan 24.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.4 Basic material properties and characteristics . . . . . . . . . . . . . . . . . 24.5 Functional properties and reported use of soluble soybean polysaccharide in foods and pharmaceutical applications . . . . 24.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Cellulosics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. C. F. Murray, UK 25.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.4 Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Bacterial cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. K. Park, Kyungpook National University, Korea, J. Y. Jung, Korea Institute of Patent Information (KIPI), Korea and T. Khan, COMSATS Institute of Information Technology, Pakistan 26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.2 Historical overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.3 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.4 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.5 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.6 Functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.7 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.8 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.9 References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Microcrystalline cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Krawczyk, A. Venables and D. Tuason, FMC BioPolymer, USA 27.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.2 Raw materials and manufacturing process . . . . . . . . . . . . . . . . . . .
677 681 683 693
693 694 694 696 700 706 706 710 710 711 711 712 714 722 724
725 725 726 728 729 730 733 736 736 740 740 741
Contents 27.3 27.4 27.5 27.6 27.7
xiii
Nutritional and regulatory information . . . . . . . . . . . . . . . . . . . . . . . Physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Food applications and functionality . . . . . . . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
743 744 745 757 758
28 Hydrocolloids for coatings and adhesives . . . . . . . . . . . . . . . . . . . . . . . A. Nussinovitch, The Hebrew University of Jerusalem, Israel 28.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.2 Today's edible protective films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.3 Novel products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.4 Inclusion of food additives in edible films . . . . . . . . . . . . . . . . . . . 28.5 Parameters to be considered before, during and after food coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.6 Hydrocolloid non-food coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.7 Film-application techniques and stages . . . . . . . . . . . . . . . . . . . . . . 28.8 Methods for testing coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.9 Market estimates for edible films . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.10 The next generation of edible films and possible research directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.11 Non-food uses and applications of adhesives . . . . . . . . . . . . . . . . 28.12 Adhesive hydrocolloid preparations: pastes and hydrogels . . . 28.13 Adhesion mechanisms of hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . 28.14 Food uses and applications of adhesives . . . . . . . . . . . . . . . . . . . . . 28.15 Uses and applications of bioadhesives . . . . . . . . . . . . . . . . . . . . . . . 28.16 Structure-function and hydrogel-adherend relationships . . . . . . 28.17 Hydrocolloid adhesion tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.18 Hydrocolloids as wet glues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.19 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.20 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
760
29 Alginates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. I. Draget, Norwegian University of Science and Technology, Norway 29.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.3 Structure and physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.5 Gels and gelling technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.6 Foods, nutrition and health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.7 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29.9 References and sources of further reading . . . . . . . . . . . . . . . . . . .
760 761 769 771 775 776 777 778 779 779 781 782 783 784 785 786 788 790 792 793 807
807 808 809 813 817 823 824 824 825
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Contents
30 Inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Meyer, Sensus, The Netherlands and J.-P. Blaauwhoed, Cosun Food Technology Centre, The Netherlands 30.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.2 Production process of inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.3 Technical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.4 Nutritional and health benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Chitin and chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. A. A. Muzzarelli, Muzzarelli Consulting, Italy and University of Ancona, Italy and C. Muzzarelli, University of Ancona, Italy 31.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 Chitosan chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.3 Properties of chitosans and derivatives . . . . . . . . . . . . . . . . . . . . . . . 31.4 Chitin as a food component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.5 Nutritional and health effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.6 Food industry applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.7 Applications in drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
829
829 831 831 838 839 843 845 845 845 849
850 851 853 860 861 867 869 876 876
32 Konjac mannan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Takigami, Gunma University, Japan 32.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2 Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.4 Technical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.5 Uses and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.6 Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
889 889 892 894 896 899 900 901
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
902
1 Introduction to food hydrocolloids P. A. Williams and G. O. Phillips, Glyndwr University, UK
Abstract: This introductory chapter provides an overview of the source, market, functional characteristics and regulatory aspects of hydrocolloids used in foods. Examples of a number of different food products are given and the role of the hydrocolloids in controlling their texture and properties is discussed. A brief introduction to the rheological properties of the various types of hydrocolloids is provided and comparisons of the viscosity and viscoelastic behaviour are given. Many hydrocolloids are able to form gels in response to changes in temperature and/or in the presence of ions. The various mechanisms by which gelation occurs are summarised. The interactions that occur in systems containing mixtures of hydrocolloids are considered and combinations demonstrating synergistic behaviour are highlighted. The role of hydrocolloids as dietary fibre is an area of increasing importance because of the associated benefits for health and this area is briefly reviewed. Key words: source of hydrocolloids, hydrocolloids, market, regulatory aspects, rheological properties, viscosity, storage and loss moduli, food products, dietary fibre, health benefits.
1.1
Introduction
The term `hydrocolloids' is commonly used to describe 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 origins are given in Table 1.1.
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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, soy protein, egg white protein, chitosan
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, clearly demonstrating the widespread application of these materials. The specific hydrocolloids used in the production of the individual products shown are: · · · · · · · · · ·
baked beans contain modified corn starch as a thickener hoi-sin sauce contains modified corn starch as a thickener sweet and sour sauce contains guar gum as a thickener 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 `light' mayonnaise contains guar gum and xanthan gum as fat replacers to enhance viscosity the yoghurt incorporates gelatin as a thickener rather than a gelling agent 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 gelling agent the fruit pie bars contain gellan gum and the blackcurrant preserve and redcurrant jelly contain pectin as gelling agents
Introduction to food hydrocolloids
3
Fig. 1.1 Examples of food products containing hydrocolloids.
· 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 ever 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, functional 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. The world
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hydrocolloids market is valued at around $4.4 billion p.a. with a total volume of about 260,000 tonnes. The market has been growing at the rate of 2±3% in recent years. Hydrocolloid selection is dictated by the functional characteristics required but is inevitably influenced by price and security of supply. It is for these reasons that starches (costing typically < US$1/kg) are the most commonly used thickening agents. It is interesting to note here, however, that xanthan gum (~US$12/kg) is becoming the thickener of choice in many applications. This is because xanthan gum has unique rheological behaviour and its increased use has led to strong competition between supplier companies ensuring that the price has remained at reduced levels. Xanthan gum forms highly viscous, highly shear 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. This explains 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 Bovine Spongiform Encephalopathy (BSE) outbreak in the UK, prices have increased significantly over recent years. There is currently considerable interest in alternative sources of gelatine, notably fish skins, and in the development of gelatin replacements. The carrageenan market has also been very competitive over recent years due to the introduction of cheaper lower refined grades (Processed Euchema Seaweed, PES), which can be used effectively where gel clarity is not important. The price differential between carrageenan and PES has decreased and the use of carrageenans has increased markedly since its use in meat and poultry products was recently approved. A further development has been the introduction of kappa/iota carrageenan hybrids which have potential to provide novel functionality. The gum arabic market has traditionally been erratic due to price fluctuations and security of supply and much effort has been directed at finding alternatives. A number of starch-based substitutes (for example, succinylated starch) were introduced some years ago as alternatives in the emulsification of flavour oils and recent work has been concerned with using pectin (notably sugar beet pectin) as a gum arabic replacement. It has been shown that the emulsification properties of gum arabic and pectin are due to the small amount of protein (2±3%) which is present as in integral part of their structure. This has led to significant interest in the development of polysaccharide±protein complexes (gum arabic lookalikes) which can be formed by a mild heat treatment through the Maillard reaction and by electrostatic interaction. Gellan gum was approved for food use in Japan in 1988 but much later in the USA and Europe, and is beginning to establish its own niche markets. An overview of the hydrocolloids market is given in Figs 1.2(a) and 1.2(b).
Introduction to food hydrocolloids
5
Fig. 1.2 (a) Value of world market for individual hydrocolloids; (b) Volume of world market for individual hydrocolloids.
1.2
Regulatory aspects
Food hydrocolloids do not exist as a regulatory category in their own right, rather they are regulated either as a food additive or as a food ingredient. With the exception of gelatin, however, the vast majority of food 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). JECFA was first established in the mid-1950s by the 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 aims 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 subcommittee, 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
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Handbook of hydrocolloids
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 for 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 The 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 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,
Introduction to food hydrocolloids
7
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 Eucheuma Seaweed (E407a) (see Directive 96/85/EC of 19 December 1996), a second amendment was introduced which affected many hydrocolloids. Member States were required to implement the provisions of this Directive in the year 2000. Thus at this time the new authorisation introduced by the current Directive 98/72/EC came 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
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Handbook of hydrocolloids
Chemicals Codex (FCC) also has currency. The FCC 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 (FDA). The current specification of hydrocolloids are 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 The international numbering system for food additives (INS) INS has been prepared by the Codex Committee on Food Additives in order to be able to identify food additives in ingredient lists as an alternative to the declaration of the specific name. The INS is intended as an identification system for food additives approved for use in one or more member countries. It does not imply toxicological approval by Codex. 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 list of INS numbers is given in Tables 1.2 and 1.3.
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 thirty years or so, particularly through the development of controlled stress and controlled strain rheometers capable of measuring to very low shear rates ( 100 nm). Xanthan gum has a unique rheological profile with a very high viscosity at low shear rates (providing good suspending properties) but exhibiting extensive shear thinning characteristics
Fig. 1.6
Viscosity±shear rate profiles for 1% xanthan gum, 1% CMC, 1% guar gum, 20% dextran and 30% gum arabic.
Introduction to food hydrocolloids
Fig. 1.7
13
Disaccharide repeat units showing 1,4 (top) and 1,6 (bottom) glycodisic linkages. , ' and ! represent the bonds of rotation.
(readily flows on shearing). This behaviour is probably due to weak associations formed between the molecular chains. CMC consists of linear chains of 1,4linked glucose residues while guar gum consists of linear chains of 1,4-linked mannose residues with approximately half having a galactose residue attached through 1,6-linkages. Both adopt relatively stiff ribbon-like conformations (q ~ 10±30 nm). They have typical viscosity±shear rate profiles exhibiting a high viscosity Newtonian plateau at low shear and shear thin above a critical shear rate. Dextran consists of linear chains of 1,6-linked glucose residues with some branching (linked 1,3 and 1,4). It has a very flexible compact structure since the (1,6) glycosidic linkage has three bonds of rotation between the glucose residues rather than two as is the case for other glycosidic bonds (Fig. 1.7). Significant interpenetration of molecular chains does not occur even at concentrations of 20% and hence the viscosity±shear rate profile exhibits Newtonian characteristics. Similar behaviour also occurs for gum arabic which has a very highly branched structure. It should be noted, however, that although the viscosity of dextran and gum arabic is much less than the viscosity of the 1% xanthan, CMC and guar solutions at low shear, they are greater at high shear rates. Charged polymers have a higher viscosity than non-ionic polymers of similar molecular mass because their molecular coils are expanded as a consequence of 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. The main hydrocolloid thickeners used in food products are listed in Table 1.4.
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Handbook of hydrocolloids
Table 1.4
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.
1.4
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, G0 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.
Fig. 1.8
G0 and G00 of 0.5% and 2.0% guar gum solutions as a function of frequency.
Introduction to food hydrocolloids
15
This is illustrated in Fig. 1.8 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 three-dimensional 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. 1.9(a) which shows the
Fig. 1.9 (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.
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Handbook of hydrocolloids
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 over this frequency range (Fig. 1.9(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, for example, will form gels at concentrations as low as 0.2%, while for acid-thinned starch, a concentration of ~15% is 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 in Table 1.5 and a comparison of their relative gel textures is illustrated in Fig. 1.10. An increase in brittleness is usually accom-
Fig. 1.10
Qualitative comparison of the textures of gels produced by different hydrocolloids.
Introduction to food hydrocolloids Table 1.5
17
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 coil-helix 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 hydroxypropylmethyl 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.
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Handbook of hydrocolloids
panied 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 phase separation. The various effects that can occur are summarised schematically in Fig. 1.11. 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) will associate and may form soluble or insoluble complexes depending on the pH, mixing ratio and ionic strength of the solution. There is evidence to show that some stiff polysaccharide molecules (xanthan gum, carrageenan, etc.) will associate with galactomannans and glucomannans leading to 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 hom*ogeneous 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 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.
Fig. 1.11 Schematic representation of the interactions that occur in solutions containing mixtures of hydrocolloids.
Introduction to food hydrocolloids
1.6
19
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 health-conscious consumer for reduced fat and enhanced fibre foods of all types. If this can be achieved using materials which 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 US$2.83 million and is set to grow significantly to meet the health aspirations of the consumer. 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, which 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 recognise the healthgiving 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 properties of such soluble and insoluble fibre allow them to perform both in a physical role and also to ferment through colonic microflora to give shortchain 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 described in this handbook fall into this category. 1.6.1 The physical effect To be effective dietary fibre 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
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Handbook of hydrocolloids
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 SCFAs 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 SCFAs 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 SCFAs, several health advantages are now established. Increasing fibre (20±30 grams per day in humans) can eliminate constipation through increased faecal bulking and waterholding. The fermentation to produce SCFAs 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 could lead 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. SCFAs stimulate water and electrolyte absorption by the mucosa and enhance
Introduction to food hydrocolloids
21
their transport through improving colonic blood flow. 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 SCFAs 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 gives greater resistance to infectious bacteria.
1.7
Future trends
The introduction of totally new hydrocolloids for food use is restricted by the large financial investment required to obtain the necessary legislative approval. Probably the last hydrocolloid to go through this process was gellan gum. There are certain hydrocolloids, however, that have a long history of use in food in other parts of the world that have potential for use as food additives in the USA and Europe. A typical 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. This material has only recently gained approval for use as an additive in the West. When dissolved in water konjac mannan 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 increasing aware of their nutritional benefits. Many hydrocolloids (e.g., locust bean gum, guar gum, konjac mannan, gum arabic, xanthan gum and pectin) for instance, have been shown to reduce blood cholesterol levels. Others
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Handbook of hydrocolloids
(e.g., inulin and 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
and VAN VLIET, T. (2003) Food Colloids: Biopolymers and Materials. Royal Society of Chemistry, Cambridge. DOXASTAKIS, G. and KIOSSEOGLOU, V. (2000) Novel Macromolecules in Food Systems. Elsevier Science BV, Amsterdam. DUMITRIU, S. (2005) Polysaccharides: Structural Diversity and Functional Versatility, 2nd edition. Marcel Dekker, New York. MCKENNA, B.M. (2003) Texture in Food. Woodhead Publishing Ltd, Cambridge. STEPHEN, A.M., PHILLIPS, G.O. and WILLIAMS, P.A. (2006) Food Polysaccharides and their Application. CRC Taylor and Francis, Boca Raton, FL. SUNGSOO CHO, S. and DREHER, M.L. (2001) Handbook of Dietary Fibre. Marcel Dekker, New York. WILLIAMS, P.A. and PHILLIPS, G.O. (2000) Gums and Stabilisers for the Food Industry 10. Royal Society of Chemistry, Cambridge. WILLIAMS, P.A. and PHILLIPS, G.O. (2002) Gums and Stabilisers for the Food Industry 11. Royal Society of Chemistry, Cambridge. WILLIAMS, P.A. and PHILLIPS, G.O. (2004) Gums and Stabilisers for the Food Industry 12. Royal Society of Chemistry, Cambridge. WILLIAMS, P.A. and PHILLIPS, G.O. (2006) Gums and Stabilisers for the Food Industry 13. Royal Society of Chemistry, Cambridge. WILLIAMS, P.A. and PHILLIPS, G.O. (2008) Gums and Stabilisers for the Food Industry 14. Royal Society of Chemistry, Cambridge. DICKINSON, E.
2 Hydrocolloids and emulsion stability E. Dickinson, University of Leeds, UK
Abstract: This chapter describes the physico-chemical principles underlying the functional role of hydrocolloids in oil-in-water emulsions. Basic terms such as emulsifier and stabilizer are defined, and the origins of electrostatic and steric interaction potentials are explained. The chapter discusses the main factors controlling flocculation, creaming, coalescence and Ostwald ripening, distinguishing between the differing effects of adsorbing and nonadsorbing hydrocolloids. The reader's attention is specifically directed towards new understanding concerning the rheological and microstructural control of emulsion stability by non-adsorbing hydrocolloids, and the great potential of electrostatic protein±polysaccharide interactions at the oil±water interface for enhancing emulsion properties. Key words: emulsion stability, depletion flocculation, rheology and microstructure, hydrocolloid emulsifiers, protein±polysaccharide complexes.
2.1
Introduction
An emulsion is a dispersion of one liquid (the dispersed phase) as small spherical droplets in another immiscible liquid (the continuous phase). The two main types of emulsions are oil-in-water (O/W) and water-in-oil (W/O). Food emulsions of the oil-in-water type include, for example, milk, cream, salad dressings, sauces and beverages; examples of the water-in-oil type are butter and margarine. The oil phase is either a flavour oil (in soft drinks) or a triglyceride oil (in dairy emulsions). In the latter case the oil phase is semi-solid at ambient temperatures due to the presence of dispersed fat crystals. Some emulsion products also contain other kinds of dispersed phases ± gas bubbles (in whipped cream), starch granules (in cake batter) and ice crystals (in ice-cream). Despite the wide diversity of structures, compositions and textures of food emulsions,
24
Handbook of hydrocolloids
there are some basic underlying principles that can be reliably used to understand and predict their general behaviour. One generally important factor influencing their stability properties is the presence of hydrocolloids. Emulsions are thermodynamically unstable. With time they tend to break down into their constituent oil and aqueous phases. The term `emulsion stability' therefore refers to the ability of an emulsion to resist this breakdown, as indicated by growth in average size of droplets or change in their spatial distribution within the sample. The more slowly that these properties change, the more stable is the emulsion. In practice, stability is a relative term which depends on the context. For some food emulsions, such as cake batters or cooked sauces, the required time-scale for stability is only a few minutes or hours. But for other products, such as soft drinks and cream liqueurs, emulsion stability must be maintained over a period of several months or years. A hydrocolloid ingredient may act as an emulsifying agent, as a stabilizing agent, or in both of these roles. An emulsifying agent (emulsifier) is a surfaceactive ingredient which adsorbs at the newly formed oil±water interface during emulsion preparation, and it protects the newly formed droplets against immediate recoalescence. Given that polysaccharides are predominantly hydrophilic in molecular character, and most hydrocolloids are not surface-active, they cannot act as primary emulsifying agents. There is really only one hydrocolloid ± namely, gum arabic ± which is commonly employed as an emulsifying agent. The main emulsifying agents used in food processing are the proteins, especially those derived from milk or eggs. A stabilizing agent (stabilizer) is an ingredient that confers long-term stability on an emulsion, possibly by a mechanism involving adsorption, but not necessarily so. In O/W emulsions, the stabilizing action of hydrocolloids such as xanthan, carboxymethycellose, carrageenan, etc., is traditionally attributed to the structuring, thickening and gelation of the aqueous continuous phase. In the context of this chapter, the expression `emulsifying agent' is to be preferred over the more concise `emulsifier'. This is because the latter term normally implies membership of the class of small-molecule surfactants, comprising lipid-based ingredients such as monoglycerides (e.g., GMS), phospholipids (lecithin) and polysorbates (Tweens). The functional role of these smallmolecule emulsifiers in food technology is typically not for emulsion making, but for other reasons: controlling fat morphology and crystallization; promoting shelf-life through interaction with starch; and destabilizing emulsions by competitive protein displacement from the oil±water interface (Dickinson, 1992). Figure 2.1 illustrates the four main kinds of instability processes exhibited by O/W emulsions: creaming, flocculation, coalescence and Ostwald ripening. (For W/O emulsions, the processes are the same, except that sedimentation replaces creaming.) Flocculation is probably the most subtle and complicated phenomenon to control, because it can be triggered by so many different factors, and the resulting emulsion properties can be quite different depending upon whether the flocculation is weak or strong. In dairy-type O/W emulsions, at or below ambient temperature, instead of the full coalescence of liquid droplets, we have
Hydrocolloids and emulsion stability
25
Fig. 2.1 Schematic representation of the key mechanisms of O/W emulsion instability: (a) stable dispersion of droplets; (b) creaming; (c) flocculation (weak); (d) flocculation (strong); (e) coalescence; (f) Ostwald ripening.
so-called `partial coalescence' (`clumping') of semi-crystalline globules. In practice, two or more of the phenomena shown in Fig. 2.1 may happen at the same time, and the presence of one mechanism (e.g., flocculation) may trigger or enhance another (e.g., creaming or partial coalescence). Emulsion phase inversion, as in the shear-induced transformation of cream (O/W) into butter (W/O), is a multi-mechanism process. Detailed theoretical analysis of these primary stability mechanisms is beyond the scope of this chapter. Readers looking for such an in-depth treatment are referred to the many standard texts (Dickinson and Stainsby, 1982; Hunter, 1986; Dickinson, 1992; Evans and Wennerstrom, 1994; Hiemenz and Rajagopalan, 1997; Walstra, 2003; Friberg et al., 2004; McClements, 2005a; Leal-Calderon et al., 2007). For any researcher embarking on a new experi-
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Handbook of hydrocolloids
mental investigation of the stability properties of emulsions, the recent critical overview of techniques and methodologies by McClements (2007) is recommended reading.
2.2
Principles of emulsion stability
A stable emulsion is one where the droplets remain sufficiently small and well separated that Brownian motion alone keeps them evenly dispersed throughout the continuous phase. The physico-chemical principles of O/W emulsion stability are based on the classical colloid theories of electrostatic and steric stabilization (Dickinson, 1992; McClements, 2005a). Electrostatic stabilization arises from the presence of electrical charge on the surface of the droplets, or more usually on the adsorbed stabilizer layer at the surface of the droplets. The greater the charge density at the surface, and the lower the ionic strength (electrolyte concentration) of the continuous phase, the more stable is the emulsion. Steric stabilization arises from the presence of a polymeric (steric) barrier at the droplet surface. To confer long-term stabilization, this polymer must be present at sufficient concentration to cover the oil±water interface completely, and it must remain permanently attached to the surface, with at least part of the molecule projecting away from the surface into the aqueous medium. Steric stabilization is increasingly supplemented by electrostatic stabilization in emulsions containing adsorbed proteins at pH values well away from the protein's isoelectric point (pI) (Damodaran, 2005). Whether emulsion droplets will remain dispersed or will tend to stick together depends on the nature of the interparticle pair potential between the droplet surfaces. Generally speaking, colloidal stability requires that the interparticle repulsion should be of sufficient range and strength to overcome the combined effects of gravity, convection, Brownian motion, and the ubiquitous short-range attractive forces, which together drive the system towards its final and inevitable phase-separated equilibrium condition. Figure 2.2 shows two possible forms of the interaction potential (measured in units of the thermal energy, kT) as a function of the surface-to-surface separation (measured in arbitrary units of the order of nanometres). The shape of the potential U(d) at any separation d tells us about the nature and strength of the interaction force between the surfaces. That is, the force F is equal to the (negative) derivative of the potential, i.e., F ÿdU d=dd. Potential A in Fig. 2.2 corresponds to a stable system where the interaction force is repulsive or zero (F 0) at all droplet separations. Potential B corresponds to a more complicated situation in which the interaction force is strongly attractive at close separations (F < 0, d < 5), repulsive at medium separations (F > 0), and weakly attractive or zero at larger separations (F 0, d 12). In this latter case, the system is stable with respect to coagulation (i.e., coalescence of emulsion droplets) but unstable with respect to reversible flocculation. In any particular emulsion, what determines whether the potential U(d) is of type A or type B, or if it has some other
Hydrocolloids and emulsion stability
27
Fig. 2.2 Theoretical interaction potential between a pair of spherical emulsion droplets. The energy U(d) (in units of kT) is plotted as a function of surface-to-surface separation d (arbitrary units). Curve A corresponds to a pure repulsive interaction. Curve B corresponds to a DLVO-type potential having a potential energy barrier more than sufficient to confer colloidal stability (pmin = primary minimum, pmax = primary maximum, smin = secondary minimum). See text for further details.
functional form, is the delicate balance between the various types of molecular forces contributing to the overall interaction.
2.2.1 Electrostatic interactions An important contribution to the stabilization of many food O/W emulsions arises from interdroplet electrostatic repulsion. As two identically charged spheres approach, there is an increase in the concentration of small ions of opposite charge (counterions) in the space between the surfaces. The consequent increase in local osmotic pressure in this gap leads to a repulsive force between the surfaces. As this repulsion has its origin in the overlap of the electrical double-layers surrounding the spheres, it is commonly referred to as the (electrical) double-layer repulsion. For a pair of spheres of radius a, the doublelayer potential has the form UR d 2r 0 a
2 0
ln 1 exp ÿd;
2:1
where r is the dielectric constant of the aqueous medium, 0 is the permittivity of free space, 0 is the surface potential, and ÿ1 is the Debye length (a measure of the thickness of the double-layer). This equation is properly valid for systems with moderate surface potentials (j 0 j 30 mV) which is typically the case for food emulsions. For a simple 1:1 electrolyte (e.g., NaCl), the double-layer thickness is given by ÿ1 =nm 0:30 I=mol dmÿ3 ÿ1=2 ;
2:2
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where I is the salt concentration (ionic strength). With increasing salt concentration, the double-layer becomes thinner, and the electrostatic repulsion becomes weaker. Strong forces of attraction occur between pairs of colloidal particles at close separations. These forces can be considered to arise from a sum of all the van der Waals forces acting between individual molecules in the different particles. For spheres of radius a, the resulting net van der Waals attraction is given by UA d ÿAH a=12d;
2:3
where AH is the Hamaker constant (5 10±21 J for oil droplets in water). For very fine oil droplets, van der Waals attractive forces are important only at very close separations, but for coarse droplets they can be of quite long range (tens of nanometres). In the absence of any opposing repulsive forces (e.g., for uncharged surfaces, or in aqueous media of high ionic strength), dispersed droplets become spontaneously aggregated (coagulated) under the influence of these ubiquitous van der Waals forces. This situation is highly unstable, leading to immediate droplet coalescence. In the classical theory of electrostatic stabilization, the double-layer energy UR (equation 2.1) and the van der Waals energy UA (equation 2.3) are added together algebraically to produce an overall pair potential: U d UR d UA d:
2:4
Equation 2.4 is commonly referred to as the DLVO potential after the four scientists (Derjaguin, Landau, Verwey and Overbeek) most closely associated with the theory. Due to the different functional forms of equations 2.1 and 2.3, the term UA(d) tends to be more important at smaller and larger values of d, and the term UR(d) is relatively more important at intermediate separations (d 2ÿ1 ). Potential A in Fig. 2.2 corresponds to the case where the double-layer repulsion is entirely predominant. Such a situation would correspond to a fine dispersion of highly charged droplets in an aqueous medium of low ionic strength. Under such conditions, the term UR(d) is sufficient to overcome UA(d) at all separations d, and the emulsion can be considered to be electrostatically stabilized. In estimating UR(d) from DLVO theory, it is common practice in formulae such as equation 2.1 to replace the surface potential 0 (more correctly, the Stern potential ) by the so-called zeta potential (-potential), i.e., the value of the potential at the plane of shear. The -potential is a convenient and popular quantity because it is readily measurable in the laboratory. For most food emulsions, however, this measurement is open to misinterpretation because the classical double-layer model is not properly applicable to polymer-coated surfaces. In particular, the presence of adsorbed polymer tends to shift the plane of shear considerably away from the droplet surface; so we actually have . Nonetheless, the value of can be quite useful as an indicator of the likely importance of electrostatic contributions to emulsion stability, especially when it is small, or its sign changes from ve to ÿve (or vice versa), as may
Hydrocolloids and emulsion stability
29
occur following adjustment of pH or introduction of a new species (e.g., hydrocolloid) into the aqueous medium. As a general rule, for an emulsion containing droplets with < 15 mV, one cannot explain stability entirely in terms of double-layer repulsion. This is true even for aqueous media of very low ionic strength, despite such solution conditions being rarely encountered in food formulations. Potential B in Fig. 2.2 corresponds to the situation where the electrostatic repulsion is predominant at intermediate separations. Such a situation would correspond to the case of moderately large charged droplets (a > 1 m) in an aqueous medium of low ionic strength. Curve B is characterized by a primary minimum (pmin) at small separations (d 2 nm), a primary maximum (pmax) at d 6 nm, and a secondary minimum (smin) at d 12 nm. Here the magnitude of the primary maximum (Umax 100 kT) is very much more than sufficient to prevent the droplet pair from aggregating into the primary minimum under the influence of the short-ranged van der Waals forces. As a rough rule of thumb, a barrier height Umax of at least 15±20 kT is required to provide long-term colloidal stability with respect to aggregation in the primary minimum (and associated emulsion droplet coalescence). However, a system described by potential B would still be unstable with respect to flocculation in the secondary minimum. This is especially the case for coarse emulsion droplets (a few micrometres in diameter) since UA increases linearly with the droplet size (see equation 2.3).
2.2.2 Steric interactions When protein or hydrocolloid is present in an adsorbed layer around the emulsion droplets, there is an additional repulsive contribution to the interparticle pair potential due to the specific polymeric character of the stabilizer layer. There are three main requirements for a polymer to be effective as a steric stabilizer: (i) the adsorbed layer should be sufficiently thick; (ii) the coverage of the interface should be complete; and (iii) the polymer should be strongly (irreversibly) attached to the interface. Theory and experiment have demonstrated (Dickinson, 2006) that an adsorbed layer formed from a biopolymer, or a mixture of biopolymers, should be at least several nanometres in thickness to provide effective steric stabilization. This implies that part of the stabilizing macromolecule should be rather hydrophilic, with a tendency to distribute itself preferentially away from the oil±water interface. In other words, the aqueous continuous phase should be a `good solvent' for part of the adsorbed polymer in order for it to be able to act effectively as a steric stabilizer. Reduction of this solvent quality (e.g., by change in temperature or pH, or by adding a cosolvent like ethanol) is detrimental to effective steric stabilization. Steric repulsion arises from the entropically unfavourable overlap and compression of the adsorbed layers when polymer-coated droplets approach close together. The overlap of polymer layers is unfavourable because it leads to a local osmotic pressure gradient associated with the free energy of mixing of
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solvent and polymer segments in the interdroplet overlap zone. The compression of layers is also unfavourable because it restricts the volume available to each polymer chain, leading to a statistical bias away from the most-probable (equilibrium) distribution of configurations, in an analogy with the elastic deformation of a rubber-like polymer network. The overall distance-dependent free energy change is given by GS d Gm d Gvr d;
2:5
where Gm is the `mixing' (osmotic) free energy arising from polymer layer overlap and Gvr is the `volume restriction' (elastic) free energy arising from polymer layer compression. By analogy with equation 2.4, the overall pair potential is obtained by combining the steric free energy with the van der Waals energy: U d GS d UA d:
2:6
For sterically stabilized droplets of small size ( 1 m) under good solvent conditions, the steric free energy predominates at all separations, and the form of the overall potential U(d) is qualitatively similar to that of curve A in Fig. 2.2. Assuming that the polymer stabilizer stays irreversibly adsorbed, the highly repulsive term Gvr always dominates the overall interaction at very close separations, even under poor solvent conditions. Hence, in contrast to the electrostatic stabilization case, the sterically stabilized system has no primary minimum. This means that, as long as the oil±water interface is fully covered by the thick polymer layer, the droplets are extremely stable with respect to coalescence. On the other hand, large sterically stabilized droplets can be susceptible to flocculation due to the presence of an energy minimum in U(d) somewhat analogous the DLVO secondary minimum of curve B in Fig 2.2. Invariably, in a food O/W emulsion, the droplets carry a significant electrical charge, and the droplet surfaces are also coated with adsorbed biopolymers. Hence most food emulsions are stabilized by a combination of electrostatic and steric mechanisms. In fact, in the same colloidal system there is intimate coupling of factors affecting both mechanisms. That is, for the most effective stabilizing biopolymers, it is the presence of hydrated charged regions of the macromolecules which confers substantial thickness to the adsorbed layer, as well as significant charge density at the plane of shear. At the same time, the adsorbed biopolymer layer affects the DLVO-type potential in two different ways: (i) by changing the value of the effective Hamaker constant in UA (see equation 2.3), and (ii) by changing the ionization of surface groups and partitioning of mobile ions between bulk phase and interface, causing perturbation of the double-layer repulsion UR. This complexity makes the identification of the main mechanism of stabilization difficult in specific cases. Nevertheless, generally speaking, for a protein-stabilized emulsion at a moderate electrolyte concentration and a pH not too far away from pI, steric stabilization is likely to be the predominant mechanism.
Hydrocolloids and emulsion stability
31
2.2.3 Flocculation Flocculation is defined as the coming together of emulsion droplets without rupture of the protective stabilizing layer. It occurs when the overall pair interaction energy at some separation d becomes appreciably negative, i.e., jU dj has a value of the order of kT (weak and reversible flocculation) or is much larger (strong and irreversible flocculation). An emulsion may become flocculated in different ways: · Lowering of the surface charge density. Flocculation may be induced by pH change or specific binding of ions (e.g., Ca2+). This is most likely in a system where electrostatic repulsion makes an overriding contribution to U(d), or where the configurations of the stabilizing polymers are especially sensitive to the charge distribution. · Increasing the ionic strength. The addition of simple salts to the aqueous phase reduces the thickness of the electrical double-layer and so diminishes the range of the electrostatic repulsion. · Lowering of the solvent quality. Flocculation is induced under thermodynamic conditions favouring reduced solubility of the adsorbed stabilizer in the aqueous medium, e.g., arising from pH change or ethanol addition. · Formation of polymer bridges during emulsification. Bridging flocculation (`clustering') arises when insufficient emulsifier is readily available during emulsification to saturate the droplet surfaces. Hence adsorbed polymer molecules or particles (e.g., casein micelles) become shared between neighbouring droplets. · Formation of polymer bridges after emulsification. Bridging may arise from cross-linking of adsorbed protein molecules on different droplets as a result of heat treatment. Bridges may also be formed through the development of associative interactions between a hydrocolloid additive and protein adsorbed at the oil±water interface (see Section 2.4.2). · Influence of non-adsorbed species. The presence of various non-adsorbed species (micelles, nanoparticles or polymers) causes depletion flocculation. The effect can be explained in terms of an extra attractive (entropic) contribution to U(d) arising from the exclusion of these species from the narrow gap between closely approaching droplet surfaces (see Section 2.3.2).
2.2.4 Creaming Creaming is the movement of oil droplets under gravity to form a concentrated cream layer at the top of an O/W emulsion. Stokes' Law tells us that the creaming speed v of an isolated rigid droplet of density and radius a is v 2a2 0 ÿ g=90 ;
2:7
where 0 and 0 are respectively the density and (Newtonian) viscosity of the continuous phase, and g is the acceleration due to gravity. Equation 2.7 is strictly
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applicable to an emulsion that is dilute, monodisperse and non-flocculated. For such a system, creaming may be slowed down in three main ways: 1. Reduction of droplet size. The mean droplet size is mainly determined by the emulsifier concentration and the efficiency of the emulsification equipment. Using a high-pressure hom*ogenizer with an adequate emulsifier/oil ratio under favourable emulsifying conditions (e.g., with milk protein at neutral pH), it is relatively straightforward to prepare fine emulsions (< 1 m) that are non-flocculated and reasonably monodisperse. Nevertheless, some creaming is inevitable because it is impossible in practice to eliminate completely the small fraction of larger droplets. 2. Reduction of density difference between phases. The density difference between liquid milk fat and water is 1 102 kg mÿ3 at ambient temperatures. Apart from triggering the (partial) crystallization of the fat phase in the oil droplets, which reduces the density difference (and hence the creaming speed) by about 20%, there is little that can be done to influence the density difference in dairy emulsions. In flavour oil emulsions prepared for the soft drinks industry, however, it is common practice to incorporate an oil-soluble `weighting agent' (e.g., ester gum, damar gum) in order to adjust the density of the citrus oil droplets towards that of the aqueous continuous phase (Tan, 2004). 3. Increase in effective viscosity of aqueous medium. Rheological control of the aqueous phase is the main function of hydrocolloids in food formulations. To make meaningful predictions of the effect of hydrocolloids on creaming stability, the viscosity 0 appearing in equation 2.7 should be the apparent viscosity of the solution measured at very low shear stress (10ÿ2 Pa). This corresponds to the stress level exerted on the surrounding medium when gravity acts on micron-sized oil droplets in water. Away from the dilute Stokes limit, the creaming rate is lower because of hydrodynamic interactions between the droplets. Therefore concentrated emulsions are inherently more stable to creaming than dilute emulsions. In fact, creaming becomes inhibited altogether when the oil volume fraction is sufficiently high that the droplets become close-packed (as in mayonnaise). A semi-empirical equation describing the creaming of a moderately concentrated emulsion is
v vS 1 ÿ = k ;
2:8
where vS is the Stokes speed from equation 2.7, is a parameter ( 0:6) approximating the close-packing limit for monodisperse spheres, and k ( 8) is an adjustable parameter. Even though it may not be readily evident to the naked eye, some creaming must inevitably occur during extended quiescent storage of a moderately concentrated emulsion. For instance, Fig. 2.3 shows the oil volume fraction as a function of height in a sample of surfactant-stabilized emulsion (18 vol%
Hydrocolloids and emulsion stability
33
Fig. 2.3 Creaming profile of stable surfactant-stabilized emulsion (18 vol% mineral oil, 2 wt% Tween 20, mean droplet size d32 = 0.65 m, height 25 cm) as determined with the Leeds Acoustiscan ultrasonic profiler (Povey, 1998). Oil volume fraction is plotted against height for an unstirred emulsion sample at 30 ëC: , after 3 hours; ·, after 188 hours. (Experimental data of Ma et al. (1994), as supplied by Prof. Macolm Povey.)
mineral oil, 2 wt% Tween 20) containing small droplets (mean droplet diameter 1 m) stored at 30 ëC (Ma et al., 1994). These data were obtained by ultrasound velocity scanning, which is an automated non-invasive technique allowing the determination of the local oil droplet density as a function of height for emulsion samples that are highly opaque (Povey, 1998). The freshly prepared emulsion has a vertically uniform oil content, but after one week of undisturbed storage there is a decrease in near the bottom of the sample tube and a corresponding increase in towards the top. The creaming behaviour shown in Fig. 2.3 is typical of that of a fine, unflocculated, low-viscosity emulsion in the absence of hydrocolloid stabilizer. Emulsion creaming is strongly affected by the state of flocculation. Provided they do not get in each other's way, flocs move faster than individual droplets because of their greater effective size. Hence flocculation increases the creaming rate at low or medium . This effect is amplified by increased polydispersity due to the differential creaming speeds of smaller and larger droplets causing them to approach and associate more often than in a more monodisperse system. In a concentrated emulsion, flocculation tends to retard creaming due to the formation of an extensive network of interconnected flocs which are prevented from readily moving past one another under the influence of gravity. Phase separation may then occur only as a result of rearrangements and consolidation of the gellike structure, with liquid continuous phase being exuded from gaps in the
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flocculated droplet network by a process of syneresis rather than by conventional droplet creaming. The cross-over volume fraction from the dilute regime (enhanced creaming) to the concentrated regime (retarded creaming) is sensitively dependent on the microstructure of the flocs.
2.2.5 Coalescence The merging of two or more emulsion droplets to form a larger single droplet is called coalescence. In the mouth, during eating, droplet coalescence has a positive role in enhancing flavour release. But coalescence perceived during emulsion storage is invariably considered as highly unacceptable in a food emulsion product. Droplet merging during product storage leads to enhanced creaming due to shifting of the overall distribution towards larger droplet sizes. Coalescence is also more probable for larger droplets, and so the process tends to be self-accelerating, leading on to the formation of a separate layer of free oil (`oiling off'). The rate depends on the nature of the interdroplet forces at very close surface±surface separations. Once prepared without bridging flocculation, fine protein-stabilized emulsions are usually impressively stable towards coalescence. When it does occur, the phenomenon is most commonly found in dense cream layers or flocculated systems, especially when the adsorbed protein layer is weakened by the presence of surfactant (emulsifier), and/or when the emulsion is subjected to aeration or intense stirring. Partial coalescence (`clumping') is the process whereby two or more partially crystalline oil droplets join together to form an irregularly shaped aggregate (Boode and Walstra, 1993; Walstra, 1996, 2003). It occurs when a solid fat crystal from one droplet penetrates the liquid oil region of another droplet. Each clump is composed of a continuous network of aggregated fat crystals held together by `necks' of liquid oil. Partial coalescence is influenced by many of the same factors as affect normal coalescence. Two important additional factors are: (i) the temperature cycling of cream layers, which causes growth and melting of fat crystals, and (ii) agitation or stirring, which greatly increases the likelihood of collision-induced crystal penetration between droplets. Partial coalescence is a key mechanism during the churning of cream into butter, and also in the stabilization of air cells during ice-cream manufacture.
2.2.6 Ostwald ripening Ostwald ripening is the growth of larger droplets at the expense of smaller ones due to mass transport of soluble dispersed material through the continuous phase. Because of the moderately high solubility of flavour oils in water, this is an important instability mechanism in soft drink emulsions. Unlike the other main instability mechanisms, Ostwald ripening is relatively insensitive to oil volume fraction or rheology of the continuous phase. The thermodynamic driving force for Ostwald ripening is the increasing
Hydrocolloids and emulsion stability
35
chemical potential of a dispersed phase component with decreasing droplet size (Dickinson, 1992; McClements, 2005a). This leads to a greater tendency for dissolution from smaller droplets than from bigger ones. According to the classical Lifsh*tz±Slezov±Wagner theory, the coarsening rate follows the relationship dhai3 =dt / K S1 D;
2:9
where hai is the mean droplet radius, t is the time, S1 is the thermodynamic solubility of the solute in the continuous phase for a droplet with infinite curvature (a ! 1), D is the diffusion coefficient of the solute in the continuous phase, and K is a characteristic length scale which is proportional to the interfacial tension . Equation 2.9 is strictly applicable to a dilute emulsion under steady-state conditions, where the shape of the droplet-size distribution is independent of time. For such a system, Ostwald ripening may be slowed in various ways: · Increase of droplet size. Because the solubility of dispersed phase increases with decreasing droplet radius, the ripening rate becomes greater the smaller the mean droplet size. Hence the preparation a very fine emulsion ± with the intention of reducing creaming (and flocculation/coalescence) ± is not a successful strategy for ensuring stability if the rate of Ostwald ripening is significant. · Increase in monodispersity. Because the thermodynamic driving force is related to differences in droplet size, the narrower the size distribution, the slower the rate. In theory, a perfectly monodisperse emulsion does not exhibit Ostwald ripening. · Reduction in interfacial tension. Substantial lowering of (e.g., by adding surfactant) may be difficult to achieve in practice without adversely affecting the solubility of the dispersed material in the continuous phase. However, growth or shrinkage of individual droplets can be stopped altogether by the presence of an adsorbed elastic layer which mechanically resists the expansion or compression of the droplet surface area. · Incorporation of insoluble material into dispersed phase. In practice, this is the most successful strategy. Entropy of mixing favours a similar composition for all dispersed droplets. So the mixing of another insoluble (or poorly soluble) component into the dispersed phase reduces the coarsening tendency by providing a large counteracting thermodynamic driving force in direct opposition to the Ostwald ripening effect.
2.3 Effect of non-adsorbing hydrocolloids on emulsion stability A major function of hydrocolloids in food emulsions is to act as a thickening agent of the aqueous medium. The intention is to reduce or inhibit creaming by modifying the rheology of the continuous phase. In addition, the hydrocolloid
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may impart a desirable texture and smooth mouthfeel to the product. This type of additive is functionally effective at very low concentrations (< 0.5%) due to its high molecular weight and extended macromolecular structure.
2.3.1 Rheology of hydrocolloid solutions The effective hydrodynamic size of a non-gelling hydrocolloid in aqueous solution is indicated by its intrinsic viscosity []. Under highly dilute conditions where the solution is Newtonian, the intrinsic viscosity is defined by limc!0 sp =c;
2:10
where c is the polysaccharide concentration and sp is the specific viscosity, defined as sp r ÿ 1 =s ÿ 1:
2:11
Here r is the relative viscosity, is the viscosity of the hydrocolloid solution, and s is the viscosity of the pure solvent (c 0). The intrinsic viscosity increases with the weight-average molecular weight Mw according to the Mark± Houwink relationship: K 0 Mw ;
2:12
where K 0 and are constants (Tanford, 1961; Launay et al., 1986). For linear flexible polymers in a good solvent (e.g., aqueous solutions of dextran, amylose or locust bean gum), the exponent has a value in the range 0.5±0.8. For highly branched polymers (like amylopectin) the value of a lies below 0.5. Conversely, the value of is greater than unity for stiff linear polymers and polyelectrolytes (especially at low ionic strength). Each flexible polymer molecule in a dilute solution does not interact significantly with the other polymers. But as the polymer content increases, a certain concentration is reached (c c ) beyond which the neighbouring polymer coils begin to overlap. In this semi-dilute solution (c > c ) each individual polymer segment (being mainly surrounded by solvent) `sees' the solution as still dilute, but each whole polymer molecule `sees' the solution as concentrated, since it is entangled with, and hence is interacting with, other polymer molecules in the solution (de Gennes, 1979). Hydrocolloids differ considerably in their values of c . In general, the higher the molecular weight of the polymer, the lower is the overlap concentration. But for the same molecular mass, [] is much smaller for a highly compact branched hydrocolloid like gum arabic than for an extended linear hydrocolloid such as carrageenan. Hence, whereas a 10 wt% gum arabic solution may be regarded as being still dilute (c < c ), a carrageenan solution of 1 wt%, or a xanthan gum solution of only 0.1 wt%, is in the semi-dilute regime (c > c ). With non-gelling hydrocolloids whose flow properties are determined by nonspecific repulsive chain±chain interactions, the relationship between the limiting zero-shear-rate viscosity 0 and the polymer concentration c changes rather
Hydrocolloids and emulsion stability
37
Fig. 2.4 Concentration-dependent viscosity of non-gelling hydrocolloid. The logarithm of the specific viscosity sp is plotted against the logarithm of c[], where c is the concentration and [] is the intrinsic viscosity defined by equation 2.10. The cross-over point between the dilute regime (c < c , slope 1.3) and the semi-dilute regime (c > c , slope 3.3) occurs at sp 10 and c 4=.
abruptly at the overlap concentration. That is, when represented as a double logarithmic plot of specific viscosity sp ( _ ! 0) against c, the experimental data fall on a master curve as shown in Fig. 2.4, with straight lines of slopes 1.3 and 3.3 representing the dilute and semi-dilute regimes, respectively (Morris, 1984). The transition point occurs at the concentration c 4=, corresponding to a viscosity of 0 c 10 mPa s (i.e., sp 10). More complex behaviour than that indicated in Fig. 2.4 is found with some hydrocolloids (e.g., guar gum or locust bean gum) whose molecules have gel-like attractive interactions between specific sequences on different chains. In these cases the onset of the semi-dilute regime occurs at lower c and the slope of the log±log plot of sp against c beyond c c is higher. Whereas the dilute solution is (nearly) Newtonian in its flow behaviour, the semi-dilute solution is shear-thinning. The dependence of the apparent viscosity on the shear-rate _ can be described, for instance, by the semi-empirical Cross equation (van Aken, 2006): 1 0 ÿ 1 = 1 _ m :
2:13
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Handbook of hydrocolloids
The quantities 0 and 1 are the limiting viscosities at very low and very high shear rates, respectively. The parameter is a characteristic time constant corresponding to the average time of interpenetration/interaction of the deformed coil, and the exponent m has a value of 0.76. The value of increases with the hydrocolloid concentration and the molecular weight. For the hydrocolloid thickener of first choice in food emulsions, i.e., xanthan gum, the value of 0 (as measured at _ 1 sÿ1) is typically several orders of magnitude higher than 1 (as measured at _ 103 sÿ1). This allows the thickened emulsion to flow readily under the moderately high-shear conditions encountered during product formulation, carton filling/emptying and eating. At the same time, the high value of 0 provides the emulsion continuous phase with an extremely viscous environment, which resists the movement of individual droplets against the combined influences of gravity, interdroplet forces and Brownian motion.
2.3.2 Depletion flocculation and serum separation One of the most common types of instability in food O/W emulsions is depletion flocculation caused by the presence of a low concentration of non-adsorbing hydrocolloid. Depletion flocculation is also induced by nanoparticles such as surfactant micelles and casein sub-micelles (Radford and Dickinson, 2004). The depletion contribution to the pair potential U(d) is associated with an osmotic pressure difference between the interdroplet gap region depleted of polymer molecules (or nanoparticles) and the continuous phase beyond the interdroplet gap region. For the idealized case of flocculation of large hard spheres of radius a by small hard spheres of diameter ds, the Asakura±Oosawa depletion potential has the form 8 3 > < ÿ3kTas =ds ds ÿ d; d < ds 2:14 Ud d > : 0; d ds
where s is the volume fraction of the small spheres. The interaction is relatively weak. For instance, for two emulsion droplets of radius a 0:5 m in a solution containing nanoparticles of diameter ds 10 nm at a concentration of s 0:02 (i.e., 2 vol%), equation 2.14 gives a maximum depletion attraction at contact (d 0) of Ud ÿ3kT. The depletion interaction arising from flexible non-adsorbed polymers is greater than that from spherical nanoparticles because the configurational entropy of the chains is substantially reduced near the particle surface (Vincent et al., 1986). A useful theoretical expression is Ud d ÿ2a ÿ 12 d1 2=3a d=6a;
2:15
where is the osmotic pressure of the polymer solution. The quantity is the depletion layer thickness, which is approximately equal to the radius of gyration of the polymer in the dilute regime. Dilution of the continuous phase with
Hydrocolloids and emulsion stability
39
solvent leads to lowering of and hence Ud(d), which means that this type of weak flocculation is completely reversible with respect to dilution by the continuous phase. Therefore its presence is not detectable with the conventional light-scattering equipment (e.g., Malvern Mastersizer) used for routine dropletsize analysis. Depletion flocculation can have an enormous effect on creaming stability (Dickinson, 1993, 2003, 2004). At very low hydrocolloid concentrations, the entropy loss linked to droplet association outweighs the depletion effect and so the system remains stable. Beyond a certain critical concentration, however, the presence of depletion flocculation is manifest in rapid serum separation. Due to their large excluded volumes, polysaccharides with stiff extended backbones (e.g., xanthan gum) are especially effective at inducing depletion flocculation. Figure 2.5 shows the influence of various concentrations of xanthan on the plot of serum layer thickness against time for samples of sodium caseinate-stabilized emulsions (10 vol% oil, 0.5 wt% protein, pH 7) stored quiescently at 25 ëC in glass tubes of height 10 cm (Cao et al., 1990). The reference emulsion without added xanthan shows no visible serum separation over the storage period of 72 h, but the presence of just 0.025 or 0.05 wt% xanthan is sufficient to induce rapid serum separation (complete within 1±2 h) of the low-viscosity liquid-like
Fig. 2.5 Influence of xanthan gum on creaming of protein-stabilized emulsion (10 vol% oil, 0.5 wt% sodium caseinate, pH = 7, ionic strength = 0.005 M, total sample height = 10 cm). Serum layer height H is plotted against storage time t at 25 ëC for various polysaccharide concentrations: t, 0 wt% and 0.125 wt%; l, 0.025 wt%; , 0.05 wt%; ú, 0.065 wt%; 4, 0.1 wt%. (Reproduced with permission from Cao et al., 1990.)
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Handbook of hydrocolloids
emulsion, with the serum (non-cream) layer occupying 65±70% of the sample volume. At 0.0625 wt% xanthan, the same degree of phase separation takes 48 h; at 0.1 wt% xanthan, there is only 10% serum separation after 72 h; and, at 0.125 wt% xanthan, the system is as stable as in the absence of xanthan. Inhibition of creaming at high xanthan concentrations is conventionally attributed to the immobilization of the emulsion droplets in a weak gel-like network (very high 0 ). Figure 2.6 shows creaming profiles (volume fraction vs height) for a surfactant-stabilized emulsion (18 vol% oil, 2 wt% Tween 20) containing 0.17 wt% xanthan (Ma et al., 1994). The data were obtained by ultrasound velocity scanning on the same emulsion system (no added salt) as that shown in Fig. 2.3. The sample shows no change in oil content for several hours, but after 1 day there evolves a pronounced continuous gradient in from the bottom to the top of the sample. After 2 days there is a distinct boundary at height 150 mm between a lower emulsion phase ( 0:05±0.1) and an upper emulsion phase ( 0:35±0.4). After 4 days the lower serum has become completely depleted of oil droplets, and after 1 week the upper cream layer has reached an oil content equivalent to close-packed monodisperse hard sphere ( 0:63). For more concentrated emulsions (say, 0:3), the change in emulsion creaming behaviour caused by addition of non-adsorbing polysaccharide tends to correlate better with the rheology of the emulsion as a whole, rather than with
Fig. 2.6 Creaming of surfactant-stabilized emulsion (18 vol% mineral oil, 2 wt% Tween 20, mean droplet size d32 = 0.65 m, height 25 cm) containing 0.17 wt% xanthan (low ionic strength). Oil volume fraction is plotted against height for an unstirred emulsion sample at 30 ëC: , after 11 h; s, after 20 h; , after 30 h; n, after 45 h; ú, after 99 h; l, after 188 h. (Experimental data of Ma et al. (1994), as supplied by Prof. Macolm Povey.)
Hydrocolloids and emulsion stability
41
the rheology of the hydrocolloid solution making up the continuous phase (Dickinson, 2003, 2004). This implies that stability is controlled by the properties of the long-lasting metastable network of flocculated droplets held together by the short-range depletion interactions induced by the added hydrocolloid (Parker et al., 1995; Dickinson, 2006). A manifestation of the early stages of the system's evolution is phase separation on the microscopic scale. This phenomenon can be considered to be somewhat analogous to the phase separation observed in mixed solutions of two polymers exhibiting thermodynamic incompatibility (Tolstoguzov, 1991). Figure 2.7 shows some microscopic images of a caseinate-based emulsion (30 vol% oil, 1.4 wt% protein, pH = 7, mean droplet size 0:5 m) containing
Fig. 2.7 Influence of xanthan gum on microstructure of protein-stabilized emulsion (30 vol% oil, 1.4 wt% sodium caseinate, pH = 7, mean droplet size d32 ~ 0.5 m). Confocal images (250 250 m) were captured 10 minutes after sample stirring had stopped. Polysaccharide concentrations in the aqueous phase: (a) 0.01, (b) 0.02, (c) 0.04 and (d) 0.1 wt%. (Reproduced with permission from Moschakis et al., 2005.)
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Handbook of hydrocolloids
various concentrations of xanthan in the aqueous phase (Moschakis et al., 2005). The images were recorded 10 minutes after stopping stirring. We can see microscopic phase separation into separate oil-rich and xanthan-rich regions, the latter appearing as dark blobs against the lighter background. In the hydrocolloid concentration range 0.02±0.05 wt%, the samples were observed to exhibit complex dynamic behaviour, including blob shape relaxation processes, microstructural coarsening and microphase coalescence phenomena. The elongated shapes of the xanthan-rich domains arise from the hydrodynamic shear forces applied during sample preparation and mixing. Once stirring ceases, the interfacial tension drives the blob shape towards spherical at a rate which sensitively depends on the viscoelastic properties of the blob and its surrounding local environment. The blob shape relaxation time is a strongly increasing function of xanthan content. For the system illustrated in Fig. 2.7, no microstructural evolution was observed over the experimental time-scale (several hours) for xanthan concentrations above 0.06 wt%. Analysis of the diffusion coefficient of individual probe particles has demonstrated that, in this low polysaccharide concentration range, the effective viscosity of the oil-droplet-rich regions is up to 103 times larger than that of the coexisting xanthan-rich regions (Moschakis et al., 2006). Furthermore, the inferred low-stress viscosity of the flocculated oildroplet-rich region increases dramatically with the xanthan concentration. So, as a result of changes in microstructure and microrheology caused by the hydrocolloid addition, the original liquid-like protein-stabilized emulsion is transformed into a stable, concentrated, viscoelastic emulsion containing `blobs' of hydrocolloid-structured water.
2.4
Effect of adsorbing hydrocolloids on emulsion stability
2.4.1 Hydrocolloid emulsifying agents Any hydrocolloid that is substantially surface-active has the potential for acting as both an emulsifying agent and a stabilizing agent. The effectiveness of the emulsifying agent is related to the minimum amount of it that is required to generate and stabilize small droplets during hom*ogenization. A useful quantity in this context is the surface load, i.e., the mass of emulsifying agent per unit area of oil±water interface (typically a few mg mÿ2). The mass adsorbed per unit volume of emulsion (Cs) is equal to the initial concentration of emulsifying agent minus that remaining in the aqueous phase after hom*ogenization, as determined following centrifugation or filtration. The surface load ÿ is calculated from ÿ Cs d32 =6;
2:16
where d32 is the volume±surface mean diameter. Knowledge of the surface load enables the calculation of the minimum amount of emulsifying agent required to prepare an emulsion of known volume fraction and droplet size. For the milk protein -casein, the theoretical minimum amount lies quite close to the amount
Hydrocolloids and emulsion stability
43
actually needed in practice. But for a less efficient emulsifier (e.g., gum arabic) a substantial excess of emulsifying ingredient is required in order to achieve the desired effect. The reason for this is that the gum is a complex mixture of polymers, and not all of the macromolecular species are capable of adsorbing and stabilizing the interface (Randall et al., 1988). Moreover, even those fractions of the gum that are capable of adsorbing do not necessarily have time to do so during the very rapid process of high-pressure hom*ogenization (10ÿ4 s). Consequently, when employing gum arabic as emulsifying agent, it is necessary to use a rather high gum/oil ratio (1:1) in order to produce fine stable emulsion droplets (d32 1 m) (McNamee et al., 1998). Even though acacia gums typically contain just a few per cent protein, this fraction seems to play a crucial role in terms of the interfacial functionality (Dickinson et al., 1988; Dickinson, 2003). For acacia gums of variable nitrogen content, there is a good correlation between the protein content and the steady-state interfacial tension at the oil±water interface, and also between the emulsifying capacity and the rate of change of tension with time. However, the nitrogen content alone is not a reliable indicator of the effectiveness of acacia gums for emulsification. Other factors also appear to be important, such as the average molecular weight of the carbohydrate polymers, and the distribution of the proteinaceous fraction amongst the different sized polymer species. The special emulsifying and stabilizing properties of gum arabic (Acacia senegal) is associated with a high-molecular-weight fraction representing less than 30% of the total hydrocolloid (Randall et al., 1988, 1989; Ray et al., 1995). The protein is covalently bound to the carbohydrate in the form of a mixture of arabinogalactan±protein complexes each containing several highly branched polysaccharide units linked to a common protein core. The protein chain firmly anchors the complex to the oil±water interface, and the charged polysaccharide units attached to the protein chain provide a barrier against flocculation and coalescence. Gum arabic is a polyelectrolyte, and so the emulsion droplets carry a net negative charge under beverage emulsion conditions. However, the value of the zeta potential is rather low (10±20 mV), indicating that the main stabilization mechanism is steric in character. Certain types of pectin have also been shown to be effective for making emulsions. Again the interfacial functionality is mainly attributed to the presence of bound protein. In the case of citrus pectin, the accessibility of the protein fraction for adsorption at the oil±water interface can be substantially enhanced by depolymerization (Akhtar et al., 2002). Whereas for citrus or apple pectin the bound fraction of protein is low (1.5±3%), it is significantly higher (up to 10%) for sugar beet pectin. In fact, due to its higher protein content, sugar beet pectin is even more surface-active than gum arabic, being effective in stabilizing fine emulsions at the relatively low emulsifier/oil ratio of 1:10 (Williams et al., 2005).
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2.4.2 Protein±polysaccharide interactions: conjugates and complexes In searching for a replacement for the expensive and occasionally scarce gum arabic, there has been a move towards preparing composite protein±polysaccharide emulsifiers by alternative methods. One straightforward and practical way to achieve conjugation of protein with polysaccharide is by using the Amadori rearrangement steps in the Maillard reaction carried out under controlled dry-heating conditions (Kato et al., 1990; Dickinson and Galazka, 1991). During this complex sequence of reactions, the terminal and side-chain amine groups on the protein become linked to the reducing end of the polysaccharide. Conjugation of carbohydrate to protein improves solubility under unfavourable solution conditions of low pH and high ionic strength, with consequent benefits for protein emulsification properties. For instance, a whey protein±maltodextrin conjugate made from commercial whey protein isolate and maltodextrin (molecular weight ~ 104 Da) can produce fine emulsion droplets (< 1 m) with triglyceride oil or orange oil under neutral or acidic conditions. As a possible replacement for gum arabic, the protein±polysaccharide conjugate emulsifier can be used at relatively low emulsifier/oil ratios to prepare low-pH beverage-type emulsions with a shelf-life of several weeks. Furthermore, in contrast to the corresponding emulsion based on protein alone, the conjugatebased formulation exhibits no precipitation or phase separation on mixing with colouring agents, either before or after extensive dilution (Akhtar and Dickinson, 2007). Polysaccharides can form various types of physical complexes with proteins depending on the pH, the ionic strength, and the biopolymer charge distributions. The formation of such complexes implies the dominance of short-ranged attractive forces. Attractive protein±polysaccharide interactions may be strong (long-lasting) or weak (reversible). These non-specific protein±polysaccharide interactions arise as a result of averaging in time and space over all the individual specific chemical interactions (ionic, hydrogen bonding, hydrophobic, etc.) on the different macromolecules (Dickinson, 1998). Electrostatic forces are especially important at low ionic strength, and complete dissociation of complexes usually occurs on addition of excess electrolyte. Strong attractive electrostatic interactions are typically found with positively charged proteins (pH < pI) and negatively charged polysaccharides. Weaker reversible complexes tend to be formed between uncharged (pH pI) or negatively charged proteins (pH > pI) and anionic polysaccharides. On adjustment of pH and/or electrolyte concentration, the net protein±polysaccharide interaction may change substantially, even switching from net attractive to net repulsive. Some hydrocolloids can achieve interfacial functionality during or after emulsion formation by forming an associative interaction with an adsorbed protein layer. Whilst the charged polysaccharide by itself is not surface-active, the electrostatic complex of protein + polysaccharide has a strong tendency to adsorb at the oil±water (or air±water) interface (Dickinson, 1995, 1998). The overall effect of the interacting polysaccharide on the emulsion stability and microstructure is dependent on the concentration of added polymer, as illu-
Hydrocolloids and emulsion stability
45
Fig. 2.8 Schematic representation of the effect of attractive protein±polysaccharide interactions on a protein-stabilized emulsion: (a) no added polysaccharide; (b) bridging flocculation at low polysaccharide concentration; (c) steric stabilization of droplets with saturated coverage of polysaccharide; (d) emulsion gel with droplets immobilized in entangled polysaccharide network.
strated schematically in Fig. 2.8. Addition of a small amount of polymer to an initially stable emulsion (a) leads to bridging flocculation (b). When sufficient polymer is present to saturate the droplet interface, the emulsion is stabilized electrostatically by the secondary layer of charged polysaccharide (c). And when the hydrocolloid content in the aqueous phase is sufficiently high (c > c ), the emulsion droplets may be immobilized in a polymer gel network (d). All these stages of (in)stability behaviour have been reported (Dickinson and Pawlowsky, 1997) for emulsions based on bovine serum albumin (BSA) at pH 6 and low ionic strength in the presence of various concentrations of -carrageenan, a sulfated polysaccharide of high charge density. Direct experimental evidence of the presence of protein±polysaccharide interactions at the oil±water interface
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Handbook of hydrocolloids
can be readily indicated by surface rheological measurements (Dickinson and Galazka, 1992; Dickinson et al., 1998). The structure of any composite interfacial layer comprising both protein and polysaccharide depends to some extent on the procedure used to make the emulsion. One way is to prepare a mixed solution of the biopolymers, and then use the protein±polysaccharide complex as the emulsifying agent during hom*ogenization. The alternative approach involves first preparing the emulsion with protein as the emulsifying agent, and afterwards adding the interacting polysaccharide to the aqueous phase to produce an emulsion containing droplets coated with a protein±polysaccharide bilayer. The so-called `layer-by-layer' approach has attracted much attention recently because of its potential for encapsulation technology and its success in protecting emulsions against severe environmental stresses (Guzey and McClements, 2007). In practice, though, it is not straightforward to use the layer-by-layer approach to make fine stable emulsions containing adsorbing hydrocolloids. A major issue is the tendency for extensive flocculation even under conditions where the protein surface should be saturated with polysaccharide (McClements, 2005b). Bridging flocculation occurs when the polysaccharide content is such that droplet collisions occur faster than the rate of polysaccharide saturation of the protein-coated droplet surface, and depletion flocculation and/or gelation may occur if the system contains too high a concentration of unadsorbed polysaccharide. Typically, with intense mechanical agitation and a low oil content ( 0:05±0.1), there is a window of polysaccharide concentrations within which fine stable emulsions can be prepared. But for high oil contents, even with vigorous agitation, there is no useful range of polysaccharide content over which unflocculated emulsions are accessible. For this reason, it can be more convenient to prepare emulsions containing protein±polysaccharide complexes by simply mixing together the two biopolymer ingredients in the aqueous phase prior to hom*ogenization (Dickinson et al., 1998; Jourdain et al., 2008).
2.5
Summary
The stability and acceptability of liquid-like oil-in-water emulsions is influenced by a number of mechanistic processes: creaming, flocculation, (partial) coalescence and Ostwald ripening. These processes are closely interrelated. In particular, the creaming stability is very sensitively correlated to the state of flocculation. In physico-chemical terms the properties of an individual emulsion are determined by the structure of the adsorbed layer, which in turn influences the nature of the interaction pair potential between the dispersed droplets. The two main stabilization mechanisms are electrostatic and steric. The basic theory underlying these two mechanisms gives useful practical insight concerning the design of emulsifier properties and the emulsion conditions favouring long-term stability.
Hydrocolloids and emulsion stability
47
Whilst food proteins are typically the primary emulsifying ingredients, a few surface-active hydrocolloids can also act as effective emulsifying agents. More generally, added hydrocolloids affect emulsion stability by modifying the viscosity and non-Newtonian rheology of the continuous phase. The presence of low concentrations of non-adsorbing hydrocolloids leads to depletion flocculation, microscopic phase separation and macroscopic serum separation. Whilst the association of polysaccharides with adsorbed protein can be a source of unwanted bridging flocculation, under optimized conditions these same interactions in electrostatic protein±polysaccharide complexes can also be used to improve emulsion stability.
2.6
References
and DICKINSON, E. (2007) `Whey protein±maltodextrin conjugates as emulsifying agents: an alternative to gum arabic', Food Hydrocolloids, 21, 607± 616. AKHTAR, M., DICKINSON, E., MAZOYER, J. and LANGENDORFF, V. (2002) `Emulsion stabilizing properties of depolymerized pectin', Food Hydrocolloids, 16, 249±256. BOODE, K. and WALSTRA, P. (1993) `The kinetics of partial coalescence in oil-in-water emulsions', in Dickinson, E. and Walstra, P., Food Colloids and Polymers: Stability and Mechanical Properties, Cambridge, Royal Society of Chemistry, pp. 23±30. CAO, Y., DICKINSON, E. and WEDLOCK, D. J. (1990) `Creaming and flocculation in emulsions containing polysaccharide', Food Hydrocolloids, 4, 185±195. DAMODARAN, S. (2005) `Protein stabilization of emulsions and foams', Journal of Food Science, 70, R54±R66. DE GENNES, P.-G. (1979) Scaling Concepts in Polymer Physics, Ithaca, NY, Cornell University Press. DICKINSON, E. (1992) An Introduction to Food Colloids, Oxford, Oxford University Press. DICKINSON, E. (1993) `Protein±polysaccharide interactions in food colloids', in Dickinson, E. and Walstra, P., Food Colloids and Polymers: Stability and Mechanical Properties, Cambridge, Royal Society of Chemistry, pp. 77±93. DICKINSON, E. (1995) `Emulsion stabilization by polysaccharides and protein± polysaccharide complexes', in Stephen, A. M., Food Polysaccharides and their Applications, New York, Marcel Dekker, pp. 501±515. DICKINSON, E. (1998) `Stability and rheological implications of electrostatic milk protein± polysaccharide interactions', Trends in Food Science and Technology, 9, 347±354. DICKINSON, E. (2003) `Hydrocolloids at interfaces and the influence on the properties of dispersed systems', Food Hydrocolloids, 17, 25±39. DICKINSON, E. (2004) `Effect of hydrocolloids on emulsion stability', in Williams, P. A. and Phillips, G. O., Gums and Stabilisers for the Food Industry ± 12, Cambridge, Royal Society of Chemistry, pp. 394±404. DICKINSON, E. (2006) `Colloid science of mixed ingredients', Soft Matter, 2, 642±652. DICKINSON, E. and GALAZKA, V. B. (1991) `Emulsion stabilization by ionic and covalent complexes of -lactoglobulin with polysaccharides', Food Hydrocolloids, 4, 281± 296. AKHTAR, M.
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and GALAZKA, V. B. (1992) `Emulsion stabilization by protein/polysaccharide complexes', in Phillips, G. O., Wedlock, D. J. and Williams, P. A., Gums and Stabilisers for the Food Industry ± 6, Oxford, IRL Press, pp. 351±362. DICKINSON, E. and PAWLOWSKY, K. (1997) `Effect of -carrageenan on flocculation, creaming and rheology of a protein-stabilized emulsion', Journal of Agricultural and Food Chemistry, 45, 3799±3806. DICKINSON, E. and STAINSBY, G. (1982) Colloids in Food, London, Applied Science. DICKINSON, E., MURRAY, B. S., STAINSBY, G. and ANDERSON, D. J. (1988) `Surface activity and emulsifying behaviour of some Acacia gums', Food Hydrocolloids, 2, 477±490. DICKINSON, E., sem*nOVA, M. G., ANTIPOVA, A. S. and PELAN, E. G. (1998) `Effect of highmethoxy pectin on properties of casein-stabilized emulsions', Food Hydrocolloids, 12, 425±432. EVANS, D. F. and WENNERSTROM, H. (1994) The Colloidal Domain, New York, WileyVCH. È BLOM, J. (EDS) (2004) Food Emulsions, 4th edn, New FRIBERG, S. E., LARSSON, K. and SJO York, Marcel Dekker. GUZEY, D. and MCCLEMENTS, D. J. (2007) `Formation, stability and properties of multilayer emulsions for application in the food industry', Advances in Colloid and Interface Science, 128±130, 227±248. HIEMENZ, P. C. and RAJAGOPALAN, R. (1997) Principles of Colloid and Surface Chemistry, 3rd edn, New York, Marcel Dekker. HUNTER, R. J. (1986) Foundations of Colloid Science, vol. 1, Oxford, Oxford University Press. JOURDAIN, J., LESER, M. E., SCHMITT, C., MICHEL, M. and DICKINSON, E. (2008) `Stability of emulsions containing sodium caseinate and dextran sulfate: relationship to complexation in solution', Food Hydrocolloids, 22, 647±659. KATO, A., SASAKI, Y., FURUTA, R. and KOBAYASHI, K. (1990) `Functional protein/ polysaccharide conjugate prepared by controlled dry heating of ovalbumin/dextran mixtures', Agricultural and Biological Chemistry, 54, 107±112. LAUNAY, B., DOUBLIER, J. L. and CUVELIER, G. (1986) `Flow properties of aqueous solutions and dispersions of polysaccharides', in Mitchell, J. R. and Ledward, D., Functional Properties of Food Macromolecules, London, Elsevier Applied Science, pp. 1±78. LEAL-CALDERON, F. L., SCHMITT, V. and BIBETTE, J. (2007) Emulsion Science: Basic Principles, 2nd edn, New York, Springer. MA, J., DICKINSON, E. and POVEY, M. J. W. (1994) `Creaming of concentrated oil-in-water emulsions containing xanthan', Food Hydrocolloids, 8, 481±497. MCCLEMENTS, D. J. (2005a) Food Emulsions, 2nd edn, Boca Raton, FL, CRC Press. MCCLEMENTS, D. J. (2005b) `Theoretical analysis of factors affecting the formation and stability of multilayered colloidal dispersions', Langmuir, 21, 9777±9785. MCCLEMENTS, D. J. (2007) `Critical review of techniques and methodologies for characterization of emulsion stability', Critical Reviews in Food Science and Nutrition, 47, 611±649. MCNAMEE, B. F., O'RIORDAN, E. A. and O'SULLIVAN, M. (1998) `Emulsification and encapsulation properties of gum arabic', Journal of Agricultural and Food Chemistry, 46, 4551±4555. MORRIS, E. R. (1984) `Rheology of hydrocolloids', in Phillips, G. O., Wedlock, D. J. and Williams, P. A., Gums and Stabilisers for the Food Industry 2, Oxford, Pergamon, pp. 57±78. MOSCHAKIS, T., MURRAY, B. S. and DICKINSON, E. (2005) `Microstructural evolution of DICKINSON, E.
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viscoelastic emulsions stabilized by sodium caseinate and xanthan gum', Journal of Colloid and Interface Science, 284, 714±728. MOSCHAKIS, T., MURRAY, B. S. and DICKINSON, E. (2006) `Particle tracking using confocal microscopy to probe the microrheology in a phase-separating emulsion', Langmuir, 22, 4710±4719. PARKER, A., GUNNING, P. A., NG, K. and ROBINS, M. M. (1995) `How does xanthan stabilize salad dressing?', Food Hydrocolloids, 9, 333±342. POVEY, M. J. W. (1998) `Ultrasonics of food', Contemporary Physics, 39, 467±478. RADFORD, S. J. and DICKINSON, E. (2004) `Depletion flocculation of caseinate-stabilized emulsions: what is the optimum size of the non-adsorbed protein nano-particles?', Colloids and Surfaces A, 238, 71±81. RANDALL, R. C., PHILLIPS, G. O. and WILLIAMS, P. A. (1988) `The role of the proteinaceous component on the emulsifying properties of gum arabic', Food Hydrocolloids, 2, 131±140. RANDALL, R. C., PHILLIPS, G. O. and WILLIAMS, P. A. (1989) `Fractionation and characterization of gum from Acacia senegal', Food Hydrocolloids, 3, 65±75. RAY, A. K., BIRD, P. B., IACOBUCCI, G. A. and CLARK, B. C., JR (1995) `Functionality of gum arabic: fractionation, characterization and evaluation of gum fractions in citrus oil emulsions and model beverages', Food Hydrocolloids, 9, 123±131. TAN, C.-T. (2004) `Beverage emulsions', in Friberg, S. E., Larsson, K. and Sjo È blom, J., Food Emulsions, 4th edn, New York, Marcel Dekker, pp. 485±524. TANFORD, C. (1961) Physical Chemistry of Macromolecules, New York, Wiley. TOLSTOGUZOV, V.B. (1991) `Functional properties of food proteins and role of protein± polysaccharide interaction', Food Hydrocolloids, 4, 429±468. VAN AKEN, G. A. (2006) `Polysaccharides in food emulsions', in Stephen, A. M., Phillips, G. O. and Williams, P. A., Food Polysaccharides and their Applications, 2nd edn, Boca Raton, FL, CRC Press, pp. 531±539. VINCENT, B., EDWARDS, J., EMMETT, S. and JONES, A. (1986) `Depletion flocculation in dispersions of sterically stabilized particles (``soft spheres'')', Colloids and Surfaces, 18, 261±281. WALSTRA, P. (1996) `Disperse systems: basic considerations', in Fennema, O. R., Food Chemistry, 3rd edn, New York, Marcel Dekker, chap. 3. WALSTRA, P. (2003) Physical Chemistry of Foods, New York, Marcel Dekker. WILLIAMS, P. A., SAYERS, C., VIEBKE, C., SENAN, C., MAZOYER, J. and BOULENGUER, P. (2005) `Elucidation of the emulsification properties of sugar beet pectin', Journal of Agricultural and Food Chemistry, 53, 3592±3597.
3 The health aspects of hydrocolloids C. A. Edwards and A. L. Garcia, University of Glasgow, UK
Abstract: The health effects of hydrocolloids are dependent on how they are incorporated into food. The contribution of each hydrocolloid is small and the health benefits of these compounds cannot be separated from those of other non-digestible carbohydrates. The impact of hydrocolloids on gastric emptying and small intestinal digestion and absorption is well established. More recent work has explored new sources and their mechanisms of action related to their fermentation to short chain fatty acids and their impact on the intestinal microbiota along with potential actions the short chain fatty acids may have on plasma lipids and satiety. This is of increasing importance given the current global epidemic in obesity. Key words: food hydrocolloids, dietary fibre, health, fermentation, short chain fatty acids, plasma lipids, satiety.
3.1
Introduction
The health effects of food hydrocolloids are dependent on how they are incorporated into foods and in the diet. There are many hydrocolloid carbohydrates naturally present in plant foods as part of the cell wall, such as hemicelluloses and pectin, or with other more specific roles within the plant such as storage polysaccharides like guar gum, exudates like gum acacia, and husk polysaccharides such as ispaghula. There are also alginates and bacterially produced hydrocolloids such as gellan and xanthan. However, the contribution of each individual hydrocolloid in the diet is small and epidemiological studies cannot identify and separate the health benefits of these compounds from those of other non-digestible carbohydrates such as insoluble non-starch polysaccharides, resistant starch and oligosaccharides. Hydrocolloids can also be incorporated in small amounts into food products
The health aspects of hydrocolloids
51
as stabilisers, emulsifiers and fat substitutes. Guar gum levels of 100 > 100 50 52 60 70 82 89 > 100
particular, calcium, in solution will inhibit the hydration of LA gellan gum as shown in Table 9.1. It is therefore necessary, in most circ*mstances, to use a sequestrant to bind the soluble calcium and so aid hydration. Typically, between 0.1 and 0.3% of a sequestrant such as sodium citrate is sufficient to allow complete hydration at 90±95 ëC in water of up to 600 ppm 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 9.2 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 9.3 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 Table 9.2
Effect of sodium citrate on the hydration of 0.25% LA gellan gum
Water hardness (ppm CaCO3) 0 100 300 600 900
Sodium citrate (%)
Hydration temperature (ëC)
0.00 0.05 0.10 0.20 0.40
75 25 65 65 68
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Table 9.3
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
citrate (less than 0.3%) is required to bind the free calcium often present in sugar syrups. LA gellan gum will not fully hydrate below pH 3.9. 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 pH 3.5, LA gellan gum can be held for up to 1 h at 80 ëC with only a minimal loss in gel quality. In neutral conditions solutions can be held at 80 ëC for several hours. LA gellan gum is readily dispersible in milk and reconstituted milk systems and will hydrate upon heating to approximately 80 ëC 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±95 ëC is sufficient to fully hydrate the gum in both water or milk systems. As a dispersion of HA gellan gum is heated, it swells rapidly at approximately 40±50 ëC to form a thick, pasty suspension. With continued heating the suspension loses viscosity suddenly at approximately 80±90 ëC signifying complete hydration. The swelling stage can be avoided by adding the gum directly to hot water (>80 ëC) 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. As with LA gellan gum, HA gellan gum will not hydrate below pH 4.0. Similar care must also be taken under hot acidic conditions to avoid hydrolytic breakdown and loss of gel quality.
9.4.3 Gelation The proposed gelation mechanism of gellan gum is based on the domain model which assumes the formation of distinct junction zones and disordered flexible polymer chains connecting adjacent junction zones.9 As a hot solution cools, gellan gum undergoes a disorder±order transition. This transition has been
Gellan gum
209
attributed to a coil-helix transition.10 In the case of LA 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. Recent studies using atomic force microscopy have challenged this and proposed a fibrous model in which network structures develop through the formation of non-associated fibres or strands via either elongation or branching.11 The acyl substituents have a profound effect on the structure and rheological characteristics of gellan gum gels. The gellan gum undergoes a similar disorder to order transition as the solution is cooled but according to the domain model further aggregation of the helices is limited by the presence of the acetyl group.8 According to the fibrous model the acyl groups inhibit end-to-end type intermolecular associations through a type of steric hindrance, resulting in a decrease in the degree of continuity and hom*ogeneity of the gelled system.11 The subsequent gels of HA gellan gum 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+).10,12 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 9.2 compares the effect of both mono- and divalent ions on the modulus of 0.5% LA gellan gum gels in water and in 60% sucrose. Below pH 3.0 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 pH 2.8±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 pH 2.5±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 results 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 9.4 and 9.5 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
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Fig. 9.2 Effect of calcium (squares), sodium (triangles) and potassium (circles) ion concentration on the modulus of 0.5% LA gellan gum gels in water (open symbols) and 60% sucrose (closed symbols).
Table 9.4 Properties of 0.5% LA gellan gum gels in optimum conditions for gel formation in water Gelling ion
Ion concentration (mM)
Modulus (Ncmÿ2)
Brittleness (%)
Setting temperature (ëC)
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 9.5 Properties of 0.5% LA gellan gum gels in optimum conditions for gel formation in 60% sucrose Gelling ion Calcium Sodium Potassium Acid
Ion concentration (mM) 0.5±1.0 25±35 8±10 pH2.5±2.7
Modulus (Ncmÿ2)
Brittleness (%)
Setting temperature (ëC)
1.66 3.13 1.71 7.74
61.5 54.1 65.6 43.9
38 47 43 64
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211
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.13 In most conditions LA gellan gum gels are not thermally reversible below 100 ëC. 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 self supporting gels at concentrations as low as 0.05% gum. LA 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 and 80 ëC and show no thermal hysteresis, i.e., they melt at the same temperature at which they set. The setting temperature increases with increasing cation concentration. For example, the temperature increases from approximately 71 ëC to 80 ëC as the calcium increases from 2 to 80 mM. A similar increase is seen when sodium or potassium concentration is increased from 10 to 200 mM.14 HA gellan gum is capable of forming self supporting gels at concentrations above approximately 0.2% gum. HA gellan gum gels do not synerise.
9.4.4 Texture of gellan gum Texture is generally regarded as a multifarious property.15 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 · 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
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Fig. 9.3
Schematic comparison of the gel texture of HA and LA gellan gum with other common gelling agents.
hydrocolloid gels. A more detailed account of the technique can be found in a review by Pons and Fiszman.16 HA and LA 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. 9.3 shows schematically how gellan gum gels compare with other
Fig. 9.4
Texture profile of HA (ÐÐ) and LA (-----) gellan gum gels measured on 1% gels at 70% strain.
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213
Fig. 9.5 Effect of HA and LA gellan gum blend ratio on the modulus and brittleness of gels prepared at 0.5% total gum concentration.
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. 9.4. 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. 9.5). It has been demonstrated by differential scanning calorimetry (DSC), and rheological measurements that mixtures of the HA and LA forms exhibit two separate conformational transitions at temperatures coincident with the individual components.8,13 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 HA and LA molecules has been found.17 Properties of the blended system can be varied through control of the blend ratio and level of ions in the mixture.18,19 At low ionic concentrations the HA form predominates, but as the ionic concentration increases the contribution of the LA form to the texture increases.
9.5
Uses and applications
Before discussing the main applications of gellan gum, an overview of the key properties of both the HA and LA forms is given in Table 9.6. This provides a useful frame of reference from which existing applications can be understood and new opportunities visualised.
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Table 9.6
Comparison of the key properties of HA and LA gellan gum
Hydration Sequestrants Viscosity Gelling ions Setting temperature Melting Clarity Texture
Low acyl gellan gum
High acyl gellan gum
> 80 ëC Yes Low Yes (mono or divalent or acid) 25±60 ëC No (except low ionic strength and in milk) Clear Firm, brittle
> 70 ëC No High Not required 70±80 ëC Yes Opaque Soft, elastic
9.5.1 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 9.1 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 600 ppm (as CaCO3). The pH and solids vary according to the juice used, but are typically pH 3.5±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. Formulation 9.1
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±45 ëC and the use of chilled fruit juice with dry-mix desserts ensures a rapid set.
Gellan gum Formulation 9.2
215
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.
LA gellan gum can also be used to modify the properties of traditional gelatin dessert jellies and an example is given in Formulation 9.2. The LA gellan gum in this formulation raises the initial set temperature of the dessert to around 35 ëC, 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 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 (pH 4.5±5.5). The extent of the interaction will depend on the pH and the ratio of gelatin to gellan gum. 9.5.2 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 hom*ogeneous, pourable systems often referred to as `fluid gels'.20 To produce smooth hom*ogeneous 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
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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. 9.6. 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 stirring, hom*ogenisation, 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. Gellan gum fluid gels can be used to produce shelf stable suspensions in a variety of beverage products.21 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 9.7. Example formulations for beverages
Fig. 9.6
Outline of potential processes for the preparation of gellan gum fluid gels.
Gellan gum Table 9.7
217
Guidelines for formulation of LA 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
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
(40±60%)
using LA or HA gellan gum which can be used to suspend gelled beads or fruit pulp are given below. Formulation 9.3 provides a starting point for a beverage type fluid gel. It has a pH of 2.9 and a setting temperature of approximately 15 ëC. It can be used to suspend jelly beads and is prepared as outlined in method 1 of Fig. 9.6 by shearing after the weak gel has been allowed to form. Formulation 9.4 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.
Formulation 9.3
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±80 ëC 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 15 ëC undisturbed. 5. Gently agitate the sample to form a fluid gel.
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Formulation 9.4
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 90 ëC to hydrate the gum. 3. At 90 ëC add the remaining dry ingredients and the fruit juice. 4. Cool to room temperature whilst mixing to form the fluid gel.
9.5.3 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 80 ëC 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 95 ëC. 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 70 ëC. It is recommended to cool the gellan/milk mixture to between 55 and 65 ëC 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 sequesterants 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 400 ppm (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. Milk beverages As described in Section 9.5.2, HA gellan gum at low concentrations is able to form a very weak gel network often referred to as a fluid gel. These fluid gels have extremely good suspension properties and can be used in a range of neutral dairy and soya based products such as chocolate milk. Development of this application was initially limited due to creation of off-flavours as a result of
Gellan gum
219
residual enzyme activity in the native, HA gellan gum acting on the milk. The off-flavour, which is reminiscent of cleaning chemicals, renders the product unpalatable and is linked to the development of para-cresol. Development of a process in which the HA gellan gum is pre-treated with a denaturing agent that is thought to act on the residual enzymes in the gellan gum has led to a new grade of gellan gum for this application.22 KELCOGELÕ HM-B is a standardised product containing the pre-treated HA gellan gum and can be used at 0.1±0.12% for stabilisation of cocoa in chocolate milk. The HA gellan gum is tolerant to a wide range of UHT conditions and products can be filled at higher temperatures than with carrageenan, the traditional hydrocolloid for this application. It provides excellent suspension of cocoa and long-term stability. It is functional in reconstituted milk, fresh milk, whey substituted beverages and low protein milk beverages. Yogurt The standard yogurt process can be followed when using LA gellan gum for both set and stirred yogurt. There are various ways in which yogurt containing LA gellan gum can be made depending on the usual manufacturing process and on the desired properties of the final yogurt. 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, hom*ogenising 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 41 ëC. 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 yogurt systems, a broken gel may result which can lead to unsatisfactory lumps in the final product. Typical use levels for LA gellan gum in yogurt are 0.04%. It can be used in combination with other stabilisers such as starch depending on the final texture required.
9.5.4 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 preventing 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 LA 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. The
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Fig. 9.7
Effect of sucrose concentration on the modulus ( ), hardness (ú) and brittleness (n) of 0.5% LA gellan gum gels.
presence of 40% w/w sugar approximately halves the calcium required for maximum gel modulus, from 8±10 mM in water gels to 4±5 mM in the sugar gels. Addition of 60% w/w sugar results in an approximately ten-fold reduction in the requirement for calcium, with only 0.5±1.0 mM added calcium required for maximum gel modulus. Similar reduction in the requirements for sodium and potassium are also seen (Fig. 9.2). Secondly above approximately 40% sugar gels become less firm and less brittle, i.e., softer and more elastic (Fig. 9.7). These effects are believed to be the result of the sugars inhibiting the aggregation step of the gelation process.23,24 These effects are also influenced by the type of sugar. Sucrose has a greater inhibitory effect than glucose, fructose or corn syrups (Table 9.8).25 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 crystalisation in confectionery manufacture, results in firmer, more brittle gels.26 Effect of sugars on HA gellan gum Less is known about the specific effects of sugars on HA gellan gum. However, addition of sugars to HA gellan gum gels generally results in an increase in the force required to break the gel. Setting and melting temperature also increase with increasing sugar concentration. In the presence of high levels of sugar (70±
Gellan gum
221
Table 9.8 Effect of sugars at 40% and 60% w/w on the textural properties of 0.5% w/w LA 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
80%), HA gellan gum 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 9.5 and 9.6. When prepared with LA gellan gum as the sole gelling agent (Formulation 9.5), the jellies are firm with a short, clean bite and flavour. The jellies can be removed from the starch moulds after about 2 h but are usually stoved for up to 72 h before demoulding. Addition of a thin boiling starch as outlined in Formulation 9.6 results in a chewier texture. Combinations of LA gellan gum with carrageenan can be used to produce gelatin-free confectionery which is suitable for halal. 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 9.9 provides a guide to preventing pregelation in gellan gum confections. 9.5.5 Fruit preparations This application covers a wide variety of systems from 30 to 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 9.10 shows that the ionic composition varies considerably between different fruits with most fruits containing significant levels of potassium ions.27 These ionic concentrations become increasingly significant in
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Formulation 9.5
Recipe for jelly sweets using LA gellan gum
Ingredients
Weight (g)
(%)
Sucrose Glucose syrup (42DE) Water Citric acid anhydrous Tri sodium citrate dihydrate KELCOGELÕ F 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 1.0 g of tri sodium citrate dihydrate and 40 g 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 90 ëC. 4. Cook the liquor to 80±82% total solids then cool to 90 ëC. 5. Dissolve the citric acid and remainder of the tri sodium citrate dihydrate, colour and flavour in 20 cm3 of water and stir into the liquor. 6. Deposit at 76±78% total solids into starch moulds. 7. Stove to final solids as required.
Formulation 9.6 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 50 g of water. 2. Blend the LA gellan gum, calcium hydrogen orthophosphate and tri sodium citrate dihydrate with 40 g 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.
Gellan gum Table 9.9
223
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
Add water to lower soluble solids
Pre-gelation before acid added
Table 9.10 Fruit Apple Blackcurrant Raspberry Strawberry Apricot Peach
Ionic composition of raw fruits27 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
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 9.7 is an example of a low solids (36%) jam which can be formulated to give 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 9.8 is slightly higher in solids than Formulation 9.7 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 (pH 3.9, soluble solids 40%). Finally, Formulation 9.9 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%).
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Formulation 9.7
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 9.8
Recipe for a peach yogfruit using LA gellan gum
Ingredients Peach pureÂe Diced peach Glucose syrup 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 90 ëC with constant stirring. 3. Hold for 1 minute then cool, with stirring, to 60 ëC. 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.
9.5.6 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
Gellan gum Formulation 9.9
225
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.
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.
9.6 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 has given gellan gum an Acceptable Daily Intake (ADI) of `not specified'. Combinations of HA and LA 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'.
9.7
Future trends
The current commercial HA and LA gellan gum products can be considered as being at opposing ends of the textural spectrum available with hydrocolloid gelling agents (Fig. 9.3). Blends of the two types enable some intermediate textures to be created but the mixed system retains the high setting temperature of the HA product and the ion sensitivity of the LA product. Far more interesting are gellan gums of intermediate acyl content as these show much more variation in texture and a single hom*ogeneous setting behaviour.28,29 Methods for creating these partially acetylated products exist but they are yet to be produced on a commercial scale.30 Realisation of this control over the degree of acylation of gellan gum could lead to a truly universal gelling agent.
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9.8
Sources of further information and advice
www.CPKelco.com Imeson, A. (1999) Thickening and Gelling Agents for Food, 2nd edn. Aspen Publishers Inc., Gaithersburg, MD.
9.9 1.
2.
3. 4. 5.
6.
7.
8.
9.
10. 11.
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. CHANDRASEKARAN, R., PUIGJANER, L.C., JOYCE, K.L. 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 L-glycerate groups on the gellan double helix', Carbohydr. Polym., 12, 431±442. CHANDRASEKARAN, R., LEE, E.J., RADHA, A. and THAILAMBAL, V.G. (1992) `Correlation of molecular architectures with physical properties of gellan related polymers', in Frontiers in Carbohydrate Research ± 2, ed. R. Chandrasekaran. Elsivier 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) `Cation-specific aggregation of carrageenan helices: domain model of polymer gel structure', J. Mol. Biol., 138, 349. GRAZDALEN, H. and SMIDSRéD, O. (1987) `Gelation of gellan gum', Carbohydr. Polym., 7, 371±93. O'NEILL, M.A., SELVENDRAN, R.R.
NODA. S., FUNAMI, T., NAKAUMA, M., ASAI, I., TAKAHASHI, R., AL-ASSAF, A., IKEDA, S.,
and PHILLIPS, G.O. (2008) `Molecular structures of gellan gum imaged with atomic force microscopy in relation to the rheological behaviour in aqueous systems. 1. Gellan gum with various acyl contents in the presence and absence of potassium', Food Hydrocolloids, 22, 1148±59. 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. NISHINARI, K.
12.
13.
KASAPIS, S., GIANNOULI, P., HEMBER, M.W.N., EVAGELIOU, V., POULARD, C., TORT-
and SWORN, G. (1999) `Structural aspects and phase behaviour in deacylated and high acyl gellan systems', Carbohydr. Polym., 38 145±54.
BOURGEOIS, B.
Gellan gum 14.
15. 16. 17.
18. 19.
20. 21. 22. 23.
24.
25.
26. 27. 28.
29.
30.
227
and SWANSON, B.G. (2004) `Gelling temperatures of high acyl gellan as affected by monovalent and divalent cations with dynamic rheological analysis', Carbohydr. Polym., 56 27±33. 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. MATSUKAWA, S. and WATANABE, T. (2007) `Gelation mechanism and network structure of mixed solution of low- and high-acyl gellan studied by dynamic viscoelasticity, CD and NMR measurements', Food Hydrocolloids, 21, 1355±61. MAO, R., TANG, J. and SWANSON, B.G. (2000) `Texture properties of high and low acyl mixed gellan gels', Carbohydr. Polym., 41, 331±8. HUANG, Y., TANG, J., SWANSON, B.G. and RASCO, B.A. (2003) `Effect of calcium concentration on the textural properties of high and low acyl mixed gellan gels', Carbohydr. Polym., 54, 516±22. SWORN, G., SANDERSON, G.R. and GIBSON, W. (1995) `Gellan gum fluid gels' Food Hydrocolloids, 9, 265±71. VALLI, R. and JACKSON, P.H. (2004) `Shelf stable suspensions: gellan gum blend forms fluid gels in beverages', Food and Beverage Asia, April, pp. 54±57. VALLI, R.C. and MORRISON, N.A. (2002) `Methods of making sterilized milk compositions comprising native gellan gum'. Patent No. PCT/WO 02/060268 A2. 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', Carbohydr. Res. 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 and Professional, Glasgow, pp. 227±49. MCCANCE and WIDDOWSON'S The Composition of Foods, 5th Edition, RSC and MAFF, 1991. BAIRD, J.K., TALASHEK, T.A. and CHANG, H. (1992) `Gellan gum: Effect of composition on gel properties', in Gums and Stabilisers for the Food Industry 6, eds. G.O. Phillips, P.A. Williams and D.J. Wedlock. IRL Press, Oxford, pp. 479± 87. MORRISON, N.A., SWORN, G. CLARK, R.C., TALASHEK, T. and CHEN, Y-L. (2002) `New textures with high acyl gellan gum', in Gums and Stabilizers for the Food Industry 11, eds. G.O. Phillips and P.A. Williams. IRL Press, Oxford, pp. 297±305. SWORN, G., CHEN, Y-L., MORRISON, N.A., TALASHEK, T. and CLARK, R. (1999) `Modified gellan gum composition process for preparation of same and use thereof'. Patent No. PCT/WO 99/64468. HUANG, Y., SINGH, P., TANG, J.
10 Galactomannans W. C. Wielinga, retired in 2000 from Meyhall AG, Kreuzlingen, Switzerland, which was subsequently acquired by Danisco A/S
Abstract: The introduction provides information about the worldwide availability of important seed gums. The following section describes the raw materials and structure of seed galactomannans, including description of the composition of the seeds of the carob tree, tara shrubs and the guar plant, and the amount of recoverable endosperm in fenugreek seeds. For the four types of gum discussed, the amount of overall galactose content is indicated, and the fine structure of the different galactomannans is briefly described, as far as information is available. There is also brief discussion of a possible biosynthesis route for galactomannans, derived from the literature. Attention is also drawn to EC and FAO/WHO specifications for the molecular weights of carob bean gum (E410) and guar gum (E412). The manufacture of carob bean gum/tara gum and guar gum is described and a manufacturing abstract for fenugreek gum is derived from two recent patents. Brief reference is also made to the development of novel technologies for the purification of guar endosperm. The various galactomannan gums are characterized in the usual way. The relationship of molecular weights of galactomannans of straight and depolymerized carob bean gum and guar gum and the usual Brookfield viscosities are determined. This kind of viscosity is examined in relation to the concentration of aqueous solutions, using regression equations. Uses and applications of galactomannans are very briefly discussed and attention is drawn to other publications in this respect. Potential future developments are also briefly examined. Key words: water soluble seed gums, carob bean gum, tara gum, guar gum, fenugreek gum, E-numbers, availability, production, galactose-mannose ratios, properties, depolymerized gums, application.
Galactomannans 229
10.1 Introduction Galactomannans are multifunctional macromolecular carbohydrates found in various albuminous or endospermic seeds. The seed galactomannans from the carob tree (Ceratonia siliqua L.) and from the guar plant (Cyamopsis tetragonoloba L.) are widely used, while the kernels from the tara shrub (Cesalpinia spinosa L.) are used to a much lesser extent. The fenugreek plant (Trigonella foenum graecum L.) is another legume that supplies a further kind of galactomannans. Ground endosperms are the source of trade galactomannan gums. The recovered endosperm halves, of the required purity, of carob, tara and guar, and the endosperm parts of fenugreek seeds, are ground to obtain a fine off-white powder. World production of carob pods is estimated at about 300,000±350,000 t/ year, yields depending on cultivar, region and farming practice. The carob pod is called St. John's bread and has two main constituents: · 90 wt% of pulp, containing a high amount of sugars (48±56 wt%) with low molecular weight · 10 wt% of seeds or kernels (undigestible). The carob kernels are composed of 30±35 wt% of hull, 20±25 wt% of germ and 40±45 wt% of endosperm on an absolute dry basis. The annual consumption of carob bean gum in the food industry is 9,000±10,000 t. The approximate composition of tara seeds is: ca. 38 wt% of hull, ca. 40 wt% of germ and ca. 22 wt% of endosperm on an absolute dry basis. About 1,500± 2,000 t of tara gum per year are available to the food industry. The average total quantity of guar seeds worldwide is estimated at about 500,000 tons p.a. However, large fluctuations of annual availability of seeds occur, mainly due to weather conditions. The seeds are composed of 20±22 wt% of hull, 44±46 wt% of germ and 32±36 wt% of endosperm on an absolute dry basis. The annual consumption of guar gum in the food industry is about 55,000 t. For fenugreek, the annual worldwide seed volume is approximately 30,000± 50,000 t, mainly used for spice production. This volume could easily be increased on demand (see `Fenugreek has a role in south-eastern Australian farming systems' by Kate McCormick et al. ([emailprotected]). Fenugreek seeds yield about 25 wt% of gum. For ease of comparison, Table 10.1 illustrates the annual consumption in 1990 of different food additives in grams per year per capita in various countries.
10.2 Raw materials and structure These polysaccharides are strongly hydrophilic, enabling the endosperm to imbibe water to protect the embryo against subsequent drought, during and before germination. They become metabolized after germination.1 During
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Table 10.1 Annual consumption of certain 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
germination the endosperm absorbs up to 75 wt% of water, based on the dry weight of the seed. This allows the diffusion of enzymes required for the germination process and enables the transport of metabolic low molecular weight end-products, needed for the growth of the plant. The carob tree bears pods with an average yield of 50±70 kg, which can be collected from orchards with minimum management, producing 2,000±3,000 kg/ ha. In irrigated orchards an average of 250±300 kg of pods per tree is possible, yielding 12,300 kg of pods/ha. The kernels are released in a process called kibbling. The pods are broken between two rollers with special geometry, and the freed seeds are then separated from the rest of the pods using special screens. Due to its high sugar content, the pod pulp is a staple in the diet of farm animals. It is also used to produce alcohol and is sold in fine powder form as a cacao substitute. People ate carob pods in times of famine and children still like them as snacks. The main interest, however, lies in the kernels. The kernel is oval or oblong in shape, 8±10 mm long, 7±8 mm wide and 3±5 mm thick. The glossy brown testa is hard and smooth. A kernel weighs ca. 0.2 g with small deviations and was used as a weight unit for gold and precious stones (carat is derived from the Latin word ceratonia). The endosperm of Ceratonia siliqua seeds or kernels consists of living cells, which can synthesize the enzymes to hydrolyse the galactomannans (galactosidase, EC.3.2.1.22, endo- -mannanase, EC.3.2.1.78 and -mannosidase, EC.3.2.1.25). The guar plant is bush-like with a height of 57±105 cm, depending on the variety. 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
Galactomannans 231 Indian continent, especially in the north-western part of India and north-eastern part of Pakistan usually provides the right amount of rainfall at the right time of year 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±8 cm long, almost round in shape and ca. 1 cm 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 can no longer be eaten and empty pods have no commercial value. The yield per ha can be as high as 1,800 kg of pods. On the subcontinent harvesting is done manually and the seeds are recovered by mobile threshing machines. Guar seeds as such are not exported, neither from India nor Pakistan. 100 seeds weigh 2.4 to 3.7 g. The seed yield per plant is a maximum of 50 g.2 On its convex periphery, the guar endosperm of Cyamopsis tetragonoloba seeds has four layers of aleurone cells with a total thickness of 0.1 mm, the only living cell layers of the endosperm (Fig. 10.1), which can also synthesize enzymes similar to those mentioned above for the carob endosperm.3 The endosperm of fenugreek (Trigonella foenum graecum) seeds also has an aleurone layer as the outer cell layer of the endosperm, which can synthesize the required enzymes for germination. The endosperm of tara (Cesalpinia spinosa) seeds is morphologically similar to that of the carob kernel, but the cell structures differ and can be distinguished microscopically. The hard, compact endosperm of all four seeds contains more than 88 wt% of galactomannans on a dry basis. The endosperm of the guar seed, for instance, develops alongside the embryo or germ and completely envelops it. The endosperm itself is protected by a seed coat (see Fig. 10.2). The germ of the carob kernel is sandwiched between two endosperm halves, but without being completely enclosed by a thin film of endospermic cells. The hard coat of the carob kernel provides the desired protection, and the same is also true for the tara kernel. The seed endosperm contains very little cellulose and no lignin. The galactomannans derived from these four seed sources are composed entirely of linear (1!4)- -D-mannan chains with varying amounts of single Dgalactose substituents linked to the main backbone by (1!6)--glycosidic bonds (Figs 10.3 and 10.4). These polydisperse galactomannans can easily be distinguished from each other by their overall mannose-galactose ratios between 1.1:1 and ca 3.5:1. The galactomannans of carob bean gum have a galactose content of 17±26 wt%, those of tara gum ca. 25 wt%, guar gum 33±40 wt% and fenugreek 47±48 wt%. The large amount of galactose side stubs of about 20±48 wt% prevents strong cohesion of the main backbones of different neighbouring macromolecules, so that no extensive crystalline regions can be formed. Water at room or elevated temperatures can thus easily penetrate between the single molecules to hydrate or dissolve the accessible gum. Substitution of the mannan chain by more than 12 wt% of galactose makes the galactomannan water soluble.4,5 The fine structure of these galactomannans can be quite irregular with respect to the distribution of the galactose units. As many as five mannose units without
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Fig. 10.1 Photos ((a)±(c)) show magnified details of aleurone cell layers (1000, 2000, 1000). The first magnification photos below ((e)), illustrates one detail of the peripheral aleurone layer (1000); the other two enlargements ((f) and (g)) show inner endosperm cells of a fractured guar split (2000).
Galactomannans 233
Fig. 10.2 A schematic cross section of guar seed. The endosperm consists of aleuronic cell layers attached to the inner endosperm. The number of mechanical purifications determines the yield of the split quality, each purification abrades more parts of the splits away, i.e. of SPS, DPS and of TPS (as imagined by the hypothetic intersection lines within the lower endosperm half, indicated by the widths between the arrows).
galactose side stubs in a row may occur in certain galactomannans of guar gum, and as many as 10±11 in specific galactomannans of carob bean gum. The fine structure of the polysaccharides of tara gum lies most probably between that of carob bean gum and guar gum. Figure 10.5 shows a possible model for a galactomannan of carob bean gum with about 20 wt% of galactose, with a molecular weight of 68,000 Daltons. (A similar model with a molecular weight of 3 million Daltons would require about 11 pages of this book.) The fine structure of the galactomannans of fenugreek gum does not pose a major problem since almost all mannose residues are substituted by galactose. The biosynthesis of galactomannans is very complex. The following brief cascading steps describe to some extent the probable route: · Step 1. Galacto- and manno-kinases i.e. phosphotransferases catalyze the phosphorylations of D-galactose to D-galactose-1-phosphate and of D-
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Fig. 10.3 Dreiding model of galactomannan with 20% by weight of D-galactose free electron pairs, indicated by 2 2 dots at oxygen atom. Hydrogen bond between OH-group at C3 of D-mannose residue and oxygen of pyranose ring of neighbouring D-mannose unit (on the left side).
Fig. 10.4
Theoretical repeating units of four different galactomannans.
Galactomannans 235
Fig. 10.5
Schematic galatomannan molecule with a molecular weight of 68,000 daltons with about 20% by weight of galactose with a random distribution.
mannose to D-mannose-1-phosphate. These mono-phosphated esters are strongly activated intermediates. The esterified C1 atom of the mannose-1phosphate is the site to be linked to the growing non-reducing end of the mannan chain and the C1 of galactose-1-phosphate reacts with a primary OH group of the mannose unit, each liberating thereby the phosphate ester group. · Step 2. Uridine triphosphate reacts with D-galactose-1-phosphate to uridinediphosphate(UDP)-galactose and guanodine triphosphate with D-mannose-1phosphate to form guanodine-diphosphate (GDP)-mannose. Both esters set pyrophosphate free at this reaction. UDP and GDP are considered to be hexose transferring coenzymes to form glycosides. · Step 3. Now two different Golgi membrane-bound transferase enzymes come into action. One has probably two distinct binding sites for the coenzyme guanodine-diphosphate (GDP)-mannose, which is called the mannan synthase, and together with the co-factor, Mg-ions first elongate the nascent mannan chain each time by mannobiose. The second enzyme, a galactosyltransferase with docking sites for the coenzyme uridine-diphosphate (UDP)± galactose cooperates with the Mn-ions to introduce the side stub onto this mannan backbone, releasing the UDP and GDP for the process again. Every mannose residue in the growing -1,4-linked mannan backbone is rotated or inverted 180ë with respect to its neighbouring residue, and therefore the sites for two guanosine-diphosphate-mannose molecules, inverted by 180ë, must be available at the right position in the mannosyl-transferase. And two sites inverted by 180ë for the uridine-diphosphate-galactose units must also be present within the galactosyltransferase to enable the substitution of galactose residues to mannose residues to the growing mannan backbone. The highly specific galactosyltransferase introduces one - D-galactosyl unit only to one mannose residue, at or close to the non-reducing end of the growing chain, and another possibly to the next mannose unit, or to the following freshly condensed mannose units, inverted by 180ë, if required.6 · Step 4. The galactomannans formed are deposited in layers on the endosperm cell walls of the seeds, according to genetically programmed size and
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architecture. It is not yet clear how this deposition should be visualized. Perhaps an electron micrograph of a fractured guar split could offer some insight (Fig. 10.1). No information has been found in the literature about how the molecular weight distribution of the galactomannans is accomplished during this reproducible synthesis. Many other questions also remain to be answered, including: `How many different galactomannan chains are synthesized simultaneously in one seed?' and `Does the time of day and temperature affect the size of the synthesized macromolecules?'. · Step 5. After the complete biosynthesis of the four different beans over several weeks, they are allowed to air dry naturally while still in the fields, before the pods are harvested. Synthetic and natural high molecular weight substances consist of blends of macromolecules with different degrees of polymerization. The distribution of molecular weights of these substances is therefore very important. Figure 10.6 shows these Mw distributions for carob bean gum, guar gum and xanthan gum. It
Fig. 10.6
GPC chromatogram of xanthan, guar and carob bean gum.
Galactomannans 237 is not uncommon for polymeric materials to contain macromolecules ranging in molecular weights from a few hundred to several million Daltons.7,8 In 1948 Kubal and Gralen published the average molecular weight of carob bean gum as 310,000 Daltons, which was determined at 20 ëC with ultracentrifugation.8 However, the investigated non-industrial sample of their test substance of carob bean gum in 1948 was prepared on bench scale in their laboratory and had an intrinsic viscosity [] of 5.0 dl/g. Current industrial carob bean gum products (E410) now on the market have intrinsic viscosities of about 12.0 dl/g, thus significantly higher than that used by Kubal and Gralen. Hui determined a molecular weight of 1,900,000 Daltons for a selected quality of guar gum, according to the same ultracentrifugation method at 25 ëC.9 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. Industrial guar products (E412) now on the market show intrinsic viscosities of 0.7±15.0 dl/g, which correspond to apparent viscosities of aqueous solutions at 2 wt% concentration (based on 10% of moisture) of about 5 to 100,000 mPa.s, if measured with a Brookfield RVT viscometer, at 20 rpm at 25 ëC after full hydration. Since 1998 EC and FAO/WHO specifications for the molecular weight of carob bean gum (E410) used as a food additive have required a range of approx. 50,000±3,000,000 Daltons. For guar gum (E412) these specifications call for molecular weights between 50,000 and 8,000,000 Daltons.
10.3 Manufacture Commercial gum products do not always consist of pure endosperm, but may contain residual hull and germ parts. Trade products are specified mainly according to moisture content, viscosity of aqueous solutions, protein content, acid insoluble residue (an indication of the residual hull content) and particle size distribution. To extract the endosperms from the seeds, the hull must first be removed as carefully as possible, either by mechanical or physical means. If the seed coat is removed chemically, as might be the case for carob and tara kernels, an efficient washing step with water is needed, followed by a drying process. Then the airdry peeled seeds are split. In guar seed technology, the seeds are already cracked, after which most of the friable germ and other fines can be screened off. Additional purification cycles are normally needed for the guar split. Seed varieties, weather conditions during growth and harvesting, geographical influences, different morphological structures of the endosperm, and processing conditions make an exact definition of trade products difficult. 10.3.1 Carob bean gum The evergreen carob tree can be planted in semi-arid or subtropical zones and grows in calcareous soils. These trees are important for the vegetation of the
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entire Mediterranean area, preventing too much erosion of the soil, especially in Morocco and Portugal. The carob tree can grow as high as 10±15 m and its roots can reach a depth of 25 m. It 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.10 The carob tree normally yields fruits after 8±10 years and the fruits, i.e. the pods, can be harvested once a year. The pods are 10±30 cm long, 1.5±3.5 cm wide and 1.0 cm thick. They are dark brown in colour, and straight or curved in shape. The pods contain 8±12 seeds or kernels, but exceptionally up to 15 kernels. The fruits are collected when they have a moisture content of 12± 18 wt%. They are shaken from the trees with long stakes. The fruits are then dried. The dry pods are leathery. The kernels are released in a kibbling process, as described in Section 10.2. The hull is without much value. The germ contains about 50 wt% of protein and is used as cattle feed. It is also 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' before separating the fragile germ. There are two different peeling processes: 1.
2.
Chemical peeling process: The hull is carbonized by concentrated sulphuric acid solutions at high temperature. The advantage of this technology is an even peeling effect, allowing the production of white powders. 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. The disadvantage of this technique is the effluent problem. Thermal mechanical peeling process: The kernels are roasted at temperatures of up to at least 550 ëC, so that the hull pops off to a large extent. The residual hull fragments are rubbed off mechanically. As this also leads to a simultaneous cracking of some endosperm halves and germ parts, a clean separation of endosperm halves and germ/hull parts becomes more 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 these carob bean gums show a somewhat lower quality and lower viscosity, all having a very light-yellowish colour due to a small amount of residual germ particles.
The endosperm halves of the carob kernel recovered by both these techniques are then ground to the desired fineness. Special technology produces coldswelling carob bean gum, attaining at 25 ëC at least 60% of the viscosity of solutions prepared at 86±89 ëC for 10 minutes. Four such cold-swelling products have been developed with viscosities between 20 and 1,800±2,000 mPa.s, dissolved in water at 25 ëC, and with their `hot' viscosities between 25 and 3,000 mPa.s, dissolved at 86±89 ëC for 10 minutes, at 1% concentration. The same techniques used for carob kernels can also be used for tara seeds. The yield of high-grade tara gum is only 21±22 wt%, due to stringent food law
Galactomannans 239 specifications with respect to protein content. The EU specification for the protein content of tara gum (E417) is 3.5 wt% (N% 5.7), thus much lower than the specifications for carob bean gum of 7 wt% and guar gum of 10 wt% (both N% 6.25). Tara gum, therefore, is the most purified of the three sources and one consequence of this is that a lower yield must be accepted. The above-mentioned 22 wt% of endosperm in the composition of the tara kernel can most probably be increased to about 28 wt%, with the hull and germ content adjusted as well. (The analysis of these galactomannan seeds is very difficult when one takes into account the manual separation of the seed components after the swelling of the seeds in water, followed by dehydration in an organic solvent.) The tara tree or shrub is native to the Cordillera region of Bolivia, Peru and northern Chile and also grows in Ecuador, Colombia, Venezuela and Cuba.
10.3.2 Guar gum Guar gum is the name of the ground endosperm halves, called guar splits, which are recovered 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. 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', in which `gau' means cow and `ahar' indicates food. Unlike the seeds of the carob tree, which are called kernels, guar seeds are in fact called seeds. The appearance of guar seeds and the guar pod can be seen in Fig. 10.7. The colour of the seeds varies from 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 the manufacturing stage with regard to specks and the colour of the finished powder. The average total quantity of guar seeds worldwide is estimated at about 500,000 tonnes p.a. However, large fluctuations of annual availability occur, mainly due to weather conditions. In order 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 have been 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 is economic conditions. Guar seeds are composed of 20±22 wt% of hull, 44±46 wt% of germ and 32± 36 wt% of endosperm on an absolute dry basis. Table 10.2 provides an analysis of guar seed components with regard to protein content, ash and moisture content, acid insoluble residue (AIR), matter extractable in ether and calculated
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Fig. 10.7 Guar pods and seeds.
gum content, which is defined as [100 wt% ÿ (wt% protein wt% moisture wt% AIR)]. The AIR is determined after hydrolysing the products for 6±8 h in 0.4N H2SO4 at boiling temperature. The germ contains about 50±55 wt% of protein and is sold as protein-rich cattle fodder, after reduction of the amount of trypsin inhibitor to an acceptable level. Figure 10.8 shows swollen pure guar seed components. 10.3.3 Production of guar splits and guar gum powder The whole seeds can be fed into an attrition mill or any other type of mill with two grinding surfaces travelling at different speeds. The seed is split into the endosperm halves covered with hull, called the crude crack and fine germ material, which can later be sifted off. The crude crack is heated to soften the Table 10.2 Composition and chemical analysis of guar seed components Composition of each portion Seed component
Weight fraction, dry matter
Hull Endosperm Germ
20±22 32±36 44±46
Protein
Ash
Moisture
AIR
%
Ether soluble %
%
%
%
Gum content %
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
Galactomannans 241
Fig. 10.8 Main seed components of guar seed. Germs are seen in upper left corner and swollen endosperm halves and overlapping edges in upper right corner. Hand-picked hull fragments are shown in the lower middle.
hull, after which it is fed into a mill which can either abrade the hull from the endosperm, or into a hammer mill, where the hull is shattered away. Any remaining germ particles are pulverized during this step and after a further sifting the resulting splits are essentially pure endosperm. The fine material, also containing the thin part of the endosperm which overlaps the small edges of the two cotelydon leaves and the radicles, is called guar meal and is marketed as cattle feed. It should show a minimum protein content of 35 wt% (N% 6.25). The guar endosperm or guar splits are processed to commercial powdered products by hydration, flaking if needed, milling-drying and screening techniques. Single-, double- and triple-purified splits are available on the market. The endosperm of the guar seed consists of solid cell material and a split for high-grade guar products weighs 4±6 mg, whereas the endosperm half of the carob kernel shows a weight of 50±60 mg. The latter has a dense fibrous cell structure, significantly different from guar splits. The guar splits are hydrated to different extents and might be flaked and then flash ground. The powdered product becomes sifted and the coarse fraction is recycled to a mill. The finer fractions are blended in order to meet the specifications for particle size distribution, protein, AIR, ash content and viscosities. Guar is derivatized to supply the appropriate technical industries with hydroxyl-propyl-, hydroxyl-ethyl-guar, carboxy-methyl-hydroxyl-propyl guar, carboxy-methyl guar and cationic guar, as well as cationic hydroxyl-propyl guar, hydrophobic guar and guar phosphates (Fig. 10.9). Guar is depolymerized with heat and acid, with alkaline hydrogen peroxide, enzymatically by electron
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Fig. 10.9 Guar derivatives.
beams, by alkali in the presence of air at elevated temperatures and by irradiation with gamma rays. Patented economical processes show that the quality of current guar products can be improved considerably. Such products can be used if high clarity aqueous solutions are required. 10.3.4 Production of fenugreek gum Fenugreek (Trigonella foenum graecum L.) is an annual plant species with a height of up to 60 cm, belonging to the Fabaceae family. It is a multi-purpose legume that can be grown for the spice and pharmaceutical markets, or for green manure. It produces seeds which contain another kind of water-soluble galactomannans. These polysaccharides consist of mannan chains, of which almost all mannose units are substituted with single galactose residues. The weight of an Australian fenugreek seed is 13±17 mg and that of Canadian seeds 18±22 mg, whereas guar seed weight varies from 24 to 37 mg. Fenugreek seeds are 4±6 mm long and about 2±3 mm wide with a deep groove in the middle, giving them a hooked appearance. The ground seeds have a bitter taste and a special smell. Dry toasting of the seeds can enhance the flavour and reduce the bitterness. Small amounts of toasted ground fenugreek seeds should be found in any good curry powder. The fenugreek plant is native to the Mediterranean region. It has been grown in the Near and Middle East, Africa and India, and today is grown all over the world.
Galactomannans 243 Patent number WO2004048419 filed by Air Green Co. Ltd (JP) in 2004 shows that the recovery of fenugreek endosperms can be achieved exclusively by physical procedures. The seed hull and germ are separated from the endosperms mechanically, presumably in a similar way to the production of the guar split, but also bearing in mind that the germ in the fenugreek seed is much more fragile than its hard endosperm. Morphologically, the embryonic radicle occupies a great deal of space within the seed and is covered by the hull and a thin endospermic film, which most probably becomes cracked away from the rest of the seed. The hull remaining on the main endosperm might also be partially torn off at the first grinding step. The different components of the seed can then be classified by screening. The crude endosperms can be further purified to meet the required specifications and the ground endosperm produces the desired grade of fenugreek. Another technique, described in Patent No. CA2206157 (1998-11-26) by Emerald Seed Products LTD (CA), uses extraction of the galactomannans from the endosperm in water, then clarifying the extract and precipitating the dissolved gum with an alcohol, like ethanol. The alcohol-wet precipitate can be dehydrated and purified with more alcohol, then dried and ground to the desired fineness. Approximately 25 wt% of Canadian fenugreek seed is recoverable pure endosperm gum.
10.4 Technical data The functionality of galactomannans is mainly due to their ability to change the rheology of aqueous systems. All four types described above are very efficient thickening agents in aqueous systems and can interact, if present, with agar-agar, Danish agar, carrageenans and xanthan gum to become more efficient with respect to thickening power, or to fortify three-dimensional stabilizing structures. The thickening power of galactomannans depends, of course, upon the size or length of the dissolved substituted mannan chains. Guar gum has the highest thickening power, followed by tara gum. Both carob bean gum and fenugreek gum show a somewhat lower viscosity than tara gum. Guar gum containing galactomannans with galactose contents of 33±40 wt% is soluble in water at 25 ëC, provided the gum is accessible to the solvent water. The majority of galactomannans of carob bean gum with galactose contents of about 17±21 wt% need heat treatment of 10 minutes at 86±89 ëC, while stirring to dissolve in water. If the galactose content of the galactomannans of guar gum is decreased enzymatically to less than 12 wt%, the final products become insoluble even in hot water. All natural galactomannans are non-ionic. The hydroxyl groups can be derivatized and thus can provide non-ionic, anionic, cationic and amphoteric derivatives. The primary and secondary hydroxyl groups show practically more or less the same reactivity. Random distribution of substituents is usually obtained. The galactose side stubs each have one primary and three secondary
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OH-groups, the substituted mannose unit two secondary OH-groups, and the unsubstituted mannose has two secondary and one primary OH-group. The maximum average degree of substitution (DS) is, therefore, three. The introduction of more than one substituent to one hydroxyl group leads to a molar substitution. Galactomannans are susceptible to strong acids, organic acids like citric, acetic and ascorbic acid, for instance, to alkali in the presence of air and strong oxidizing agents, especially at elevated temperatures, and also to electron beams and towards irradiation with -rays, so that depolymerizations of the galactomannans can be achieved to different extents or may occur randomly. Both mannose and galactose contain vicinal secondary cis-hydroxyl groups, on 2, 3 and 3, 4 respectively. These vicinal cis-hydroxyl groups form cyclic complexes with appropriate reagents (such as borate). Semi-dry processes, therefore, allow the production of derivatives and modified products, which can be water-washed if required. The removed impurities and dissolved gums in the effluent can be treated anaerobically to produce methane, which is a welcome additional energy source for the factory. Figure 10.9 illustrates some of the guar derivatives available. New patented 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 non-derivatized guar products are available for food applications. Dissolved in water, they also yield solutions of excellent clarity. More than 90% by weight of the available carob bean gum and tara gum are no longer derivatized. These products are used as straight gums as food additives in the food and pet-food industries. Table 10.3 summarizes some characteristics of two high-grade carob bean gums. The powdered guar products are specified for the viscosity of the fully hydrated solutions, according to a specified method (but not an optimal one) and mesh analysis. The other specifications of wt% of H2O, wt% of protein, wt% of AIR, wt% of ash and pH of 1 wt% aqueous solutions are respectively 12, 5, 3, 1 wt% and 6.5 for guar M100, M175, M200 and M225. Guar CSA 200/50 has slightly less protein and less AIR. The minimum viscosity of these guar products is respectively 3,000 mPa.s for the first three gums, and 3,600 and 5,000 mPa.s for the other two. The viscosity of the aqueous solutions, prepared at 25 ëC, of the two tara gums M175 and M200 at 1 wt% concentration are both 3,000 mPa.s. Prepared at 86±89 ëC for 10 minutes, the coarser product has a higher viscosity of 4,400 mPa.s compared to the finer M200 type of 4,000 mPa.s. If 1 wt% solutions of a regular CBG M200 and a cold swelling CBG M200, prepared at 86±89 ëC for 10 minutes while stirring at low shear rates, are spun out at 35,000 g for 30 minutes, respective quantities of insolubles of 17 wt% and 14 wt% are found. The 1 wt% solutions of these two products, prepared at 25 ëC, contain respectively 39 and 70 wt% soluble galactomannans. Additionally, heat treatment at 86±89 ëC solubilizes 44 wt% of galactomannnans for the regular CBG and, as might be expected, much less, i.e. only 16 wt% of galactomannans,
Galactomannans 245 Table 10.3
Typical analysis of two carob bean gum
Specification
High grade CBG M175
High grade CBG M100
% H2O % protein (N% 6.25) % AIR 1% viscosity, 10 min at 86±89 ëC RVT Brookfield, 20 rpm, 25 ëC mPa.s ± M80 (max) ± M200 (max) Trace elements As, ppm Pb, ppm Cu, ppm Zn, ppm Cd, ppm
10.0±12.0 6.5 2.0 min. 3,000
10.0±12.0 6.5 2.0 min. 3,000
99% 25%
99% 10%
0.2 ca. 0.03 2.5 5.6 ca. 0.05
0.2 ca. 0.03 2.5 5.6 ca. 0.05
for the cold swelling product. This means that the latter product can stabilize systems at much lower temperatures than regular CBG, which is very important during a heat process. Certain galactomannans of carob bean gum in aqueous solutions selfassociate under defined conditions, such as at a freezing process when nanocrystalline regions of 3±5 nm can be formed, which alternate with much bigger amorphous regions. Nevertheless, it was found that a test system of this kind yields a weak gel upon thawing, and therefore carob bean gum is used as an excellent ice cream stabilizer. The company Emerald Seed Products (101 Wood Mountain Trail East, Box 149 Avonlea, SK, Canada, S0H 0C0) produces Canafen Gum, a fenugreek gum from seeds grown in Canada. Table 10.4 shows the chemical analysis of fenugreek seeds. The applications of this fenugreek gum are considered to be the same as for the other gums described in this chapter. It might be a better candidate as a healthy food ingredient. The galactomannans of fenugreek endosperms are much more resistant towards enzymatic breakdown in the digestive tract, providing the most effective fibre in retaining viscosity. The mannan chains are protected by the side stubs of the galactose units, since the mannose residues in this gum are almost completely substituted. Acid hydrolysis apparently does not reduce the viscosity of the aqueous solutions of fenugreek gum to the same extent as in the other three types: carob bean gum, tara gum and guar gum. Fenugreek gum, therefore, is an ideal ingredient for functional foods with reduced glycemic indices. The Japanese company Air Green (http://www.airgreen.co.jp/fenugreek/ index_e.html) has three grades of fenugreek on the market with different galactomannans content, i.e. one over 86 wt%, the second with 80±86 wt% and the lowest grade containing 60±80 wt%. The specifications of these products are summarized in Table 10.5.
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Table 10.4 Chemical analysis of fenugreek seeds (source: November 2006, University of Saskatchewan) Component
% by weight
Dietary fibre Insoluble dietary fibre Soluble dietary fibre Protein Oil Ash Starch Sugar
45.4 32.1 13.3 36.0 6.0 3.2 1.6 0.4
Table 10.5 Specifications of three fenugreek products, varying in gum content Description
Over 86% gum content
80±86% gum content
Colour white or slight yellow light yellow Smell slight original smell slight original smell AIR % 3 3 Water insoluble matter % 3 3 Heavy metal as Pb ppm 20 20 ppm As 2 2 10 10 H2O % Viscosity, 1%, 25 ëC 3,400 mPa.s 3,000 mPa.s Granularity M70 M70 Protein % 5 5 Starch negative negative Fat % 1 1 Fibre % 86 80 Caloric value kcal/g 1 1 Ash % 1.5 1.5 Galactomannans % 86 80 counts per g Bacteria 300 300 counts per g Fungi 300 300 counts per g E. Coli negative negative
60±80% gum content light brown slight curry smell 5 5 20 2 10 1,000 mPa.s M100 5 negative 1 70 1 1.5 60 300 300 negative
The fact that guar is freeze-thaw stable had already been shown in 1984, whereas carob bean gum solutions generally form gels after a freezing process. A blend of these two galactomannan gums, 1:1, tends to form gels as well rather than being freeze-thaw stable.11 Details of depolymerized guar products can be found in a publication from 1990.12 The molecular weights, M, of depolymerized guar products in kDa, based on limiting viscosity data and characterized by their Brookfield RVT viscosity of 1 wt% solutions, Br 1 after full hydration, measured at 20 rpm, at 25 ëC in mPa.s, show a rather good fit with a squared correlation coefficient of 0.9874 with the following power law equation:
Galactomannans 247 M 99:072 Br 1 0:3674
10:1
The guar products investigated showed Brookfield viscosities, as described above, of ca. 17±5,000 mPa.s. A pocket calculator can be used to calculate the molecular weights in a simple way. This also holds true for the other relationships determined, described below, for the different regression parameters. Cold swelling CBG with defined Brookfield viscosities of 25±3,000 mPa.s at 1 wt% concentration were characterized by SEC (size exclusion chromatography) to determine the peak, Mw and Mn molecular weights, called M in equation 10.2.13 These molecular weights in Da (Y-axis) were plotted against the usual Brookfield viscosities Br 1 (X-axis) and the curves followed an excellent fit with polynomial equations, at least in the above-mentioned viscosity range. The general polynomial regression equation is: M a Br 1 2 b Br 1 c
R2 1
10:2
Table 10.6 summarizes the different equation coefficients and the squared correlation coefficients for the above-mentioned polynomial equation. The Brookfield RVT viscosity of high-grade carob bean gum M100 solutions in mPa.s, prepared at 86±89 ëC for 10 minutes, while stirring with concentrations, c wt%, based on 10 wt% moisture, of about 0.5 wt% to about 2.5 wt%, fits the following power law equation: Br 2992:1 c3:7507
R2 0:9979
10:3
The same potential relationship was also tested for cold swelling carob bean gum in the concentration range of 0.4±2.0 wt%, prepared at 86±89 ëC and at 25 ëC, leading to the following equation values, i.e., the theoretical viscosity at 1 wt% in mPa.s, the exponent n and the squared correlation coefficient summarized in Table 10.7. Similar data for the different depolymerized and straight guar products are also found in Table 10.8, where the word `cold' between brackets means dissolved at 25 ëC and `hot' means 86±89 ëC for 10 minutes. The curve for P150 (cold), range of concentration 0.3±3.0 wt%, was evaluated by a polynomial regression, giving a better fit, and the following regression equation was obtained: Br 1401 c3 6050 c2 ÿ 2896 c 313
R2 1
10:4
Table 10.6 Different coefficients of the polynomial equations and the squared correlation coefficients for four solutions of cold swelling cbg of different viscosities at 1% by weight, dissolved at 86±89 ëC for 10 minutes Type of Mw Peak Mw Mn
Coefficient a
Coefficient b
Coefficient c
Coefficient of correlation R2
ÿ0.0262 ÿ0.0558 ÿ0.110
353.89 432.41 186.71
146,120 206,146 97,701
0.9996 0.9999 0.9997
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Handbook of hydrocolloids
Table 10.7 Power law data for solutions of cold swelling carob bean gum at 1% by weight in water Temperature of dissolution ëC 25 86±89
Table 10.8 water Product type
P150 (cold) P120 (cold) P90 (cold) P60 (cold) P30 (cold) P150 (hot) P120 (hot) P90 (hot) P60 (hot) P30 (hot) P7 (hot) P50 (hot) P100 (hot) P200 (hot)
Calculated viscosity of 1% solution in mPa.s
Exponent (n)
Correlation coefficient R2
1571 2790
3.9202 3.8125
0.9999 0.9996
Power law data for different guar products, dissolved in demineralized Calculated viscosity of 1% solution, mPa.s 2237 735 74 16 5642 4141 998 137 20 0.32 648 1215 2115
Exponent n
Squared correlation coefficient R2
Concentration range in % by weight
3.4577 3.5225 3.8009 3.5414 2.6188 2.7484 3.4231 3.2220 3.3844 3.4268 3.6788 3.6865 3.4673
0.9953 0.9981 0.9992 0.9875 0.9995 0.9998 0.9996 0.9896 0.9916 0.9901 0.9980 0.9976 0.9973
0.3±3.0 0.3±4.0 0.6±5.0 0.9±5.0 0.5±3.0 0.5±3.0 0.4±4.0 0.5±5.0 0.5±5.0 3.0±15.0 0.4±2.0 0.4±2.0 0.4±2.0
Fig. 10.10 Acid stability of different guar solutions at certain pHs prepared at 86±89 ëC for 10 minutes, while stirring, expressed as rest viscosity in %, based on viscosity at pH 4 times 100%.
Galactomannans 249 The acid stability of depolymerized and straight guar products, specified by the Brookfield viscosity at 25 ëC, 20 rpm at 1 wt% solutions, was tested at different pH values. The products were dissolved at the required pH at 86±89 ëC for 10 minutes and the rest viscosity was expressed by the quotient of the viscosity measured at the defined pH and the viscosity at pH 4 times 100% (see Fig. 10.10). The guar products which were purposely depolymerized at acidic pH and with heat are more stable towards acidic hydrolysis in the final applications than modified guar products obtained after an alkaline oxidation. In a liquid food system with a pH below 3.5 one must expect a certain depolymerization of the guar galactomannans, while heating at elevated temperatures. An increase of temperature of a liquid system from 20 to 80 ëC, stabilized with galactomannans, decreases the viscosity at 80 ëC by about 50%, but upon cooling to original temperature, the viscosity returns.
10.5 Uses and applications These thickening and gelling agents are widely used as additives in food products, mainly to make them appealing and attractive to the consumer. Furthermore, they should also improve shelf-life by binding water, control the texture, influence crystallization, prevent creaming or settling, improve the freeze-thaw behaviour, prevent syneresis and the retrogradation of starch products, maintain turbidity in soft drinks and juices and act as dietary fibres. This means that these food additives find applications mainly 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 found in the textile industry (carpet dyeing and textile printing), and in the paper, mining, explosive, drilling, construction, oil field and chemical industries. The functional properties of regular carob bean gum are listed in Table 10.9. Similar tables can also be created for guar gum, tara gum and fenugreek gum. Further examples of applications are to be found in references 11 and 12. The work of P.H. Richardson et al. is particularly informative about the behaviour of carob bean gum and guar gum in aqueous sucrose solutions.13
10.6 Regulatory status Apart from fenugreek gum, the other three gums are approved food additives with the following E-numbers: E410 for carob bean gum, E412 for guar gum and E417 for tara gum. Fenugreek gum is on the GRAS list in the USA. It is not quite clear whether fenugreek gum should be considered as a food ingredient.
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Table 10.9 Functional properties of carob bean gum Function
Example
Adhesion Binding agent Body agent Crystallization inhibitor Clouding agent Dietary fibre Foam stabilizer Gelling agent Moulding Protective colloid Suspending agent Swelling agent Synergistic agent Thickening agent
Glazes, 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 products Jams, pie fillings, sauces, baby food
10.7
Use level (%) 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 10 min) and then heating the dispersion in a tubular heat exchanger to 70 ëC followed by spray drying.33 In an extrusion process, casein at 50% solids from a dewatering device can be fed into an extruder, and 25% NaOH added to form a viscous sodium caseinate. Caseinate may also be prepared by feeding dry acid casein into the extruder and reacting with 20±40% NaOH at temperatures up to 120 ëC. The caseinates are subsequently roller dried and ground to a powder of the required particle size.34 Compositional standards for caseins and caseinates are given in Table 13.5.
Table 13.5
Compositions of caseins and casein-derived milk protein products EU regulation 2921/90
EU Acid casein
EU Rennet casein
EU Caseinates
USDA
Codex Alimentarius Stan A-18-1995
Edible dry casein (acid)
Rennet Acid Caseinates casein casein
Annex I
Annex II
Annex I
Annex II
Annex I
Annex II
Annex III
Extra Standard grade grade
Protein (%, min)
±
±
±
±
88.00
88.00
85.00
95 dry basis
90 dry basis
84 dry basis
90 dry basis
Moisture (%, max)
12.00
10.00
12.00
8.00
6.00
6.00
6.00
10
12
12
Fat (%, max)
1.75
1.50
1.00
1.00
±
±
1.50
1.5
2.0
Ash (%, min)
±
±
7.50
7.50
±
±
±
±
Ash (%, max)
±
±
±
±
±
±
6.50
Max fat and ash
±
±
±
±
6.00
6.00
Lactose (%, max)
±
±
±
±
±
Max free acid
0.30
0.20
±
±
±
FIL-IDF 72:1974
FIL-IDF 45:1969
Caseinate
Acid casein
Extra grade
First grade
Extra Standard grade grade
88 dry basis
90 dry basis
88 dry basis
95 dry basis
90 dry basis
12
8
6.0
8.0
12
12
2.0
2.0
2.0
1.5 dry basis
1.5 dry basis
1.7 dry basis
2.25 dry basis
±
7.5
-
±
±
±
±
±
2.2
2.2
±
2.5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.00
1.0
1.0
1.0
1.0
1.0
0.5
1.0
0.20
1.0
±
±
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.
Milk proteins 307 13.3.2 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 applications such as 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. Use of individual casein fractions is also a prerequisite for the generation and recovery of casein derived bioactive peptides of high purity. Essentially all methods employed for fractionating caseins are based on the association characteristics of the individual caseins. Disruption of the caseins micellar/associated structure in part or fully is necessary for fractionation of the caseins. In a micellar casein system such as milk and in a dispersion of calcium or sodium caseinate, the individual casein molecules associate via a range of forces, including hydrophobic interactions. The strength of hydrophobic interactions is temperature dependent and at low temperatures (< ~5 ëC) when hydrophobic interactions are weaker, the -casein molecules dissociate from s-/-casein complexes and exist in solution as monomers.35 -Casein can be fractionated by renneting calcium or sodium caseinate at 4 ëC; under which conditions -casein remains soluble, while s- and para--caseins precipitates and can be separated by centrifugation. Precipitation of the -casein can be achieved by warming the supernatant to 30 ëC.36 -Casein may also be fractionated from a slurried casein feedstock by cooling to ÿ10 to 14 ëC at pH 3.5±8.0 for a sufficient time to separate the slurry into a solid phase ( -casein depleted) and a liquid -casein enriched phase.37 Terre et al.38 isolated -casein by treating milk or calcium caseinate with a calcium complexing agent which dissociates all of the casein, prior to microfiltration at 0±7 ëC. The permeate was -casein enriched and the retentate was -casein depleted. This procedure has been modified to obtain, on microfiltration at 4 ëC, a -casein enriched permeate from whole casein adjusted to pH 4.2±4.6, or from sodium caseinate treated with calcium chloride to enhance aggregation of the other caseins.39,40 Huppertz et al.41 isolated -casein from renneted skimmed milk; the rennet casein curd was recovered by centrifugation, resuspended in water and the dispersion incubated at 5 ëC to facilitate -casein dissociation; the dissociated -casein was recovered by low temperature centrifugation. Murphy and Fox42 reported a method for fractionation of -casein from a dilute sodium caseinate solution by ultrafiltration at 4 ëC. The -casein was recovered from the enriched permeate by heating it to >20 ëC to facilitate polymerisation of the -casein, following by further ultrafiltration to recover the polymerised -casein in the retentate, or by precipitation of the -casein at pH 4.9 at 20 ëC followed by centrifugation. Law and Leaver43 described a method of selective precipitation of all four caseins from skimmed milk or caseinate. The skimmed milk or caseinate was adjusting to pH 11 at 30 ëC with NaOH; calcium chloride was added to a final concentration of ~0.06 M, precipitating s-/ -casein after which the pH was readjusted to 7 with HCl and the s-/ -casein was recovered by low speed
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centrifugation or filtration. The -casein enriched supernatant was acidified to pH 3.8 to precipitate -casein which was recovered by centrifugation/filtration. The precipitated s-/ -casein was then redispersed and separated by adjusting the dispersion to pH 4.5 and 2 ëC, which yielded s-casein as a precipitate and a supernatant which on warming to 35 ëC yielded a precipitate of -casein. Chromatographic methods can be used to fractionate caseins and have been shown to be superior to precipitation techniques; 44±47 however, these chromatographic methods are not easily scaled up and require high concentrations of nonfood grade dissociating/reducing agent making them unsuitable for producing casein for human consumption. Turhan et al.48 prepared casein fractions by microfiltration of skim milk followed by anion exchange chromatography using L-cysteine as a food grade reducing agent in the eluting buffer, replacing nonfood grade reducing agents such as dithiothreitol or -mercaptoethanol.
13.3.3 Whey protein-enriched products Whey is the serum or liquid remaining after the removal of fat and casein from milk during the manufacture of cheese or acid and rennet casein. Methods for the recovery of proteins from whey have been reviewed by Marshall,49 Matthews,50 IDF,51 Morr,52 Mulvihill,16 Mulvihill and Grufferty,53 Timmer and van der Horst54 and Mulvihill and Ennis.20 These methods are summarised in Fig. 13.3 and are outlined below. 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.55 Acid whey has higher mineral/ash content (increased amounts of calcium and phosphate) 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 13.6. 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 nonhygroscopic, non-caking wettable whey powders. Following concentration to 50±60% solids, concentrates are cooled to 30 ëC, seeded with finely ground lactose monohydrate or well-crystallised whey powder, held for several hours and then cooled to 10 ëC to crystallise lactose as -lactose monohydrate which is less hygroscopic than non-crystalline lactose glass. The concentrate may then be dried by spray-drying in a single stage process or, more commonly, by multistage processes for improved functionality of the powders. In a two-stage
Milk proteins 309
Fig. 13.3
Industrial isolation of protein products from whey.
drying process the pre-crystallised concentrate is spray dried at low temperature and post-crystallisation and final drying takes place in an external vibrating fluidised bed; in a three-stage process the second drying stage is on an integral fluid bed within the spray dry chamber and final drying is in the third stage located outside the chamber. Another three-stage process involves atomisation with primary drying in a low profile chamber, post-crystallisation and two drying stages occur on a conveyor belt system at the base of the chamber. These two- and three-stage processes produce large agglomerated particles of low bulk density that are non-hygroscopic and readily rehydratable. 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
Table 13.6
Compositions of typical whey-derived milk protein products
Protein (min) Moisture (max) Fat (max) Ash (max) Lactose (min)
Codex Alimentarius Stan A-18-1995
Codex Alimentarius Stan-A-18-1995
DMV International
DMV International
Davisco Foods International
Davisco Foods International
Glanbia
Glanbia
Whey powder
Acid whey
WPC
WPC
WPI
WPH
WPC
WPI
Esprion 300
Textrion PROGEL 800
PiPro
BioZate 3
WPC 30
11 5.0 2 9.5 61.0
10 4.5 2 15.0 61.0
30 5 3.5 9.5 By difference
80 5 8 6 By difference
95.0 5.0 1.0 3.0 1.0
94.0 5.5 1.0 5.0 1.0
30.5 4 4 6.5 53.0
90 5 0.5
Carbery
Carbery
Carbery
Dairygold Food ingredients
Dairygold Food ingredients
Kerry ingredients
Kerry ingredients
Kerry ingredients
WPC
WPI
WPH
WPC
WPC
Whey powder
Carbelac 80
Isolac
Optipep 80
Protein (min) Moisture (max) Fat (max) Ash (max)
80 6 9 4
90 6 1 4
80 6 9 8
Lactose (min)
5
15% protein) gel on heating to 50±60 ëC, 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± 6.0.141 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,
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ionic species present, other non-protein components added (lipids, salts and sugars) and the presence of reducing agents also influence the gelation characteristics. Depending on these solution conditions during thermal whey protein gelation, different types of gels may be formed (e.g., fine stranded gels, particulate gels) which vary in modulus/hardness, elasticity and turbidity. WPCs and WPIs possessing different gelling properties can be obtained by careful selection of whey type and manipulation of processing conditions during manufacture. Thermal denaturation of whey proteins, in appropriate solution conditions, and subsequent appropriate treatment of the cooled solution can lead to a gelling process called `cold gelation'.142 When whey protein isolate solutions of neutral pH and low ionic strength are heated at an appropriate temperature and then cooled, denaturation and possibly minimal aggregation occurs to produce whey protein polymers but gelation does not occur due to strong electrostatic repulsive forces between the polymers formed. Acidification of this cold solution using an acid precursor like glucono-delta-lactone, that slowly reacts with water forming gluconic acid, or infusion of salts into the cold solution, alters the solvent properties reducing electrostatic repulsive forces and thus results in `cold gelation'.143 Rapid acidification results in weak brittle gels while slower acidification allows increased exposure of the free thiol groups at the surface of the polymers, increasing disulphide bond formation between aggregates and hence strengthening the gels.144±146 Enzymatic gelation of milk proteins, other than rennet (chymosin) gelation, may be induced by either protein crosslinking by transglutaminase or by specific hydrolysis using proteolytic enzymes. Transglutaminase induced gelation occurs because the enzyme catalyses the transfer reaction between the amide group of glutamine and an -amino group of lysine, crosslinking the protein molecules via the formation of covalent intra- and intermolecular isodipeptide bonds.147 Proteolytic enzyme induced gelation occurs because specific enzymatic hydrolysis of the protein is followed by ordered aggregation of the hydrolysed products to form a gel network.148 In enzymatically induced gelation of whey protein, rate of gelation and gel strength are dependent on protein concentration, pH, ionic strength and level of protein denaturation.149
13.4.4 Hydration properties The ability to hydrate and thus bind or entrap water is an important functional property of dairy proteins in food applications. The level of hydration is highly dependent on the protein composition, conformation, concentration, pH, salts, the number of exposed polar groups, surface polarity and topography as well as processing conditions, including drying procedure and storage conditions.125 The level of hydration depends on the particular products; reported hydration values for casein micelles range from 1.4 to 6.4 g H2O/g, for caseins/caseinates range from 0.7 to 3.8 g H2O/g and for individual native whey proteins range from 0.32 to 0.60 g H2O/g. Generally sodium caseinate is capable of binding more water than calcium caseinate or micellar casein particularly at high pH.
Milk proteins 321 Reported values for the water sorption capacity of several milk protein products in a model flour dough system range from 0.96 to 3.45 g H2O/g of product. Depending on the environmental conditions, thermally denatured whey proteins can have a hydration of over 10 g H2O/g; this is a result of an increase in exposed protein surface area on denaturation and thus an increased availability of hydrogen bonding sites.127 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.
13.4.5 Viscosity Caseinates form highly viscous solutions at concentrations >~15%; this is due to hydration and swelling which increase the hydrodynamic volume, and polymerpolymer interactions, both of which increase resistance to flow and thus viscosity. Below 12% w/w, caseinates are reported to exhibit Newtonian flow while above 12% w/w, flow becomes more pseudoplastic reflecting increasing protein-protein interactions.150 The viscosity of solutions containing >~20% protein is so high that processing is difficult even at high temperatures.25 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.42,141,150 Sodium caseinate viscosity is also very pH-dependent, with minimum viscosity occurring at ~ pH 7.0. By increasing the pH towards 9, an increase in viscosity is observed due to an increase in strong localised inter-molecular interactions. Above pH 9 the casein molecules tend to behave as separate entities and thus viscosity decreases rapidly. The viscosity of casein is also 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 40 ëC. Caseinates exhibit pseudoplastic rheological behaviour and are thixotropic at high shear rates. Calcium caseinate exhibits a lower viscosity than sodium caseinate at the same concentration and ionic strength indicating that the cation present has a significant effect on the viscosity of caseinate (Na > K > NH4).151 The viscosity of caseinate containing 1% calcium shows unusual temperature dependence; it decreases sharply in a curvilinear fashion with increasing temperature from 30±38 ëC, then remains constant up to ~57 ëC, above which the solution gels at pH 5.4 but not at higher pH values. The relationship between viscosity and temperature depends on protein concentration, pH and [Ca2+].141 Low levels of added calcium increase the viscosity of sodium caseinate above pH 7.0 but below this pH and at sufficient calcium addition level 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 leads to increased caseinate viscosity. Precipitation at pH values lower than normal (~ pH 3.8) and especially at higher pH values (~ pH 5.05) also increases the viscosity of caseinates. Roller dried caseinates
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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. A logimetric decrease in viscosity of medium calcium co-precipitates containing 1% calcium was reported on increasing temperature; however, the co-precipitate viscosities were far less sensitive to temperature than caseinate viscosities. Increasing the calcium concentration in co-precipitates increases the tendency for gelation at pH 8 and above at ~30 ëC.152 Solutions of non-denatured whey proteins are much less viscous than caseinate solutions. They exhibit minimum viscosity around their isoelectric point (pH ~ 4.5); while an increase in pH (6±10) slightly increases viscosity.150 The viscosity of whey protein solutions is temperature dependent and relative to water, their viscosity decreases between 30±65 ëC, but increases at higher temperatures as a result of thermal denaturation of the globular proteins. WPC solutions containing 4±12% protein are reported to exhibit Newtonian flow while at higher concentrations flow becomes pseudoplastic and at 18±20% yield values are observed. At higher concentrations (>35% w/w), WPC solutions exhibit time-dependent shear thinning behaviour (thixotropic). In general, the addition of salts has little effect on the viscosity of whey protein solutions with the exception of CaCl2 which significantly increases the viscosity.153
13.4.6 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 via complex intermolecular interactions and thus impart structural rigidity 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.154 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.155 At low protein concentrations sodium caseinate spreads as a thin film with a slightly lower surface coverage than whey protein due to the greater flexibility of the caseins compared to the globular whey proteins, which because of their globular structure tend to absorb as a thicker film on the surface. Aggregated milk protein products such as MPC and calcium caseinate have a limited ability to spread at a surface as the aggregate structures are maintained by calcium bonds and/or colloidal calcium phosphate156 and this leads to the formation of a multilayer structure which has high surface dilatational modulus/viscosity. Partially heat denatured whey proteins are capable of binding ionic calcium leading to aggregation; these aggregates are much more readily absorbed at a
Milk proteins 323 surface than native whey proteins and therefore have enhanced surface activity.157 Limited hydrolysis of milk proteins generates more flexible amphiphilic peptides with a sufficient molecular weight to form stable films at the interface.111 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.
13.4.7 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 hom*ogeniser, exhibited lower creaming stabilities than similar emulsions stabilised by either WPC or soy isolate.158 Highly dispersed sodium, ammonium and low-calcium caseinates showed higher emulsifying capacities than more aggregated highcalcium 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.159 Heat treatment (>120 ëC) of sodium caseinate solutions has been shown to reduce emulsifying properties while improving foaming properties of the caseinate.160 Caseinates generally produce foams with higher overruns but lower stability than foams produced using egg white or WPC and WPI. Whey protein-enriched products are widely used in foaming applications in foods. The foaming properties of WPC increase with solids content, with an optimum of ~10% total solids. Factors such as protein concentration, level of denaturation, ionic environment, protease peptone level, pre-heat treatment and especially the presence of lipids in the WPC or WPI influence whipping properties. The foamforming capacity is enhanced by partial hydrolysis of the proteins; however, hydrolysis tends to reduce foam stability.10 Milk protein ingredients have been specifically modified to improve their foaming and emulsifying properties. Crosslinking of milk proteins with the enzyme transglutaminase produces a polymerised protein; the degree of polymerisation is directly related to an increase in the size of droplet formed on using the modified protein as an emulsifier; however, polymerisation improves emulsion storage stability by increasing the surface shear modulus/ viscosity, thus forming a solid-like protein gel around the emulsion droplets.128
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Attachment of polysaccharide to casein/whey protein via a conjugation reaction improves the emulsifying properties of the protein; the conjugated protein molecules form a more bulky polymeric layer on the droplet surface, with the polysaccharide portion protruding outwards into the emulsion's continuous phase providing better steric stabilization.161 Limited enzymatic hydrolysis of milk proteins generates peptides that are more amphiphilic, have less secondary structure and are more flexible than the native intact protein; thus a limited degree of hydrolysis confers good foaming and emulsifying properties on protein hydrolysate ingredients. Casein hydrolysates (degree of hydrolysis, DH, 1±10%) and whey protein hydrolysates (DH, 10±20%) diffuse rapidly, and absorb at the interface reducing the droplet size on emulsion formation, but these hydrolysates are less efficient at stabilising emulsions because of their limited ability to form stable surface films.111 Emulsions prepared with hydrolysates are less stable to reduction in pH or alcohol addition but more stable to calcium ions.162 Extensive hydrolysis of milk proteins generates low molecular weight peptides and free amino acids, resulting in milk protein ingredients which decreased ability to form and stabilise emulsions in comparison to the intact native proteins.162±164
13.5
Biological activity of milk protein products
Milk proteins are a source of nitrogen and amino acids which are essential for the growth of the neonate and for the maintenance of various bodily functions; in addition, intact milk proteins have some important biological activities. Milk proteins have an ability to bind small hydrophobic vitamins such as retinol (vitamin A),165 folate166,167 and cyancobalamin (vitamin B12).168 This binding may improve the absorption of these vitamins in the intestinal tract169 as it may protect the vitamin against the possibly detrimental environmental conditions during passage through the digestive system thus improving the bioavailability of these vitamins. Milk proteins are a rich source of biologically active peptides which are encrypted in their primary sequence; these biologically active peptides are latent until released on hydrolysis of the intact proteins. These peptides show a wide variety of biological activities including opiate, antithrombotic, antihypertensive, immuno-modulating, antibacterial and mineral carrying properties.170
13.5.1 Biological activity of intact caseins In addition to providing nitrogen and essential amino acids for growth, the principle biological function proposed for casein is to transport high levels of calcium to the neonate in a manner that prevents pathological calcification during its transport through the mammary gland.171 s1-Casein enhances mitogen-stimulated proliferation of murine splenic T-lymophocytes in vitro.172 -Casein enhances the mitogen induced proliferation of ovine T and B
Milk proteins 325 lymphocytes in a dose-dependent manner.173 The glycomacropeptide of casein exhibits an immunosuppressive effect, with those macropeptides containing high levels of N-acetylneuraminic acid showing strong activity against T-lymphocytes.174
13.5.2 Biological activity of intact whey proteins Whole whey protein and intact individual whey proteins have been shown in vitro to modulate lymphocyte functions, suppressing T-lymphocyte mitogenesis in cell cultures.175,176 A whey protein diet has been shown to have a protective role against the development of tumors in the GI tract as well as selectively inhibiting cancer cell growth.177,178 The principle biological function of -la is to act as a coenzyme in the synthesis of lactose,9 while its immunomodulatory and anticarcinogenic properties have also been reported on.178,179 -Lg has been shown to act as a carrier for hydrophobic and amphiphilic molecules including retinol; it is proposed that it protects these molecules from oxidation during transportation through the stomach to the small intestine; -lg may also stimulate lipolysis by binding free fatty acids; it also shows immunomodulatory activities.180,181 -Lg chemically modified using 3-hydroxyphthalic anhydride (3-HP) has antiviral activity; it has been shown to be effective in inhibiting HIV-1 infections in humans.182 BSA functions as a carrier protein for the transport of nonpolar molecules in biological fluids.183 Bovine immunoglobulins (Igs) provide various types of immunity including passive immunity via colostrum to the neonate. Lacteal secretions containing Igs provide an immunological protection against microbial pathogens and toxins, thus protecting the mammary gland from infection.184 Immunoglobulins include the so-called lactenins, L1 and L3, which inhibit gram-positive bacteria; L1 is particularly efficient against some strains of Streptococcus pyogenes and L3 acts against some strains of Lactococcus lactis.9 Lactoferrin (LF) is an iron chelating glycoprotein which acts as a natural nonspecific defence system for the body; it modulates the immune system by promoting inflammatory, intestinal and peripheral specific antibody response and also by controlling lymphokines production.184,185 The ability of LF to sequester iron from a relatively iron-free environment is the principle factor governing the antimicrobial, antifungal, antiviral, antioxidative and anticarcinogenic activity of LF. Its ability to create an iron deprived environment and to bind to the membranes of microbes, thus disrupting the structure, function and integrity of the cell membrane allows it to modulate the intestinal microflora.186±188 Lactoperoxidase (LP) has biological activity; it provides protection against invading micro-organisms and thus it is regarded as an indigenous antimicrobial agent.184 It exhibits a broad range of activity against various bacteria, viruses, yeasts and moulds via the LP system. In the presence of H2O2, LP oxidises the thiocyanate anion (SCN±) and certain halides producing numerous intermediary oxidation products, including the hypothiocyanate anion (OSCN±) and hypothiocyanate (HOSCN) which cause structural damage to the cytoplasmic membrane of the micro-organism.189,190
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13.5.3 Biologically active peptides Milk proteins are proposed to be the main source of biologically active peptides; the biological activity of these peptides has been reviewed by Meisel,191 Fitzgerald and Meisel,117 Mater et al.,192 Kilara and Panyam,115 PihlantoLeppaÈlaÈ and Korhonen,184 Silva and Xavier Malcata,193 Gauthier et al.,194 Korhonen and Pihlanto,116 Nnanna112 and Gobbetti et al.124 Opioid peptides Milk protein derived opioid peptides are referred to as exorphins in order to distinguish them from naturally occurring enkaphalins, endophins and dynorphins.195,196 Opioid peptides (typically 5±10 amino acids) from casein are termed casomorphins or casoxins, while those from whey proteins are called lactorphins or lactoferroxins. The biological effect of the opioid peptides is dependent on the receptor type to which they bind; -receptors are said to control intestinal motility and emotional behaviour, -receptors control emotional behaviour and -receptors are linked to analgesia and satiety.117 A common structural trait in opioid peptides is the presence of a Tyr residue at the N-terminal of the amino acid sequence (except for s1-casein exorphin, casoxin 6 and lactoferroxins B and C), coupled with the presence of another aromatic residue, Phe or Tyr, in the third or fourth position which ensure the binding of the peptide to the opioid receptor.193 The opioid activity is also dependent on the localised negative potential in the vicinity of the phenolic hydroxyl group of tyrosine, as the removal of tyrosine results in the absence of activity.197 The most widely studied of the milk protein opioid peptides are the casomorphins which include fragments 60-63, 60-64, 60-65, 60-66 and 60-70 of the -casein amino acid sequence and act as -type ligands.198±201 s1-Casein derived opioid peptides, known as exorphins, correspond to fragments 90-96 (Arg-Tyr-Leu-Gly-Tyr-Leu-Glu), 90-95 and 91-96 and are -type receptor ligands.202,203 Whey protein derived peptides from -lactoglobulin (fragment 102-105 known as -lactorphin (Tyr-Leu-Leu-Phe) and from -lactalbumin (fragment 50-53, -lactorphin (Tyr-Gly-Leu-Phe)) are -type ligands but show a weak but consistent affinity for opioid receptors.198 Perpetuo et al.204 isolated a peptide from -casein, fragment 114-121 (Tyr-Pro-Val-Glu-Pro-Phe-Thr-Glu), which exhibited opiate activity in vitro. Milk proteins also contain opioid antagonist peptides which suppress the action of opiate agonists and humoral peptides enkephalin. These opioid antagonist peptides, referred to as casoxins, are derived mainly from -casein and are -type receptor ligands. Casoxins A and B are fragments 35-41 (TyrPro-Ser-Tyr-Gly-Leu-Asn), and 58-61 (Tyr-Pro-Tyr-Tyr) of -casein, respectively, and have a low antagonistic potency when compared to naloxone, a synthetic opiate receptor antagonist. Casoxin C is fragment 25-34 of -casein (Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg) and exhibits high opioid antagonistic activity.205,206
Milk proteins 327 ACE inhibitory peptides Angiotensin-I converting enzyme (ACE, peptidyldipeptide hydrolase; EC 3.4.15.1) is a multifunctional, zinc-dependent carboxypeptidase, which plays an important role in the rennin angiotensin system, by influencing the regulation of peripheral blood pressure, the kallikrein-kinin system and the immune system. ACE converts angiotension I to a potent vasoconstrictor, octapeptide, angiotension II. ACE also catalyse the degradation of bradykinin, a vasodilatory peptide, and stimulates the release of aldosterone in the adrenal cortex which decreases the renal output while increasing water retention.117,184 ACE inhibition primarily results in a hypotensive effect but also affects the immunodefence and nervous systems.207 Casein derived peptide inhibitors of ACE are known as casokinins, which have been found in s1-casein (fragments 23-24, 23-27 and 194-199), -casein (fragments 74-76, 108-113, 114-121, 177-183, 193-198, 169-174 and 193-202) and -casein (fragments 108-110).117,204 s2-Casein also contains ACE inhibition peptides (fragments 189-193, 189-197, 190-197, 198-202) but these fragments have a very low activity.208,209 Whey protein derived inhibitors of ACE are referred to as lactokinins and have been found in -lactoglobulin (fragment 142-148), -lactalbumin (fragment 105-110) and bovine serum albumin (fragment 208-216).210±212 The binding of ACE is strongly influenced by the C-terminal tripeptide sequence of the substrate. Most of the potent casokinins and lactokinins contain hydrophobic amino acid residues at each of the three C-terminal positions, with proline, lysine or arginine residues being the most efficient in enhancing substrate binding to the negatively charged catalytic site of ACE. The removal or absence of arginine from the C-terminal can result in inactive ACE-inhibitory peptides.117 Immunomodulatory peptides Casein-derived immunopeptides include fragments of s1-casein (fragment 194199) and -casein (fragments 63-68, 191-193 and 193-202), which stimulate phagocytosis activity of murine and human peritoneal macrophages in vitro and exert a protective effect against Klebsiella pneumonaie infection in mice in vivo after intravenous administration.213 Coste et al.214 showed that a peptide from the C-terminal sequence of -casein (fragment 193-209) which also contains the ACE-inhibiting peptide ( -casokinin-10) induced a significant proliferation response in rat lymphocytes. Whey protein derived immunopeptides isolated from the N-terminal of -lactorphins derived from -lactalbumin (fragments 1819, 18-20 and 50-51) has in vitro enhanced the proliferation of peripheral blood lymhocytes.215 -Casein fragment 60-66 displays immunomodulatory activity; it confers a suppressive or stimulatory effect on lymphocyte proliferation of human colonic lamina propria lymphocytes (LPL), which is dependent on concentration. The anti-proliferative effect is reversed by the opiate receptor antagonist, naloxone.215 Immunomodulatory milk peptides may also alleviate allergic reactions in atopic humans and enhance mucosal immunity in the
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gastrointestinal tract.184 It has been suggested that opioid peptides may affect the immunoreactivity of lymphocytes, because opioid -receptors for endorphins are present in lymphocytes.216 Caseinophosphopeptide The caseinophosphopeptides (CPPs) are derived from the highly phosphorylated regions of the caseins; phosphorylated peptides have been isolated from hydrolysates of s1-casein (fragments 43-58, 45-55, 59-79, 66-74 and 106-119), s2-casein (fragments 2-21, 46-70, 55-75, 126-136, 138-149) and -casein (fragments 1-25, 1-28, 2-28).217 A common motif of CPPs is that their sequence consists of three phosphoseryl residues followed by two glutamic acid residues (SerP-SerP-SerP-Glu-Glu).218,219 These phosphopeptides are stable under acidic conditions and are very resistant to further proteolysis because of the high negative charge density.170 The CPPs are capable of binding and solubilising calcium, magnesium, iron, zinc and copper.191 Their ability to sequester calcium may be considered beneficial in the prevention of osteoporosis.220 CPPs also have anticariogenic effects by promoting recalcification of tooth enamel by localising amorphous Ca2+ at the surface of the tooth, depressing demineralisation and enhancing remineralisation of the enamel.220,221 A -casein derived CPP (fragment 1-25) has been shown in vitro and in vivo to enhance the bioavailability of iron when compared to whole casein and organic salts and has been used to treat anaemia in rats.222,223 Dephosphorylated casein peptides do not bind minerals.224 Mineral binding peptides have also been found in whey protein hydrolysates (-lactalbumin, -lactoglobulin and lactoferrin). These peptides are not phosphorylated; therefore, the minerals bind to the peptides through binding sites other than phosphorylated sites; the ability of these sites to bind minerals may be influenced by peptide conformation. Also both -lactalbumin and lactoglobulin derived mineral binding peptides have shown higher affinity for iron than the native proteins.225 Antithrombotic peptides Antithrombotic peptides derived from the C-terminal, glycomacropeptide portion of -casein (fragment 106-169) are known as casoplatelins; fragment 106-116 inhibits the aggregation of ADP-activated platelets and the binding of human fibrinogen -chain to platelet surface fibrinogen receptors.115,126 Smaller peptides (fragments 106-112, 112-116 and 113-116), known as casopiastrin, do not inhibit fibrinogen binding but are capable of inhibiting platelet aggregation.226,227 Glycomacropeptide mediates the absorption of minerals (calcium, zinc or iron) and modulates the gut microflora; the carbohydrate moities (sialic acid) promotes the growth of bifidobacterium.228,229 The absence of Phe, Tyr, Trp and Cys residues in GMP makes it a suitable ingredient for the nutrition of phenylketonuria sensitive patients.19 Non-glycosylated forms of GMP, referred to as caseino-macropeptide (CMP), are reported to inhibit gastric secretions and slow down stomach muscle contractions. CMP also stimulates the
Milk proteins 329 release of cholecystokinin, the satiety hormone involved in controlling food intake and digestion in the duodenum of animals and humans.230 Antibacterial peptides Isracidin, an antibacterial peptide from s1-casein (fragment 1-23) was isolated by Hill et al.231 Isracidin strongly inhibits the growth of gram-positive bacteria at high concentrations in vitro and also confers a protective effect in vivo against S. aureus and C. albicans at low concentrations. McCann et al.232 isolated an antibacterial peptide from s1-casein (fragment 99-109), which displayed a broad antibacterial activity including activity against gram-positive and gramnegative bacteria. Casocidin-I is an antibacterial peptide isolated from s2casein (fragment 165-203) which contains a high proportion of basic amino acid residues (10 of 39) and inhibits gram-positive bacteria (S. carnosus) and gramnegative bacteria (E. coli).233 Kappacin is an antibacterial peptide found in nonglycosylated, phosphorylated CMP and has been reported to inhibit grampositive bacteria (S. mutens) and gram-negative bacteria (P. gingivalis, E. coli), as well as showing an ability to bind to enterotoxins and inhibiting bacterial adhesion and growth.234 Whey protein hydrolysates also contain peptides ( lactoglobulin fragments 15-20, 25-40, 78-83 and 92-100) which exhibit activity against gram-positive bacteria. Modification of the -lactoglobulin fragment 92100 via the substitution of Asp with Arg and the addition of a Lys residue at the C-terminus expanded the bactericidal activity to include gram-negative bacteria including E. coli.235 Lactoferricin (fragment 17-41), is an antibacterial peptide produced by enzymatic hydrolysis of lactoferrin. It inhibits a wide variety of bacteria including enterotoxigenic E. coli and L. monocytogenes, yeast and fungi by disruption of their cytoplasmic membrane.236±239
13.6 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,240 International Dairy Federation,241 Southward and Walker,242 Otten,243 Zadow,244 Hugunin,245 De Wit,246,247 Southward,248,249 Mulvihill,16 Mangino,250 Chandan,251 Mulvihill and Ennis20 and O'Connell and Flyn.21 Milk protein products have also had applications as diverse as animal feed ingredients, plastics and industrial glues but these uses are not considered here. The following are brief outlines of some reported food applications of milk protein products. 13.6.1 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,
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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.252 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 13.7. Table 13.7 Application of milk protein products in baked products Milk protein product
Application
Effect/property
High-calcium co-precipitate Low-calcium co-precipitate Casein
Pastry glaze, cake mix for diabetics, milk biscuits, cookies Cookies
Colour, shine, nutrition, cake volume, texture, appearance Nutrition, texture, appearance
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
Nutrition
Calcium caseinate Sodium caseinate
Co-precipitate
Lactic casein Acid casein
Cookies Non-fat dry milk substitute
WPC
Cookies, muffins, croissants Bread Cake
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
Milk proteins 331 13.6.2 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 13.8). Caseins, vegetable fat, salts and water are used to make imitation cheeses (cheese analogues), which result in significant costTable 13.8
Application of milk protein products in dairy-type products
Milk protein Application product
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
Coprecipitate
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, cheeselike 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
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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 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 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 squeezable 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.
13.6.3 Beverages Casein products are used for their whipping and foaming properties or as stabilisers in drinking chocolate, effervescent drinks and beverages (Table 13.9). 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. Sodium and calcium caseinates are used in the production of GlucernaTM and ResourceTM which are nutritional beverages produced for
Milk proteins 333 Table 13.9
Application of milk protein products in beverages
Milk protein Application product
Effect/property
Casein
Clarification, colour removal, stabiliser, palatability, colour stability, removal of phenolic compounds Stabiliser
Beer, wine
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
patients with abnormal glucose tolerance. 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 WPCs 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. 13.6.4 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 13.10). In the manufacture of whipped toppings the basic ingredients of vegetable fat, sugar, protein (sodium caseinate), emulsifier, stabilisers and water are blended at 38± 46 ëC and the mixture is pasteurised and hom*ogenised 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
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Table 13.10 Application of milk protein products in dessert-type products Milk protein Application product
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
replaced by 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. 13.6.5 Pasta products Milk protein products may be incorporated into the flour base for pasta manufacture to improve nutritional quality and texture (Table 13.11). 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 Table 13.11 Application of milk protein products in pasta products Milk protein Application product
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- Imitation rice calcium coprecipitate
Nutrition, texture
WPC
Flour replacer, nutrition
Pasta
Milk proteins 335 cooking. Imitation pasta-type products containing substantial proportions of milk protein have also been manufactured. 13.6.6 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 13.12). 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 replacement for 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. A milk protein hydrolysate, `Prodiet F200', has been used in chocolate formulations; it contains a bioactive peptide with relaxing properties.253 Calcium caseinate, WPC, WPI, MPI, and milk protein hydrolysate ingredients are used in the manufacture of high protein energy bars; they are used to extend bar shelf-life and minimise bar hardening, and nutritionally to optimising muscle performance during exercise. EnsureTM, GlucernaTM and Choice dmTM are speciality nutritional bars for people with diabetes; these all contain calcium caseinate. Table 13.12 Application of milk protein products in confectionery products Milk protein Application product
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 High protein energy bar
Aeration, body, mouthfeel, texture Nutrition, improve shelf-life stability
Hydrolysed casein
Aerated confection
Whippability
Coprecipitate
Protein-rich chewable bar
Nutrition, texture
Milk protein Chocolate formulations hydrolysate
Relaxation
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13.6.7 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 13.13). 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. In addition, WPI and its hydrolytic products have been used in the preparation of cooked pork patties, where they reduce cook loss as well as inhibiting lipid oxidation during refrigerated storage.254 Casein phosphopeptides have also been incorporated into ground meat products including ground beef and have been shown to enhance product stability by inhibiting lipid oxidation.255 Lactoferricin is used in the washing of animal carcasses at refrigeration temperatures to inhibit the growth of food pathogens (e.g., E. coli 0157:H7) and spoilage micro-organsims (e.g., C. viridans). 13.6.8 Nutritional/medical/pharmaceutical applications Milk protein products are used extensively in special dietary preparations for the ill or convalescing, for malnourished children and for people on therapeutic or weight-reducing diets (Table 13.14). Modified low mineral whey powders are used to produce `humanised' infant formulae with a whey protein-to-casein ratio resembling that of human milk. Infant formula is supplemented with -
Table 13.13 Application of milk protein products in meat products Milk protein Application product
Effect/property
Sodium caseinate
Liver sausage, sausage, blood, patties, low fat meat paste
Binder, decolouriser, emulsifier
Potassium caseinate
Low fat meat paste
Emulsifier
Caseinophos- Ground meat products phopeptides
Enhance product stability, prevent lipid oxidation
Sodium salt Pate sausage of coprecipitate
Nutrition, sensory, cost
WPC
Cost, improved performance, water and fat binding, water solubility at low viscosity
Meat products
Milk proteins 337 Table 13.14 Application of milk protein products in nutritional, pharmaceutical 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
Coprecipitate
Carbohydrate-free and low-lactose infant food
Nutrition
Highcalcium coprecipitate
Cake mix for diabetics
Nutrition, cake volume
WPC
Geriatric/hospital/liquid diets Infant formulae
Nutrition Nutrition, digestability
WPI
Nutritional protein bar/beverages
Improve immuno-defence and nervous system activity
Whey protein hydrolysate
Hypoallergenic infant formulae
Nutrition
Caseinophosphopeptides
Dental filling material
Mineral binding activity
Lactoferricin
Medication
Treatment of gastritis
Prevent caries
lactalbumin, providing a rich source of essential amino acids, in particular tryptophan and its metabolites such as serotonin, which is essential for brain maturation and development of other neurobehavioural regulations of food intake and sleep-wake-rhythm.256 Whey protein hydrolysates have been used in hypoallergic, peptide-based formulae. Fractionation 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 used as ingredients for the production of more `humanised' infant formulae. Lactoferrin is being added to infant formulae for its bacteriostatic and bactericidal activity and CMP is being added to contribute to improved mineral adsorption. Caseinates and coprecipitates 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
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specific renal problems. Casein hydrolysates are used in specialised foods for premature infants and in formulae for infants suffering from various intestinal disorders; casein hydrolysates, low in phenylalanine, have been proposed uses in formulae for feeding infants with phenylketonuria. The absence of Phe, Tyr, Trp and Cys residues in -casein glycomacropeptide make it a possible stable source of nutrition for phenylketonuria-sensitive patients. Casein products are added to various foods and drinks as a nutritional supplement. Caseins are used in special nutritional preparations for athletes as they enhance athletic performance which is attributed to the high glutamine levels that are important in the maintenance of muscle protein mass. Caseinates, co-precipitates and casein hydrolysates are often used in combination with whey protein concentrate or isolate in protein supplemented powders/beverages for athletes; calcium caseinate and micellar casein are used by athletes as a `slow release protein' supplement which decreases the rate of muscle degradation while dieting.21,257 Non-glycosylated caseinomacropeptide is used in weight reduction diet products (low carbohydrate yoghurts), where it stimulates the release of cholecystokinin, resulting in the production of insulin which inhibits gastric secretions that play an important role in controlling food intake and digestion in the duodenum of animals and humans.230 A casein-based diet containing high levels of TGF- (transforming growth factor beta) fed to children with Crohn's disease ameliorated chronic inflammation of the intestine.258 Whey protein enriched diets have been shown to reduce the growth of tumors in the GI tract, selectively inhibit cancer cell growth in head and neck cancer patients as well as aid the immune defence system against post-operative infections.177,178 Milk protein hydrolysates are used for intravenous nutrition for patients suffering from protein metabolism disorders, intestinal disorders, and for post-operative patients. Special casein preparations have been used as food for patients suffering from cancer, pancreatic disorders or anaemia. Biologically active peptides have shown potential applications in a wide variety of products; opiate peptides exhibit pharmacological properties similar to morphine while also influencing postprandial metabolism by stimulating pancreatic insulin and gastrointestinal somatisation release, prolonging gastrointestinal transit time and exerting antidiarrhoeal action.259±262 `BioZate', a whey protein isolate hydrolysate which contains an ACE-inhibitory peptide, has been shown to significantly reduce both systolic and diastolic blood pressure, as well as influencing immuno-defence systems and nervous system activity. This product exhibits both emulsifying and foaming properties, enabling this product to have a wide variety of applications including nutritional protein bars and beverages, meal replacers and specialised foods for sport nutrition. `Calpis'TM is a sour milk drink produced in Japan, and `Evolus', a calcium-enriched fermented milk drink produced in Finland, both contain antihypertensive peptides and have been shown to reduce blood pressure.263 Antibacterial peptides including Casocidin-I and Kappacin have a variety of applications in oral care products, due to their strong inhibition of gram-positive bacteria and gram-negative bacteria as well as showing an ability
Milk proteins 339 to bind to enterotoxins and inhibit viral and bacterial adhesions.234 Lactoferricin, which has antimicrobial and haemolytic activity, has been shown to be useful in the development of peptide antibiotics as therapeutic agents with low toxicity,191 and preparing medicines for treating acute gastritis, chronic gastritis, peptic ulcer and high stomach acidity.264 Casein derived peptides such as phosphopeptides, which have mineral binding ability have uses in the preparation of tablets, toothpaste and dental filling material.220 Sulphonated glycopeptides prepared from casein have been used for the treatment of gastric ulcers. Several other bioactive peptides may be used in pharmaceutical preparations, for example, casomorphins in the treatment of diarrhoea, casokinins (hypertension), casoplatelins (thrombosis) and immunopeptides (immuno-deficency).191 In the pharmaceutical industry, the oral route is considered the most convenient way of administering drugs to patients. Hydrophilic drugs cannot readily diffuse across the cells in the intestinal epithelium through the lipid bilayer. Milk proteins possess many of the properties required for effective drug delivery by oral administration as they are readily degraded at the low pH in the stomach and act as good microencapsulating agents. The ability of milk proteins to encapsulate a wide variety of materials suggests several promising applications in the food industry including use in the production of low calorie and reduced fat products, while masking off flavours, preventing oxidation and improving sensory characteristics; in the cosmetic industry to produce easily spreadable creams and lotions with encapsulated ingredients in both the oil and water phases and in the pharmaceutical industry to manufacture drug delivery systems.265 In the food industry, WPI or WPI/ lactose has been used to encapsulate anhydrous milk fat while also conferring good oxidative stability by preventing oxygen uptake.266 WPC may also be used in many applications as a fat replacer.267 Whey protein concentrate gels can be used as pH-sensitive hydrogels for controlled delivery of various materials including biologically active substances.268,269 Casein-dextran conjugates have been used as secondary emulsifiers in the preparation of multiple emulsions with the capability of encapsulating numerous materials (flavours, bioactive peptides and anticancer drugs) depending on their application, while also being safe for use in food, pharmaceutical and medical preparations. Multiple emulsions have been used to produce low caloric and flavoured mayonnaise.270 Semo et al.271 demonstrated that the casein micelle can be used as a nano-encapsulation vehicle for hydrophobic nutraceutical substances (vitamin D) for enrichment of non-fat or low-fat food products.
13.6.9 Convenience foods Applications of milk protein products in convenience foods are outlined in Table 13.15. 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
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Table 13.15 Application of not protein products in convenience foods Milk protein Application product
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
Coprecipitate
Potato soup with rice, vegetable cutlets
Emulsification, nutrition, texture
WPC
Salad dressing, egg replacer Quiches, egg replacer Cream-based soups
Viscosity, mouthfeel, emulsification Cost, extender Cost
Whey solids Gravy mix, dehydrated soup mix
Whitener, flavour, emulsifier, stabiliser
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.
13.6.10 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 protein-enriched snack-type food products (Table 13.16). 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 coprecipitates by renneting followed by a combination of heat treatment and extrusion or working. Microwave heating of whey protein solutions results in
Milk proteins 341 Table 13.16 Application of milk protein products in textured products Milk protein Application product
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
Coprecipitate
Dietary fibre snack
Texture
Whey protein Dietary fibre snack
Texture
WPC
Texture, cost
Surimi
simultaneous expansion and gelation to give textured products with potential for use in comminuted meats. WPCs are proposed as cost effective replacements for beef plasma protein or potato starch in the modification of surimi texture.272 13.6.11 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 WPC-based 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, 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.273
13.7 Future trends A large number of different milk protein products are presently recovered from milk and it is highly likely that this range will be further extended in the future. Cost effective commercial methods for separating individual casein and whey
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protein fractions are currently emerging as new technologies are being developed and these fractions will become more widely available in the near future. Tailoring of milk protein products, most likely by enzymatic and physical modifications, will also be further developed to produce new speciality milk protein ingredients which meet the specific physico-chemical and functional demands of specific food applications. Commercial interest in milk protein hydrolysates and biologically active peptides is likely to continue to grow and to focus on procedures for the commercial preparation, isolation and purification of these products. A small number of milk protein derived biologically active peptides are presently commercially available; it is likely that the search for new milk-derived peptides will continue and an area of future focus will be the impact of interactions with other food constituents and technological processing procedures on their biological activity. While the main area of application for milk protein products will continue to be as functional ingredients in the food industry, the expanded range of milk protein-derived products will find increased uses in nutritional and nutraceutical applications. Consumer awareness of food ingredients will grow and consumers will view milk-derived protein and peptide ingredients as natural, wholesome, nutritious and healthy constituents in foods, thus enhancing and expanding their importance.
13.8
Sources of further information and advice
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 +13127825299. · USDA/AMS, Dairy Standardisation Branch, Room 2750- South Building, PO Box 96456, Washington, DC, USA. Fax +12027202643. · Food and Agriculture Organisation of the United Nations (Codex Alimentarius), Viale delle Terme di Caracalla, 00100-Rome, Italy. Fax +390657054593. · International Dairy Federation, 41, Square Vergote, 1030 Brussels, Belgium. Fax +3227330413. 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, Clonmel Road, Mitchelstown, Co. Cork, Republic of Ireland. Fax + 353 (0) 25 44135. · Kerry Ingredients Ireland, Tralee Road, Listowel, Co. Kerry, Republic of Ireland. Fax + 353 (0) 68 21562.
Milk proteins 343 · Glanbia (formerly Avonmore-Waterford Group), Ballyragget, Co. Kilkenny Republic of Ireland. Fax + 353 (0)5688 36001. · 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. · Arla Food 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 Street 26, D-40545, Dusseldorf, Germany. Fax +49211555583.
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SOUTHWARD, C. R. and WALKER, N. J. (1982). Casein, caseinates and milk protein coprecipitates. In CRC Handbook of Processing and Utilization in Agriculture, Volume 1. Animal Products (A. Wolf, ed.), CRC Press, Boca Raton, FL, 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. International Dairy Federation, Atlanta, GA, 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.), Elsevier Applied Science Publishers, London, pp. 273±316. HUGUNIN, A.G. (1987). Applications of UF Whey Protein: Developing New Markets. Bulletin 212, International Dairy Federation, Brussels, pp. 135±144. DE WIT, J.N. (1989). The use of whey protein products. In Developments in Dairy Chemistry ± 4 (P.F. Fox, ed.), Elsevier Applied Science Publishers, London, pp. 323±346. DE WIT, J.N. (1998). Nutritional and functional characteristics of whey proteins in food products. Journal of Dairy Science, 81, 597±608. SOUTHWARD, C.R. (1986). Utilization of milk components: casein. In Modern Dairy Technology, Volume 1. Advances in Milk Processing (R.K. Robinson, ed.), Elsevier Applied Science Publishers, London, pp. 317±68. SOUTHWARD, C. R. (1989). Use of casein and caseinates. In Developments in Dairy Chemistry ± 4 (P.F. Fox, ed.), Elsevier Applied Science Publishers, London, pp. 173±244. MANGINO, M. E. (1992). Properties of whey protein concentrates. In Whey and Lactose Processing (J.G. Zadow, ed.), Elsevier Applied Science Publishers, London, pp. 231±270. CHANDAN, R. (1997). Dairy-based Ingredients. Eagan Press, St. Paul, MN. ERDOGDU-ARNOCZKY, N., CZUCHAJOWSKA, Z. and POMERANZ, Z.Y. (1996). Functionality of whey and casein in fermentations and in breadbaking by fixed and optimised procedures. Cereal Chemistry, 73, 309±16. TM F200: an innovative stress-relieving ingredient. LE FRANC, C. (2002). Prodiet Agro-Food-Industry Hi-Tech, July/August, 9±11. PEnÄA-RAMOS, E.A. and XIONG, Y. (2003). Whey and soy protein hydrolysates inhibit lipid oxidation in cooked pork patties. Meat Science, 64, 259±263. SAKANAKA, S., TACHIBANA, Y., ISHIHARA, N. and JUNEJA, L.R. (2005). Antioxidant properties of casein calcium peptides and their effects on lipid oxidation in beef hom*ogenates. Journal of Agricultural and Food Chemistry, 51, 3668±3674. BOEHM, G. (2007). Infant Formulas. In Handbook of Food Products Manufacture (Y.H. Hui, ed.), John Wiley and Sons, Chichester, pp. 667±714. BEANFRERE, B., DAGING, M. and BOIRIE, Y. (2000). The fast and slow protein concept. In Proteins, Peptides and Amino Acids in Enteral Nutrition, Volume 3 (P. Furts and V. Young, eds.), Karger, Basel. FELL, J.M., PAINTIN, M., ARNAUD-BATTANDIER, F., BEATTIE, R.M., HOLLIS, A., KITCHING,
and WALKER-SMITH, J.A. (2000). Mucosal healing and a fall in mucosal pro-inflammatory cytokine mRNA induced by a specific oral polymeric diet in paediatric Crohn's disease. Alimentary Pharmacology and Therapeutics, 14, 281±289. 259. SCHUSDZIARRA, V., SCHICK, R., DE LA FUENTE, A., HOLLAND, A., BRANTL, V. and PFEIFFER, E.F. (1983). Effect of -casomorphins on somatostatin release in dogs. Endocrinology, 112, 1948±1951. P., DONNET-HUGHES, A., MACDONALD, T.T.
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260.
and (1983). Effect of -casomorphins and analogs on insulin release in dogs. Endocrinology, 112, 885±889. DANIEL, H., VOHWINKEL, M. and REHNER, G. (1990). Effect of casein and -casomorphins on gastrointestinal motility in rats. Journal of Nutrition, 120, 252±257. DANIEL, H., WESSENDORF, A., VOHWINKEL, M. and BRANTL, V. (1990). Effect of DAla 2,4, Tyr5- -casomorphin-5-amide on gastrointestinal functions. In Casomorphins and Related Peptides (F. Nyberg and V. Brantl, eds.), Fyris-Tryck AB, Uppsala, pp. 95±104. SEPPO, L., JAUHIAINEN, T., POUSSA, T. and KORPELA, R. (2003). A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. American Journal of Clinical Nutrition, 77, 326±330. ZHU, K.F. (2004). Application of bovine lactoferricin in preparation of medicine for treating HP infected gastric disease. European Patent Application, CN 1840179. VAN DER GRAAF, S., SCHROEÈN, C.G.P.H. and BOOM, R.M. (2005). Preparation of double emulsions by membrane emulsification ± a review. Journal of Membrane Science, 251, 7±15. MOREAU, D.L. and ROSENBERG, M., (1996). Oxidative stability of anhydrous milkfat microencapsulated in whey proteins. Journal of Food Science, 61, 39±43. GIESE, J. (1996). Fats, oils and fat replacers. Food Technology, 50, 78±83. GUNASEKARAN, S., XIAO, L. and OULD ELEYA, M.M. (2006). Whey protein concentrate hydrogels as bioactive carriers. Journal of Applied Polymer Science, 99, 2470± 2476. GUNASEKARAN, S., KO, S. and XIAO, L. (2007). Use of whey proteins for encapsulation and controlled delivery applications. Journal of Food Engineering, 83, 31±40. MATSUMATO, S., KOH, Y. and MICHIURE, A. (1985). Preparation of W/O/W emulsions in an edible form on the basis of phase inversion technique. Journal of Dispersion Science and Technology, 6, 507±521. SEMO, E., KESSELMAN, E., DANINO, D. and LIVNEY, D. (2007). Casein micelle as a natural nano-capsular vehicle for nutraceuticals. Food Hydrocolloids, 21, 936±942. HSU, C.K. and KOLBE, E. (1996). The market potential of whey protein concentrate as a functional ingredient in surimi seafoods. Journal of Dairy Science, 79, 2146±51. CHEN, H. (1995). Functional properties and applications of edible films made of milk proteins. Journal of Dairy Science, 78, 2563±2583. ZALL, R.R. (1992). Sources and composition of whey and permeate. In Whey and Lactose Processing (J.G. Zadow, ed.), Elsevier Applied Science Publishers, London, pp. 1±72. FOX, P.F. and BRODKORB, A. (2008). The casein micelle: historical aspects, current concepts and significance. International Dairy Journal, 18, 667±684. SCHUSDZIARRA, V., SCHICK, R., DE LA FUENTE, A., SPECHT, J., KLIER, M., BRANTL, V.
PFEIFFER, E.F.
261. 262.
263.
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266. 267. 268.
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271. 272. 273. 274.
275.
14 Egg proteins M. Anton, INRA Nantes Unite 1268 BiopolymeÁres Interactions Assemblages, France and F. Nau and V. Lechevalier, UMR INRA Science et Technologie du Lait et de l'Oeuf, France
Abstract: This chapter deals with the chemical composition and structural characteristics of egg yolk and white in relation to three important functional properties: emulsifying, foaming and gelling properties. Key words: egg, yolk, white, emulsions, foams, gels, structure, assemblies, interfaces.
14.1 Introduction: technofunctional uses of egg constituents Hen egg was categorised by Baldwin in 1986 as a polyfunctional ingredient, as it can simultaneously realise several technological functions in the same formulated foodstuff. Its emulsifying, foaming, gelling, thickening, colouring and aromatic properties make it still today a universal basic ingredient for the domestic kitchen and the food processing industry. Whereas egg yolk is well recognised for its emulsifying properties, egg white (or albumen) is a reference in terms of foaming and both parts are used as gelling ingredient in many foods. Yolk takes part in the formation and the stabilisation of emulsions. In spite of the intensive use of yolk in formulated foodstuffs, and since the invention of mayonnaise three centuries ago, the role of its major constituents is not clear because of its complex structure. Yolk is a mixture of proteins and lipids forming natural assemblies at various scales. These natural assemblies contribute to the nano- and the microstructure of yolk. Thus, an understanding of the emulsifying properties of yolk lies in the comprehension of these various levels of structure.
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The exceptional foaming properties of the albumen are also the base of traditional recipes among which meringues act certainly as reference. Indeed, the extreme simplicity of their formula (albumen and sugar, possibly added with flavours) allows albumen to express in an optimal way its foaming properties. However, the technological parameters influence the final quality of foam obtained, and three types of meringues (traditional meringue, Swiss meringue and Italian meringue) can be distinguished, depending on whether whipping is achieved in the presence or absence of sugar, and at ambient or warm temperature. But there is also a great number of other products in which previously foamed albumen is added, which are either fat-free formulas (angel food cake) or lipid-containing formulas (spoon biscuits, `sponge' cake, blown). In such products, the complexity of the phenomena is extreme, foaming and emulsification taking place simultaneously, which makes the control of the physico-chemical and technological parameters of these operations very delicate. Concerning the gelling properties of albumen and yolk, they are related to the heat-gelation capacity of egg proteins. Then, these properties imply a cooking step during the food processing. The heat gelation of egg proteins completely conforms to the model of heat gelation of globular proteins. The corresponding mechanisms have been extensively studied, on egg proteins as well as on other ones, and the key technological parameters have now been identified. However, the addition of other ingredients in mixture with egg (polysaccharides, for example) complicates the understanding of the egg gelation behaviour, and developments with more complex models are still needed.
14.2
Physico-chemistry and structure of egg constituents
14.2.1 Egg yolk Chemical composition Yolk correspond to 36% of whole hen egg weight. Its dry matter is about 50± 52% according to the age of the laying hen and the duration of preservation (Kiosseoglou, 1989; Thapon and Bourgeois, 1994; Li-Chan et al., 1995). The compositions of fresh and dry yolks are presented in Table 14.1: the main components are lipids (about 65% of the dry matter) and the lipid to protein ratio is about 2:1. Yolk lipids are exclusively associated with lipoprotein assemblies. They are made up of 62% triglycerides, 33% phospholipids, and less than 5% cholesterol. Carotenoids represent less than 1% of yolk lipids, and give it its colour. Proteins are present as free proteins or apoproteins (included in lipoprotein assemblies). The interactions between lipids and proteins result in the formation of lipoproteins (low and high density), which represent the main constituents of yolk. Macrostructure and main constituents Yolk is a complex system with different structuration levels consisting in aggregates (granules) in suspension in a clear yellow fluid (plasma) that contains
Egg proteins 361 Table 14.1
Composition of hen egg yolk
Water Lipids Proteins Carbohydrates Minerals
Fresh yolk (%)
Dry yolk (%)
51.1 3.6 16.0 0.6 1.7
Ð 62.5 33.0 1.2 3.5
Source: Powrie and Nakai (1986)
lipoproteins and proteins. Granules consist in circular complexes ranging in diameter from 0.3 m to 2 m (Chang et al., 1977). Consequently, yolk can be easily separated into two fractions after a dilution (two times) with 0.3 M NaCl and a centrifugation at 10,000 g (30 min) according to the method of McBee and Cotterill (1979): a dark orange supernatant called plasma and a pale pellet called granules (Fig. 14.1). Granules represent 22% of yolk dry matter, accounting for about 50% of yolk proteins and 7% of yolk lipids. The dry matter content of granules is about 44%, with about 64% proteins, 31% lipids and 5% ash (Dyer-Hurdon and Nnanna,
Fig. 14.1
Fractionation of plasma and granules from hen egg yolk.
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Table 14.2 Repartition of hen egg yolk constituents Yolk D.M. (%)
Yolk lipids (%)
Yolk proteins (%)
Lipids (%)
Proteins (%)
100
100
100
64
32
Plasma LDL Livetins Others
78 66 10 2
93 61 Ð Ð
53 22 30 1
73 88 Ð Ð
25 10 96 90
Granules HDL Phosvitin LDLg
22 16 4 2
7 6 Ð 1
47 35 11 1
31 24 Ð 88
64 75 95 10
Yolk
Source: Powrie and Nakai (1986)
1993; Anton and Gandemer, 1997). They are mainly constituted by high density lipoproteins (HDL) (70%) and phosvitin (16%) linked by phosphocalcic bridges between the phosphate groups of their phosphoseryl residues (Burley and Cook, 1961; Saari et al., 1964). Low density lipoproteins (LDL) (12%) are included in the granular structure (Table 14.2). At low ionic strength, granules mainly form insoluble HDL-phosvitin complexes linked by phosphocalcic bridges as HDL and phosvitin contain a high proportion of phosphoserin amino acids able to bind calcium (Causeret et al., 1991). The numerous phosphocalcic bridges make the granule structure very compact, poorly hydrated, weakly accessible to enzymes, and lead to an efficient protection against thermal denaturation and heat gelation. At an ionic strength over 0.3 M NaCl, the phosphocalcic bridges are disrupted because monovalent sodium replaces divalent calcium. In such conditions, the solubility of granules reaches 80% because phosvitin is a soluble protein and HDL behave like soluble proteins (Cook and Martin, 1969; Anton and Gandemer, 1997). Complete disruption of granules occurs when ionic strength reaches 1.71 M NaCl. Acidification or alkalinisation similarly cause the disruption of granules and the solubilisation of these constituents by increasing the number of the positive (NH3+) or negative (COO-) charges inducing electrostatic repulsions between granule constituents. Recently, we have established (Sirvente, 2007) a phase diagram drawing the different states of granules as a function of pH and ionic strength (Fig. 14.2). Plasma comprises 78% of yolk dry matter and is composed of 85% LDL and 15% livetins (Burley and Cook, 1961; Table 14.2). It forms the aqueous phase where yolk particles are in suspension. It accounts for about 90% of yolk lipids (including nearly all the carotenoids), and 50% of yolk proteins. Plasma contains about 73% lipids, 25% proteins and 2% ash. Lipids of plasma are distributed thus: 70% triglycerides, 25% phospholipids and 5% cholesterol.
Egg proteins 363
Fig. 14.2 Physical state of granules as function of pH and ionic strength.
LDL are spherical particles (17±60 nm in diameter with a mean of about 35 nm) with a lipid core in a liquid state (triglycerides and cholesterol esters) surrounded by a monofilm of phospholipid and protein (Cook and Martin, 1969; Evans et al., 1973). LDL are soluble in aqueous solution (whatever the pH and ionic conditions) due to their low density (0.982). Phospholipids take an essential part in the stability of the LDL structure because association forces are essentially hydrophobic (Burley, 1975). Some cholesterol is included in the phospholipid film, increasing its rigidity. LDL are composed of 11±17% protein and 83±89% lipid, out of which 74% is neutral lipid and 26% phospholipid (Martin et al., 1964). 14.2.2 Egg white Egg white represents about 60% of the total egg weight. It consists of an aqueous protein solution, containing few minerals and carbohydrates (Table
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Handbook of hydrocolloids Table 14.3 Composition of hen egg white % of hen egg white Water Lipids Proteins Carbohydrates Minerals
88.0 Ð 10.6 0.8 0.6
Source: Thapon and Bourgeois (1994)
14.3). During egg storage, different physico-chemical modifications happen, among them the CO2 departure that induces a pH increase, from 7.5 at the laying moment to 9.5 after a few days. This pH modification should be the cause of the egg white liquefaction, because of the dissociation of a protein complex (ovomucin-lysozyme complex) (Kato et al., 1975). Another evolution observed concerns the ovalbumin modification toward S-ovalbumin, which is a more heat-stable form (Smith and Back, 1965), resulting from isomerisation of three serine residues (Yamasaki et al., 2003). Proteins Proteins represent more than 90% of the dry matter of egg white, but until very recently, only the major ones have been identified. However, the recent and powerful techniques for separation and analysis enabled the identification of many minor proteins (Table 14.4) (GueÂrin-Dubiard et al., 2006; Mann, 2007). The egg white proteins are predominantly globular proteins, and acidic or neutral, except lysozyme and avidin which are highly alkaline proteins. All are glycosylated, except cystatin and the major form of lysozyme. Some of them are very heat-sensitive and/or sensitive to surface denaturation, explaining their noteworthy functional properties. The major egg white protein (more than 50% of the total proteins) is ovalbumin, a 45 kDa globular and phosphorylated protein. Half of its amino acids are hydrophobic, and one-third are electrically charged, essentially negatively at physiologic pH. Ovalbumin possess six buried Cys residues, two being involved in a disulfide bridge (Cys73-Cys120). Ovalbumin is then the only egg white protein with free thiol groups, capable of inducing some rearrangements with variations of storage conditions, pH and surface denaturation. Ovotransferrin (13% of total proteins) molecular weight is around 78 kDa. This protein consists of two lobes, each containing a specific binding site for iron (or copper, zinc, aluminium) (Kurakawa et al., 1995). It is the most heatsensitive egg white protein, but the complexation of iron or aluminium significantly increases its heat stability (Lin et al., 1994). OvomucoõÈde is a highly glycosylated protein (up to 25% carbohydrates, w/w) of 28 kDa. At pH 7, its denaturation temperature is around 77 ëC, but this protein
Egg proteins 365 Table 14.4 Composition and some physico-chemical and functional properties of egg white proteins %
Mw (kDa)
pI
Ovalbumin Ovalbumin Y Ovalbumin X Ovotransferrin OvomucoõÈd Ovomucin
54 5 0.5 13 11 1.5±3.5
45 44 56 76 28 230±8300
5 5.2 6.5 6.7 4.8 4.5±5
Lysozyme Ovoinhibitor Ovoglycoprotein Flavoprotein Ovostatin Cystatin Avidin Ex-FABP Cal gamma TENP
3.5 0.1±1.5 0.5±1 0.8 0.5 0.05 0.05 nd nd nd
14.4 49 24.4 32 760±900 12.7 68.3 18 20.8 47.4
10.7 5.1 3.9 4 4.6 5.1 10 5.5 6 5.6
nd
18
6.4
Protein
Hep 21
Major biological properties Immunogenic phosphoproteine nd nd Iron binding, bacteriostatic activity Trypsin inhibitor Highly glycosylated, viral hemaglutination inhibition Lysis of Gram bacteria wall Serine protease inhibitor nd Riboflavin (vitamin B2) binding Serine protease inhibitor Cysteine protease inhibitor Biotine binding Lipocaline family Lipocaline family BPI (bactericidal permeabilityincreasing protein) family uPar/Ly6/Snake neurotoxin family
Sources: Li-Chan and Nakai (1989), Stevens (1991), GueÂrin et al. (2006).
is much more heat resistant at acidic pH (Lineweaver and Murray, 1947). Ovomucin is also a highly glycosylated protein, with a very high molecular weight (104 kDa). Electrostatic interactions can be observed between ovomucin and some of the other egg white proteins. In the freshly laid eggs (pH 7.5), the carboxylic groups of the ovomucin sialic acids especially interacts with the NH3+ of lysozyme lysine residues to form a lysozyme-ovomucin complex that may be responsible for the gel-like structure of egg white (Kato et al., 1975). Lysozyme is a small (14 kDa) globular, and strongly basic protein. Its structure is very rigid, stabilised by four disulfide bridges. Glucidic and mineral fractions The glucidic fraction of egg white consists of free glucose (0.5% w/w) and carbohydrates linked to proteins (0.5% w/w). The mineral fraction is predominantly composed of Na+, K+ and Clÿ, as free minerals, whereas P and S are essentially constitutive elements of proteins. Egg white also contains CO2, in equilibrium with bicarbonate, which plays a major role for pH control (Thapon, 1994).
14.3 Egg yolk emulsions 14.3.1 Basic principles Emulsifying activity is related to the capacity of surface active molecules to cover the oil±water interface created by mechanical hom*ogenisation, thus
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reducing the interfacial tension. Consequently, the more active the emulsifying agent, the more the interfacial tension is lowered. Emulsion stability indicates the capacity to avoid flocculation, creaming, and/or coalescence of oil droplets. Creaming and flocculation are reversible phenomena which can be avoided by a simple agitation of the emulsion. Coalescence is the irreversible fusion of oil droplets due to the rupture of the interfacial film created by emulsifying agents. This phenomenon leads to a complete destruction of the emulsion. This relates the importance of the structure and the viscoelasticity of the interfacial film.
14.3.2 Role of egg yolk constituents In researching the principal contributor to yolk emulsifying properties, numerous authors have separated yolk into its main fractions: plasma and granules. Large similarities have been observed between emulsifying properties of yolk and plasma, whereas emulsions made with granules behaved very differently (Dyer-Hurdon and Nnanna, 1993; Anton and Gandemer, 1997; Le Denmat et al., 2000). Specifically, emulsions made with granules are more coarse (more important oil droplet size) than emulsions made with yolk and plasma, and notably at acidic pH where granules are not soluble (Le Denmat et al., 2000) (Fig. 14.3). Concerning the parameters of emulsion stability (creaming), we showed (Le Denmat et al., 2000) that emulsions made with yolk and plasma had the same creaming rate, in function of the medium conditions, whereas emulsions made with granules behaved very differently (Fig. 14.3). Consequently, these studies demonstrated that yolk emulsifying power was situated in plasma. Among plasma constituents, some authors demonstrated that LDL are better emulsifiers than bovine serum albumin (BSA) (Mizutani and Nakamura, 1984) and casein (Shenton, 1979). Even though some authors suggested that, in certain conditions, HDL were more efficient than LDL to form and stabilise O/W emulsions (Hatta et al., 1997; Mine, 1998), a large number of studies confirm the prevalent role of LDL in yolk emulsions. These findings have been confirmed recently (Aluko et al., 1998; Mine and Keeratiurai, 2000; Anton et al., 2003; Martinet et al., 2003). In particular, it has been established that LDL made emulsions finer than HDL, along different conditions of pH and ionic strength (Martinet et al., 2003). The next question is how to explain the exceptional efficiency of LDL at the interfaces.
14.3.3 Importance of assemblies Given that any destructurating treatment affects the emulsifying properties of LDL, it appears that the integrity of the structure of LDL seems essential to ensure their interfacial properties (Tsutsui, 1988). Direct adsorption of apoproteins and phospholipids from LDL is not easy because of the nonsolubility of these species in water or in aqueous buffer. So the interactions between apoproteins and lipids to assemble the LDL particles are essential to
Egg proteins 367
Fig. 14.3 Mean droplet diameter (d3.2) and creaming index (Icr) in oil/water emulsions (30 : 70) prepared with yolk, plasma and granules, protein concentration: 25 mg/ml, hom*ogenisation pressure: 200 bars, n 3.
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Fig. 14.4 /A isotherms of the different lipid constituents extracted from LDL and spread at the air±water interface; neutral lipids = 85 g, phospholipids = 198 g, total lipids = 287 g, compression rate = 100 cm2/min.
transport the surfactants in a soluble form in the neighbourhood of the interface and then to release them at the interface. Using Langmuir film balance (air±water interface), three phase transitions have been detected in compression isotherms and these three transitions (19, 41 and 54 mN/m) have been attributed, respectively, to neutral lipids, apoproteins and phospholipids by comparison with films of neutral lipids, phospholipids and total lipids extracted from LDL (Fig. 14.4) (Martinet et al., 2003). The transition observed at 19 mN/m corresponds to the collapse of neutral lipids, and the transition at 54 mN/m corresponds to phospholipid collapse. These different transitions show that LDL actually break down when they come into contact with the interface to release neutral lipids, phospholipids and apoproteins from the lipoprotein core and to allow their spreading. In a recent study made with atomic force microscopy (AFM) after a Langmuir±Blodgett transfer of the layers from the air±water interface to a silica plate, it has been shown that the second transition (previously attributed to apoproteins alone) is not due to apoproteins alone, but to apoprotein±lipids complexes (Dauphas et al., 2006). So, it has been deduced that LDL serve as vectors of surfactant constituents (apoproteins and phospholipids) that could not be soluble in water, until the interface. At this step the conservation of the LDL structure is essential. Once LDL are near the interface, the structure is then broken up to release surfactant constituents at the interface (Fig. 14.5). Furthermore, comparing interfacial behaviour of LDL and liposomes (double phospholipid layer not containing proteins), it has been shown that the apoproteins situated on the LDL surface start the LDL disruption mechanism by
Egg proteins 369
Fig. 14.5 Hypothetical mechanism of LDL adsorption at an oil±water interface as compared with liposome behaviour.
their initial anchorage. This anchorage provokes an unfolding of the protein leading to the destabilisation of the external layer of the LDL. Then this phenomenon could be followed by a deformation of the particle due to the creation of a neutral lipid lens conducive to the spreading of the LDL constituents. In the case of liposomes, without external proteins, the structure remains steady at the interface and then this structure is not able to adsorb efficiently and to decrease interfacial tension (Fig. 14.5).
14.4 Egg white foams 14.4.1 Formation and stabilisation mechanisms Foam formation is a highly energetic and dynamic process, in which interfacial area is created. The ability of a protein solution, such as egg white, to foam depends on protein structure and conformation, depending themselves on extrinsic factors such as pH, ionic strength, etc. The formation mechanism of globular protein foams can be divided into three phases happening near gas bubbles: protein diffusion towards the air±solution interface, conformation changes of adsorbed proteins, and irreversible rearrangement of the protein film (McRitchie, 1991). Foams are short-lived states and there is any correlation between foam stability and protein adsorption kinetic (Dickinson, 1996). Foam stability, indeed, depends on protein association at the air±solution interface to form a
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continuous intermolecular network. Foam stability is affected by the protein film cohesion, drainage and Ostwald disproportionation.
14.4.2 Interfacial properties of egg white proteins Interfacial properties of egg white proteins are responsible for egg white's excellent foaming properties. Table 14.5 gathers some data on the kinetics of diffusion towards the air±solution interface of three major egg white proteins. Ovalbumin interfacial behaviour is well known, since a large set of data is available about its tensioactivity, adsorption kinetics, interfacial shear and dilatational rheology (de Feijter et al., 1978; de Feijter and Benjamins, 1987; Benjamins and van Voorst Vader, 1992; Benjamins and Lucassen-Reynders, 1998; Damodaran et al., 1998; Lucassen-Reynders and Benjamins, 1999; Pezennec et al.; 2000; Razumovsky and Damodaran, 2001; Croguennec et al., 2007) but also on its structure at the air±water interface (Renault et al., 2002; Lechevalier et al., 2003, 2005; Kudryashova et al., 2003). It is now known that ovalbumin forms a single layer at the air±water interface, whatever its concentration in the bulk (Renault et al., 2002). As for ovalbumin, lysozyme interfacial behaviour has been extensively studied (de Feijter and Benjamins, 1987; Damodaran et al., 1998; Razumovsky and Damodaran, 2001; Kim et al., 2002; Postel et al., 2003; Chang et al., 2005; Roberts et al., 2005; Perriman and White, 2006) as well as its structure at the air±water interface (Lechevalier et al., 2003, 2005). However, its interfacial behaviour differs as lysozyme forms films that are much thicker than a protein monolayer whereas the surface pressure is definitely smaller than the ovalbumin one (Le Floch-FoueÂre et al., 2009). These different behaviours observed on planed air±water interface result in different foaming properties. Ovalbumin foaming properties are much better than those of lysozyme, since in native state at pH 7.0, the foaming capacity of lysozyme is very weak (Townsend and Nakai, 1983), probably because of its little surface hydrophobicity and its rigidity due to its four disulfide bonds. Egg white proteins thus show different behaviour at 2D and 3D air±water interfaces. When they are in mixture, their behaviour is again different. Indeed, Damodaran et al. (1998) showed that the adsorption kinetics of egg white proteins are different depending on whether they are in single protein systems or in mixture. They suggested the formation of electrostatic complexes between positively charged lysozyme and other negatively charged egg white proteins. Moreover, the mixture ovalbumin-lysozyme forms films that are much thicker than those of both proteins in single protein systems, suggesting a synergy in interfacial adsorption between the two proteins (Le Floch-FoueÂre et al., 2007).
14.4.3 Egg white foams Egg white is the reference for foaming properties: compared with other protein ingredient of vegetable or animal origin, it still offers the best foaming properties (Vani and Zayas, 1995; Matringe et al., 1999; Pernell et al., 2002;
Table 14.5
Parameters of the kinetic of diffusion towards the air±solution interface of three major egg white proteins
Parameters
Ovalbumin
Apparent diffusion coefficient (10ÿ10 m2 sÿ1)
0.5 (C=10ÿ4% prot.) (in solution: 0.7)
Ovotransferrine
Lysozyme
Reference
0.2 (C=10ÿ4% prot.) (in solution: 1) 0.15 (C=1.510ÿ4% prot.)
De Feijter and Benjamins (1987) Xu and Damodaran (1993) Pezennec et al. (2000)
2.4 (C=10ÿ4% prot.)
De Feijter and Benjamins (1987) Damodaran et al. (1998) Pezennec et al. (2000) Croguennec et al. (2007)
0.5 to 1 (C=0.1% prot.) Surface concentration (mg mÿ2)
Surface pressure (mN mÿ1)
1.6 (C=10ÿ4% prot.) 1.5 (C=5.410ÿ4% prot.) 2.1 (native protein) to 2.9 (heat-treated protein) (C=0.01% prot.)
0.8 (C=1.210ÿ4% prot.)
1 (C=10ÿ4% prot.) 14 (C=5.410ÿ4% prot.) 24 (C=0.01% prot.)
2.5 (C=1.210ÿ4% prot.)
0.5 (C=0.3510ÿ4% prot.)
1.4 (pH 4) to 3 (pH 11) (C=0.1% prot.)
Perriman and White (2006)
3.5 (C=10ÿ4% prot.)
De Feijter and Benjamins (1987) Damodaran et al. (1998) Pezennec et al. (2000) Roberts et al. (2005)
2.5 (C=0.3510ÿ4% prot.) 8 (pH 5.6) to 14 (pH 11) (C=0.012% prot.) 9 (C=510ÿ4% prot.) to 24.5 (C=0.1% prot.)
Lag phase
YES if C 20 mM) effects during the electrostatic complexation between BSA and pectin at the surface of a quartz crystal microbalance. Such a salt content dependency of complex formation was also described for purely cognate protein±polysaccharide systems (Moss et al. 1997). Hence, it was shown that cartilage lysozyme/chondroitin sulfate complex formation under physiological pH was suppressed in presence of 140 mM NaCl, whereas only 60 mM were sufficient to prevent complex formation between the same protein and hyaluronan. The authors explained that this specific salt dependence of complex formation was at the origin of the ability for mammalian lysozyme to dissociate proteoglycan assemblies in the cartilage. Some studies have considered the interplay between the ionic strength and the critical pHc and pH. The general trend is that for a given protein to polysaccharide ratio, both pH values are generally shifted towards more acidic values in order to compensate the partial screening of the charges induced by the added microions (Grymonpre et al. 2001), i.e., the charge density of proteins needs to be increased so as to reach the same level of charge neutralization between proteins and polysaccharides. For example, pH decreased from 9 to 7 upon addition of 200 mM NaCl in the gelatine/-carrageenan system at a protein to polysaccharide weight ratio of 1:1 (Fang et al. 2006) or from 4.75 to 4.25 in whey proteins/acacia gum mixture at a ratio of 2:1 (Weinbreck et al. 2003b). To conclude this section, it is worth mentioning the very interesting results reported by Weinbreck et al. (2004b) on the importance of the type of ions
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present in the mixture upon control of ionic strength in the whey proteins/carrageenan system. It was shown that addition of sodium chloride was shifting the pH to more acidic pH, as reported for other systems. However, when calcium chloride was used, a marked shift of pH towards more basic pH values (up to pH 8 upon addition of 200 mM CaCl2) could be observed. The given explanation was that the two negatively charged biopolymers were indirectly forming complexes via calcium bridges. Thus, ionic strength dependence for complex formation between protein and polysaccharide might not only be related to salt concentration, but sometimes to the type of ion. After having considered the pH and ionic strength, the two major parameters affecting directly the number of charges on the macromolecules, we will now discuss the effects of mixing ratio that is modifying the balance between the charges at constant pH and ionic strength. Influence of protein to polysaccharide weight ratio Electrostatic complex formation and coacervation can be maximized for a given set of pH and ionic strength conditions when the optimum protein to polysaccharide mixing ratio (Pr:Ps) is found. This experimental evidence has been modelled for a globular protein±flexible polyelectrolyte system using Monte Carlo simulation and it was found that these conditions exactly corresponded to full charge compensation between the two macromolecules (Akinchina and Linse 2002). The optimum ratio can be determined from electrophoretic mobility measurements of the two biopolymers at constant weight concentration or by measuring the turbidity and scattered light intensity of mixtures upon titration (Burgess and Singh 1993; Xia and Dubin 1994; Bowman et al. 1997; Schmitt et al. 1999; Ye et al. 2006). At the complete charge neutralization ratio, the electrophoretic mobility of the complexes converges to zero, whereas the turbidity or scattered light intensity passes through a maximum indicating a maximum mass within the electrostatic complexes and/or a maximum number of formed particles. For protein to polysaccharide ratios below the neutralization point, complexes remained soluble because of insufficient charge neutralization. Interestingly, this was generally traduced by a steady state for the size of the complexes, while the scattered light intensity was still increasing as was reported by Weinbreck et al. (2003b) during whey proteins±acacia gum complex formation. The latter result clearly indicated that before phase separation occurred, an increasing number of similarly sized complexes were formed. Regarding the interplay between the protein to polysaccharide ratio and the two critical pH values, pHc and pH, it is important to note that pHc is independent from the mixing ratio (Mattison et al. 1995; Kaibara et al. 2000). Thus, as soon as protein molecules mix with the polysaccharide ones, soluble complexes start forming, independently from the initial mixing ratio. In contrast, the critical pH value leading to phase separation, pH, is strongly ratio dependent as it corresponds to full charge neutralization of the complexes, or in other words, full saturation of the polysaccharide chains by protein molecules (Kaibara et al. 2000).
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Another important external parameter influencing complex formation and coacervation is the total biopolymer concentration. Influence of total biopolymer concentration Veis et al. (1967) already took this parameter into account in their model. They assumed that once the biopolymer concentration was higher than a critical value, the energetic entropic advantage of forming electrostatic complexes to release counterions disappeared as counterions would be as concentrated in the dilute and in the coacervate phase. This would be the main reason why experimentally, the two-phase region is defined by a finite area in ternary phase diagrams and why complex coacervation can be induced from a monophasic system upon dilution with the solvent (Phares and Sperandio 1964). From the extensive study of a series of protein±polysaccharide complexes, Tolstoguzov (1986) concluded that suppression of coacervation occurred around a total biopolymer concentration of 4 wt%. In fact, it seems that this critical concentration is highly system dependent as, for example, a critical concentration of 4.5 wt% was needed to observe self-suppression of coacervation in the -lg/acacia gum system at pH 4.2 (Schmitt et al. 2000). Very interestingly, the presence of protein aggregates in the same system induced an increase of the selfsuppression concentration from 4.5 wt% up to 20 wt%, suggesting that a control of coacervation self-suppression could be obtained tailoring the size and surface properties of aggregates (Schmitt et al. 2000). In a very close system, whey proteins/acacia gum, but a pH 3.5, it was reported that complexes could be observed up to 15 wt% (Weinbreck et al. 2003b). The likely reason to explain these experimental differences was that the electrostatic entropy gain at pH 3.5 was still advantageous at 15 wt%, but not more around 4.5 wt% at pH 4.2. To conclude on the importance of the total biopolymer concentration, it is worth mentioning that it has been reported to have no effect on pHc or pH for total biopolymer concentrations below 0.5 wt% (Mattison et al. 1995, 1999; Weinbreck et al. 2003b, 2003c). Nevertheless, for higher biopolymer concentration, pH has been reported to shift to higher values as was the case in the whey proteins/acacia gum system (Weinbreck et al. 2003b). Finally, the size of the coacervates was shown to depend on the total biopolymer concentration up to 1 wt% for the -lg/acacia gum system at pH 4.2 (Schmitt et al. 2000). For higher concentration, the coacervate size remained independent of total biopolymer concentration, showing again the importance of the concentration range tested to observe or not effects. Apart from the chemical parameters described above, several physical parameters, such as the temperature, the pressure or the shearing, might influence the formation of protein±polysaccharide complexes or coacervates. Influence of temperature, shearing and pressure Temperature is known to possibly affect conformation of proteins and polysaccharides, but also to favour several non-electrostatic interactions (Tolstoguzov 1997; Schmitt et al. 1998). Low temperature is, in principle,
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Fig. 16.3 Evolution of the binding enthalpy (H) with temperature during the titration of an aqueous dispersion of total acacia gum (1.8 wt%) into an aqueous dispersion of -lg (0.6 wt%) at pH 4.2. Line corresponds to a linear fit of data. The slope represents the heat capacity changes (C p).
favourable to hydrogen bonding whereas higher temperature is favourable to hydrophobic interactions. For example, it can be seen in Fig. 16.3 that the binding enthalpy recorded in the -lg/acacia gum system decreased (became less negative) with increasing temperature. The molar heat capacity, Cp , calculated from the slope of the H vs. temperature relationship, originated from changes in the degree of surface hydration in the free and complexed molecules, and to a lesser extent from changes in molecular vibrations (Jelesarov and Bosshard 1999). The Cp calculated from Fig. 16.3 was large and positive, a typical signature of ionization/charge neutralization reactions (Ziegler and Seelig 2004; GoncËalves et al. 2005; Chung et al. 2007). A positive Cp but with a H parameter remaining favourable at all temperatures studied, i.e. H < 0, would be indicative of a significant contribution of hydrogen bonding (GoncËalves et al. 2005). The importance of hydrophobic interactions in other protein± polysaccharide systems has been shown. In some cases electrostatic binding between macromolecules was impossible unless strong hydrophobic interactions are generated by heating (Zhang et al. 2007). Other interesting results highlighted the effect of temperature on the conformational changes in BSA and the resulting formation of complexes with alginate (Harding et al. 1993). Thus, between 35 and 70 ëC, no complexes were formed at pH 6.8 at an ionic strength of 0.1 M, whereas complexes appeared above 70 ëC. Complex formation was mainly related to the conformational changes of BSA around its denaturation temperature of 55 ëC, so that additional
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hydrophobic groups could be exposed, favouring complex formation. Similar effects of temperature have been reported upon complex building between gelatine and -carrageenan (Fang et al. 2006). Temperature values above the conformational transition of both biopolymers, i.e. > 25 ëC led to complex coacervation, whereas thermodynamic incompatibility occurred below 25 ëC. In a very recent study involving complex formation between two proteins, -la and lysozyme, it was shown that below 5 ëC, precipitates were obtained whereas coacervates were formed if the mixing temperature was 45 ëC (Nigen et al. 2007). This change in the nature of the solubility of the formed complexes was due to a conformational change of the -la at temperatures higher than 27 ëC, enabling adoption of a molten-globule conformational state that was favouring exposure of hydrophobic regions. This study suggested that electrostatic interactions could be mostly important during the initial biopolymer complex formation but large-scale aggregation or coacervation would be mainly driven by hydrogen bonding or hydrophobic interactions, depending on the temperature. In a very recent paper, it was shown that temperature could also affect the structure of the polysaccharide (Kayitmazer et al. 2007). Hence, the effect of temperature (12 or 25 ëC) on the structure of BSA/chitosan was compared to BSA/poly(diallyldimethylammonium chloride) (PDADMAC). It was shown, that even if the two polyelectrolytes had very similar charge densities, different rheological properties were obtained for the coacervate phase, probably due to the fact that temperature had a stronger effect on the flexibility of chitosan, but not on that of PDADMAC. Several studies were related to the effect of temperature on pHc and pH, leading to the conclusion that these critical pH values were not affected as long as electrostatic interactions were mainly involved in the complex formation mechanism (Kaibara et al. 2000; Weinbreck et al. 2004b; Singh et al. 2007). Another possibility to control the formation of protein±polysaccharide complexes is to apply pressure to the system in order to partially denature proteins. For example, Galazka et al. (1999b) reported the formation of ovalbumin/ dextran sulfate electrostatic complexes at low ionic strength and pH 6.5 after high-pressure treatment of the mixture at 600 mPa for 20 minutes. This type of weak electrostatic complex formation upon high pressure treatment was also highlighted when ovalbumin was mixed with -carrageenan. Another important physical parameter influencing more generally the coacervation phenomenon rather than the complexation one is shearing. Most of the studies reported results carried out in non-controlled shearing devices; however, some trends could be discerned. When protein±polysaccharide mixtures were submitted to mixing (below 1000 rpm), the size of the coacervates was generally decreasing with the increase of the shearing rate (Elgindy and Elegakey 1981; Burgess and Carless 1985; Tirkkonen et al. 1994; Ovez et al. 1997; Sanchez et al. 2001). The given explanation was mainly a breakdown of the coavervates due to interfacial destabilization by the shear, as is the case for emulsions, leading to fragmentation of the coacervate phase into smaller
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droplets. For higher mixing rates (3500 rpm), an increase of the coacervate size was reported, probably because complex turbulent flow was favouring recoalescence of the coacervates (Sanchez et al. 2001). Interestingly, an additional effect of the time of shearing was noticed for low shearing rates (below 1000 rpm), as an increase of the coacervate size was demonstrated for constant shearing rate in the case of gelatine/gelatine (Burgess and Carless 1985), -lg/ acacia gum coacervation (Schmitt et al. 1998) or gelatine/chitosan (RemunanLopez and Bodmeier 1996). In these experiments, the shearing applied was likely favouring coalescence of the coacervates or not high enough to prevent it. Two additional internal parameters have been shown to control protein± polysaccharide complex formation, the charge density and the molecular weight of the biopolymers.
16.3.2 Internal parameters Influence of biopolymer charge density The charge density of the biopolymer is defined by the number of charges present for a given distance along the protein or polysaccharide chain. The charge density is of great importance in the type of phase separation obtained in protein±polysaccharide complexes as high charge density (sulphate or phosphate side chains) generally led to precipitates, whereas lower charge densities (carboxylic side chains) led to liquid coacervates (Frugier and Audebert 1994; Weinbreck et al. 2003b, 2003c, 2004b). This effect was attributed mainly to the fact that strong electrostatic interactions induced high compaction of the complexes with a high level of local dehydration of the biopolymer chains, leading to insolubilization in the form of precipitates. Interestingly, high charge density allowed the formation of soluble complexes on a larger range of ionic strengths due to the local strong electrostatic interactions that were able to overcome the screening effects induced by microions (Mattison et al. 1998; Y. Wang et al. 2000). For a given type of side groups, the charge density could lead or not to the formation of complexes as was shown in mixtures of ovalbumin with carrageenans or dextran sulphate at pH 6.5, i.e., above the IEP of the protein (Galazka et al. 1999b). Hence, formation of electrostatic complexes was shown to be possible on the `wrong' side of the IEP of the protein, i.e. when both biopolymers carry opposite charges (Park et al. 1992; Mattison et al. 1998). Such local high charge density regions were named `charges patches'. These patches can be generated on synthetic polyelectrolytes but they can already exist on proteins (de Vries et al. 2003). For example, sequence analysis of the charge distribution on the surface of -lg and -la led to the conclusion that -la could bind electrostatically to acacia gum through a single patch, whereas binding of acacia gum to -lg occurred via several patches (de Vries 2003). This could explain why -la was able to form electrostatic complexes more than one pH unit higher than its IEP (Weinbreck and de Kruif 2003). Such a type of single electrostatic patch has also been described on the surface of human serum
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albumin, allowing complex formation with hyaluronic acid (Grymonpre et al. 2001). Another way to describe this phenomenon is that ion±dipole interaction overcomes ion±ion repulsion. However, another possible explanation would be that when the polyacid/polybase is strong enough or the protein has a high enough regulation capacity, a reversal of charge may be induced on the protein (Bisheuvel and Cohen Stuart 2004). The capacitance is an intrinsic property of a protein defining its ability for charge regulation, i.e., to change their charges upon interaction with a polyelectrolyte. It was shown recently by Monte Carlo simulations and perturbation theory that -la, -lg and lysozyme displayed very strong capacitance at and near their IEP (da Silva et al. 2006). This induces an additional and strong attraction of several kT between proteins and polyelectrolytes through charge-induced charge interactions. A last internal parameter that is closely related to the biopolymer charge density is the molecular weight, which also plays an important role on the resulting biopolymer flexibility (Kayitmazer et al. 2005; Cooper et al. 2006). Influence of biopolymer molecular weight The increase of the molecular weight of the polyelectrolyte or polysaccharide was shown to favour the formation of electrostatic complexes (Zaitzev et al. 1992; sem*nova 1996). The understanding of these observations was that the volume occupied by the polyelectrolyte increased with its molecular weight, enabling a higher number of proteins to interact with it and to build complexes. Here, a very interesting effect of the polyelectrolyte and the charge density was shown when polyacrylic acid was mixed with lysozyme or ovalbumin (Shieh and Glatz 1994). No effect of the increase of the molecular weight of the polyacrylic acid was observed with lysozyme, as both polymers had close charge densities. However, a much stronger binding of ovalbumin was detected when increasing the molecular weight of the polyacrylic acid, due to the larger difference in charge density. Y. Wang et al. (2000) tested coacervation between anionic surfactant micelles and PDADMAC of various molecular weights. They came to the conclusion that a critical molecular weight of the polycation existed for every total polymer concentration and mixing ratio tested. This critical molecular weight was leading to a critical size of 45 nm for the complexes, enabling coacervation to occur. Recently, Laneuville et al. (2005b) reported the influence of the molecular weight of xanthan gum on the size and compactness (fractal dimension, df) of the formed electrostatic complexes. With low molecular weight xanthan gum, smaller complexes with high df (2.56, i.e. compact) were obtained, whereas much larger and more linear complexes (df 2.26) were obtained with the high molecular weight xanthan gum. It is worth noting that several studies have been undertaken on the modelling of the effect of polyelectrolyte molecular weight on the formation of soluble complexes or coacervates (SkepoÈ and Linse 2003; Akinchina and Linse 2003). The main conclusion was that small molecular weight polyelectrolytes generally led to soluble complexes, whereas larger ones induced further aggregation of the soluble complexes leading ultimately to
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complex coacervation. These simulations fitted reasonably with the experiments described above for real systems. After having reviewed the most important parameters controlling the formation of protein±polysaccharide complexes and coacervates, we now turn to understanding their structure formation and morphology.
16.4 Structure, morphology and coarsening of protein± polysaccharide complexes and coacervates The understanding of the structure of protein±polysaccharide complexes and coacervate structure from the molecular to the macroscopic level is probably the topic that has received the most scientific interest during the last decade (Schmitt et al. 1998; Turgeon et al. 2007).
16.4.1 Molecular level Regarding the molecular structure of the protein and the polysaccharide in the complexes and coacervate, most of the information was gained from the use of calorimetry and spectroscopy. For example, Imeson et al. (1977) reported that the denaturation temperature of BSA or myoglobin was increased upon mixing with anionic polyelectrolytes, and concluded that complexes were formed, leading to increased protein heat stability. In another study, the content of helix structure of ribulose diphosphate carboxylase was followed by circular dichroism upon complexation with pectins close to the IEP of the protein (Braudo and Antonov 1993). It was shown that complex formation was leading to a loss of -helix in the protein, enabling identification of which regions of the protein were involved in the interaction. This loss of -helix structure was also reported upon complexation of -lg with acacia gum, especially close to the EEP (Schmitt et al. 2001a; Mekhloufi et al. 2005). The likely explanation for this observation was that this region of the protein was rich in positively charged amino acids at the given pH, but also that these amino acid residues were exposed to the solvent at the surface of the protein. The same -helical region with high charge density of -lg was identified in the interaction with pectin (Girard et al. 2003a). In addition, a loss of helical structure was also reported upon complex formation between lysozyme and polystyrene sulfonate by means of Fourier transform infrared spectroscopy (Cousin et al. 2005). Nevertheless, depending on the structure of the protein, complex formation may also lead to an increase in -helix as, for example, upon complex formation between poly(Llysine) and -carrageenan (Girod et al. 2004). In the same vein, helical regions of -gliadin proteins were mainly involved in the complex formation with gum arabic, whereas -sheets were involved when pea globulin protein was tested (Chourpa et al. 2006). In another study dealing with complex formation/ coacervation processes in -lg/acacia gum system, a loss in the amount of helix protein structure after complexation and a gain in protein secondary
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structure after complex formation were shown (Mekhloufi et al. 2005). By contrast, Delben and Stefancich (1998) reported no structural changes upon complex formation between ovalbumin and glutamate glucan, which might be due to the compact structure of this protein. It seems obvious that no generalization can be drawn from these different results since both protein conformational changes and unchanged states are described in the literature. Interestingly, not only could the molecular structure of the protein be modified upon complex formation, but also that of the polysaccharide. For example, the helical structure of - and -carrageenans was shown to be completely lost upon interaction with -casein as more protein was bound to the polysaccharide chain (Burova et al. 2007). This was an indication that the electrostatic interactions between the two biopolymers were preventing the formation of the secondary hydrogen bonds that are responsible for the helical structure of the bare polysaccharide. Also conformational changes affecting a potassium pectate upon complexing with two enantiomeric forms of poly(lysine) were studied (Paradossi et al. 2001). The pectate adopts a super-helical conformation around the -helix of the poly(L-lysine) but not with poly(Dlysine), indicating a stereo-specificity of the interaction of poly(lysine) with pectate.
16.4.2 Mesoscopic level This structural level is related to the soluble complexes or soluble complex aggregates arising from the charge neutralization of the two biopolymers. Here, it should be mentioned that a number of experimental data have been reported on protein±polyelectrolyte systems, but that still scarce data are available on protein±polysaccharide complexes (Turgeon et al. 2003). However, interesting molecular simulations carried out on model polyelectrolytes/macroions complexes with different chain length, flexibility, charge or mixing ratio led to several possible structures ranging from linear to globular complexes (de Vries and Cohen-Stuart 2006; Ulrich et al. 2006). In terms of size, hydrodynamic diameters reported from light scattering experiments on various protein/ polysaccharide systems varied between 100 and 300 nm (Grymonpre et al. 2001; Weinbreck et al. 2003b; Mekhloufi et al. 2005; Schmitt et al. 2005b). An example of aggregated complexes based on the interaction between -lg and acacia gum is shown in Fig. 16.4. An interesting feature related to the structure of the electrostatic complexes close to the EEP was that the polydispersity index measure by light scattering was very low (i.e. below 0.1), indicating a very hom*ogeneous composition of all the complexes before macroscopic phase separation (Mekhloufi et al. 2005; Schmitt et al. 2005b). The internal structure of protein±polysaccharide complexes has been investigated by means of in situ acidification (GDL) coupled with time resolved static light scattering for the -lg/acacia gum, -lg/pectin and -lg/xanthan gum (Girard et al. 2004; Mekhloufi et al. 2005; Laneuville et al. 2006). Results showed that more or less compact fractal aggregates were formed
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Fig. 16.4 (a) Cryo-TEM micrographs of -lg/acacia gum aggregated complexes obtained at pH 4.0 and a protein to polysaccharide weight ratio of 2 : 1; (b) structural details of the bottom left particle after FFT filtering.
depending on the type of polysaccharide used (flexible, like acacia gum, or rigid, like xanthan gum). For acacia gum or pectin, fractal dimensions (df) ranging between 1.5 and 2.2 were found, indicating linear complexes. In the case of the xanthan gum, diffuse aggregates with df 1.8 reorganised in order to form more compact aggregate complexes with df 2.3. Interestingly, when the -lg/ xanthan system was submitted to shear, even more compact aggregates with df 2.53 were built for the same protein to polysaccharide ratio of 5:1 (Laneuville et al. 2005b). In a recent paper dealing with complex formation between lysozyme and polystyrene sulfonate with various chain lengths, small-angle neutron scattering experiments showed that three types of complexes could be formed, leading to gelled or to liquid three-dimensional structures depending on the lysozyme to PSS ratio and the PSS chain length (Cousin et al. 2005). The same group showed that when the charge ratio between the lysozyme and the PSS was close to one, the system packed into dense spheres with a radius of about 10 nm and a df 2.1 as shown by SANS and freeze-fracture microscopy (Gummel et al. 2006, 2007b). Interestingly, Leisner and Imae (2003) reported the aggregation of soluble complexes in a poly(L-glutamate)/synthetic dendrimer system into fractal ones to end up with a three-dimensional network characterizing the coacervate phase. This paper showed that the structure of the coacervate phase was very similar to that of dense aggregated complexes. In a more recent paper considering complexes built between two oppositely charged synthetic polymers, atomic force microscopy investigation allowed getting a very clear insight on the structure of the complexes adsorbed onto a mica surface (Kiriy et al. 2006). It was shown that depending on the mixing ratio, complexes were able to rearrange leading to more or less compact morphologies. The more compact ones were obtained for electrostatic neutrality. The next section considers the structural features of the coacervate phase.
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16.4.3 Microscopic level At this level of structure, liquid droplet of coacervates originating from aggregation of electrostatic complexes with various levels of hydration can be observed using light microscopy (Mekhloufi et al. 2005). However, before discussing the morphology and the coarsening of the coacervates, several studies have considered their internal structure. Upon investigation of the diffusion coefficient of protein in the BSA/PDADMAC coacervate using dynamic light scattering, two different regimes were found, indicating that the coacervate phase could behave like a heterogeneous network of polyelectrolyte chains where proteins could diffuse (Bohidar et al. 2005). Heterogeneity of structure was also reported in the -lg/pectin coacervates at various protein to polysaccharide ratios and ionic strengths (Wang et al. 2007b). It was concluded that the coacervate could be viewed as a network of pectin chains bound together electrostatically by protein molecules. In excess of proteins, some aggregates tended to form in the network. Similar description of the coacervate as a dense phase (70% of water content) was given for the whey proteins/acacia gum system after 48 hours upon small angle X-ray scattering (Weinbreck et al. 2004d). In this case, it was shown that increasing ionic strength or pH led to less hom*ogeneous structure because the strength of the electrostatic interactions between the acacia gum chains and the whey proteins was reduced. Diffusivity measurements of the whey proteins were carried out on the same system using NMR and fluorescence recovery after photo-bleaching (FRAP), leading to the conclusion that whey proteins were free to move within the coacervates, but their diffusion coefficient was reduced in conditions where electrostatic interactions were maximum (Weinbreck et al. 2004c). Recently, the meso/ microstructure of the BSA/PDADMAC coacervate phase has been probed by FRAP (using interacting and non-interacting fluorescent probes), light scattering and cryo-TEM (Kayitmazer et al. 2006). It was shown that the coacervate was composed of dense domains concentrated in protein and polyelectrolyte and more dilute domains, leading to the different diffusion coefficients that were reported previously. It is interesting to observe in Fig. 16.4 that the inner structure of the complexes is heterogeneous with regions having differing affinities for electrons, the structural pattern being close to those observed after polymer spinodal decomposition. This section dealing with the structure of protein±polysaccharide complexes will finish with some evidence on the coacervate coarsening mechanism and some morphological properties of the coacervates.
16.4.4 Coarsening mechanism and macroscopic level The charge neutralization and aggregation of protein±polysaccharide complexes leads to the formation of liquid droplets ranging from hundreds of nanometres to several microns in diameter (Kaibara et al. 2000; Schmitt et al. 2000, 2001b). These coacervates tend to coalesce to form ultimately a dense liquid phase, as mentioned previously. The time coarsening of a -lg/acacia gum system was
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Fig. 16.5 Confocal scanning laser micrograph of aggregate-free -lg/acacia gum mixture at pH 4.2 and 1 wt% total biopolymer concentration after 3±5 min at a mixing ratio of 1 : 1. Scale bar is 5 m. From Schmitt et al. (2001b) with permission of Elsevier.
followed by static light scattering and confocal scanning laser microscopy (CSLM) after labelling of the two biopolymers (Sanchez et al. 2002). It was clearly seen that the initially formed small coacervates tended to coalesce, forming large structures characterized by solvent vacuoles, leading to the socalled sponge structure that can be seen in Fig. 16.5 for the -lg/acacia gum mixture (Menger et al. 2000; Schmitt et al. 2001b). The phase separation kinetics could be scaled with a nucleation and growth model, where initial biopolymer concentration fluctuations induced by electrostatic complex formation lead to hydrodynamically driven coalescence and formation of sharp interfaces delimiting the coacervate phase from the dilute phase (Sanchez et al. 2006). The coalescence of the coacervates was already reported by Bungenberg de Jong et al. (1940) for the gelatine/acacia gum system. It can be explained by the reduction of the interfacial energy of the system because of the appearance of interfacial viscoelasticity in the phase concentrated in biopolymers. One should recall here that the coacervate phase is mainly liquid in nature as in contains around 70% water (Schmitt et al. 2000; Weinbreck et al. 2004d). The coalescence phenomenon was shown to occur very quickly (few minutes), especially in optimum conditions of pH and protein to polysaccharide ratio (Schmitt et al. 2001b). Over time, the coacervates tend to expel the water in order to increase their free energy as demonstrated by the transition from a turbid system (because of light scattering from the water vacuoles) to a clear viscous and dense phase (Weinbreck et al. 2004c).
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16.5 Functional properties of protein±polysaccharide complexes and coacervates The formation of protein±polysaccharide complexes can potentially lead to different functional properties, compared to the two biopolymers taken individually (Schmitt et al. 1998; McClements 2006). This is generally due to a synergistic combination of the functional features of both the protein (generally hydrophobic and/or hydrophilic and globular) and the polysaccharide (generally hydrophilic and branched). In this section, the solubility and rheological properties of complexes and coacervates will be discussed, followed by interfacial properties at the oil/water and air/water interfaces. 16.5.1 Solubility and rheological properties These two functional properties are intrinsically linked to the extent of interaction between the complexes and coacervates with water. Solubility The solubility of proteins was shown to be improved upon complex formation with polysaccharide, especially close to their IEP. This property was initially used in systems composed with caseins, in order to induce colloidal stability in acidic dairy products (Payens 1972; Ambjerg Pedersen and Jorgensen 1991; Matia-Merino et al. 2004). The physical explanation for this stabilizing effect was given by Tuinier et al. (2002), who demonstrated the electrosorption of pectins onto casein micelles upon pH decrease (renneting). The formation of these pectin multilayers at the surface of the casein micelles was able to prevent strong protein aggregation mediated through hydrophobic interactions. This balance between hydrophobic attractions leading to protein low solubility and electrostatic repulsions leading to solubility was also considered upon complex formation between carboxymethylcellulose (CMC) with lysozyme or ovalbumin (Clark and Glatz 1991). Lysozyme, characterized by a high charge density, was able to form a complex with CMC, preventing further aggregation close to IEP. This was not the case for ovalbumin that had a lower charge density and a larger number of hydrophobic regions. Interestingly, several papers have reported that the formation of a complex with a polysaccharide was also preventing protein association upon heating that is known to strongly promote hydrophobic interactions (Shalova et al. 2005; Chung et al. 2007; Mounsey et al. 2008). In this case, the formation of the complex between the polysaccharide and the unfolded proteins prevented the aggregation process leading to the formation of large insoluble protein aggregates. Obviously, an important parameter controlling the solubility of the protein± polysaccharide complexes is the initial mixing ratio at a given pH and ionic strength. Incomplete charge neutralization led to the formation of soluble complexes, improving protein solubility by electrostatic repulsions or steric effects. In general, these soluble complexes were obtained close to the phase separation boundaries of phase diagrams (Schmitt et al. 2000; Weinbreck et al.
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2003b; Chung et al. 2007; Plashchina et al. 2007; Feng et al. 2007; Jourdain et al. 2008). For example, complex formation between -lg and sodium alginate at pH 5.0 was shown to induce a protein solubility of 70% when 0.75 M of sodium alginate was added to 0.5 M -lg (Harnsilawat et al. 2006a). The resulting soluble complexes were characterized by a size of around 80 nm and a -potential of ÿ40 mV, leading to strong electrostatic repulsions. Lastly, it should be noticed that for some protein purification purposes, protein± polysaccharide complex insolubility could be preferred, but this will be discussed in detail in Section 16.6 (Hansen et al. 1971; Strege et al. 1990). The formation of electrostatic complexes, and further coacervates, may induce changes in the viscosity and rheological properties of the protein±polysaccharide mixtures. Rheological properties In order to get a clear picture of the rheological properties of protein± polysaccharide complexes, this section will consider first the flow behaviour of the coacervate phase under shear, then the viscoelastic properties under small deformations and finally the gelling properties induced by temperature. The flow behaviour of the coacervate phase has been investigated for several systems and the common finding was that the flow could be described as shear thinning, i.e., the viscosity decreased when the shear rate increased to values higher than 20±30 s-1 (Burgess and Singh 1993; Weinbreck et al. 2004e; Malay et al. 2007). This result clearly indicated that the coacervate was a structured phase, whose structure could be modified when submitted to a mechanical stress. Interestingly, most of the flow behaviour of the coacervate could be related to the strength of the electrostatic interactions between the two biopolymers. Weinbreck and co-workers (2004d) reported that the viscosity of a whey protein/acacia gum coacervate phase was the highest at the EEP and was almost linearly increasing with the absolute value of the product of the potential of the two biopolymers for a given pH. A remarkable flowing feature of the coacervate phase in this case was that the system was able to recover its initial viscosity at low shear rate when submitted to a flow hysteresis. However, the authors reported that the coacervate phase took some time to reorganize after deformation, which might be an indication for some diffusion controlled events, such as alternative formation/disruption of electrostatic interactions between the whey protein molecules and the acacia gum chains. If the formation of an electrostatic network between protein and polysaccharide within the coacervate phase has already been reported for gelatine-based systems (Muchin et al. 1978), it could be proven for globular proteins by submitting the system to small oscillatory deformations (Bohidar et al. 2005). It was shown that an elastic (G0 ) and a viscous shear modulus (G00 ) could be measured in the BSA/PDADMAC system. Similar behaviour has been reported for the -lg/acacia gum coacervate, where again a frequency dependence for the two shear moduli was reported, indicating that some rearrangements were occurring at various frequencies (Schmitt et al. 2005a).
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The formation of the weak electrostatic network in the coacervate phase was also demonstrated from the ionic strength dependence of G0 in the -lg/pectin system, as it was shown that a critical value of 200 mM NaCl was screening the charges between the two biopolymers (Wang et al. 2007a). In this same study, it was found that the elastic modulus developed by the coacervate phase was also dependent on the protein to polysaccharide ratio, as was the case in -lg/xanthan gum (Laneuville et al. 2006) or pectin/poly-L-lysine systems (Marudova et al. 2004). It should also be mentioned that complex formation between globular proteins and polysaccharide could be used to produce thermogels with various viscoelastic properties (Mann and Malik 1996; Cai and Arntfield 1997). For example, heat treatment of -lg/CMC coacervate led to a viscoelastic particulate network after heat denaturation of the protein at 90 ëC (Capitani et al. 2007).
16.5.2 Adsorption at oil/water interface and emulsifying properties Due to the dual hydrophobic/hydrophilic nature of protein±polysaccharide complexes, their functionality at the oil/water interface and related emulsifying properties has been the purpose of most of the studies reporting on protein± polysaccharide functionality during these last 20 years (Gurov and Nuss 1986; Dickinson 1998; Benichou et al. 2007c). The complex formation between protein and polysaccharide was shown to affect the adsorption behaviour of the protein at the oil/water interface, but also the resulting interfacial tension. Ganzevles et al. (2007a) reported that the adsorption rate at the oil/water interface for -lg/pectin complexes was lower than for the protein alone. This difference was explained by the larger hydrodynamic radius of the complexes compared to the protein, leading to a lower diffusion coefficient in the bulk. It was confirmed by the subsequent increase of the surface tension drop rate close to that of the protein when salt was added to the system. Nevertheless, the equilibrium surface tension value reached at the equilibrium was similar for the -lg and for its complexes with pectin. Interestingly, it seemed that the lower surface adsorption rate due to diffusion was not a general rule and might be modulated by the intrinsic properties of the biopolymers. For example, a faster adsorption rate of pea globulin/acacia gum coacervates to the vaselin/oil interface was reported compared to pea globulin (Ducel et al. 2005b). Such variability in the results could be explained by the lower surface hydrophobicity of -lg compared to pea globulin, leading to a larger favourable adsorption energy for the latter complexes. These differences in the protein structure on the decrease of surface tension was clearly demonstrated for the same protein, Faba pea legumin, comparing the intact protein and a partially hydrolyzed fraction that was more hydrophobic after complex formation with chitosan (Plashchina et al. 2001). Both types of complexes were able to lower significantly the interfacial tension depending on the protein to polysaccharide ratio, but complexes with hydrolysed legumin reduced it by more than two times compared to intact protein. The reason
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probably resided in an optimal conformation of the protein at the interface, exposing the hydrophobic regions to the interface, while interacting with the chitosan chains on the bulk side, as also proposed previously for the -lg/pectin system (Ganzevles et al. 2007a). Regarding the interfacial rheology of the adsorbed films composed by protein±polysaccharide complexes, these were generally characterized by an increased surface shear viscosity, indicating formation of a dense and viscous phase at the interface of the oil droplets (Dickinson and Galazka 1992). This interfacial network formation was also highlighted from interfacial dilational measurements, where it was shown that compact layers (high dilational modulus) were formed at protein to polysaccharide ratios close to electroneutrality of the -lg/pectin complexes, whereas more diffuse layers were formed (low dilatational modulus) if the complexes were initially soluble (Ganzevles et al. 2007a). Similar behaviour was reported for a given ratio, but different pH values, in the pea globulin/acacia gum system, where the higher oil/ water interfacial elasticity was obtained for neutral complexes (Ducel et al. 2005b). It should be noticed that several studies proposed not to adsorb complexes at the oil/water interface, but to create the complexes directly at the interface after the protein has been adsorbed, using the so-called layer-by-layer method (McClements 2006). These two methods led to very different physical properties of the emulsions, especially in terms of stability, and we will review some of them in the next paragraph (Garti et al. 1999; Jourdain et al. 2008). The use of BSA/dextran sulfate complexes at a 1:3 mixing ratio was shown to improve greatly the stability of a n-tetradecane 10 wt% emulsion with no changes in size distribution for more than 3 weeks, compared to the protein stabilized emulsion (changes in size distribution after 24 hours) (Dickinson and Galazka 1992). Similar improvement of emulsion stability was described when complexes of Portulaca oleracea gum/casein complexes formed at pH 3 were used (Garti et al. 1999). It seems important to note here that the protein to polysaccharide ratio played a major role in the stabilization of emulsions stabilized by protein±polysaccharide complexes formed in the bulk vs. those formed at the interface. Hence, complexes should not be formed in charge neutralization conditions, in order to maintain sufficient electrostatic repulsions between the oil droplets. In addition, the polysaccharide should not be in excess in order to avoid oil droplets bridging flocculation. These conditions of emulsion stability were clearly demonstrated in a study involving whey protein isolate/ chitosan complexes (Laplante et al. 2006). Hence, for protein/polysaccharide ratios leading to a reduction of the interfacial net electrostatic repulsions, flocculation and syneresis of the emulsion were observed. Very similar results have been reported for whey protein/pectin stabilized emulsions (Neirynck et al. 2007). For the study involving chitosan, it should, however, be added that even if the increase of the ionic strength led to a similar reduction of interfacial charge of the droplets at a given ratio, emulsion stability could still be achieved; the likely reason being that in these conditions, more chitosan was adsorbed in the
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interfacial layer, inducing steric stabilization of the droplets (Laplante et al. 2006; Speiciene et al. 2007). Recently, Jourdain et al. (2008) showed that emulsions prepared with already formed caseinate/dextran sulfate complexes (mixed emulsions) were more stable regarding pH lowering compared to layerby-layer emulsions, which could be explained by the different interfacial structures formed at the interface in both cases. In the layer-by-layer case, the interface was probably composed by a protein layer onto which dextran sulfate adsorbed electrostatically, whereas in the case of mixed emulsion, DS formed a more compact layer together with the casein molecules and was therefore less prone to desorption at low pH. The formation and stability of layer-by-layer emulsion have been extensively studied by the group of Prof. McClements, using several protein±polysaccharide pairs, and even multilayers (Guzey and McClements 2006b). Nevertheless, Dickinson and Pawlowsky (1997) already reported that it was possible to produce a network of polysaccharide stabilized oil droplets emulsified with BSA upon controlling the quantity of -carrageenan added to the emulsion. Lower and higher polysaccharide concentrations led to depletion flocculation or aggregation of the oil droplets. Very similar results were reported for whey proteins/carrageenan stabilized emulsions (Singh et al. 2003). The creaming stability of layer-by-layer emulsions stabilized by -lactoglobulin/pectin or -lactoglobulin/ -carrageenan towards pH and ionic strength environmental stresses was shown to be improved by secondary emulsification by ultrasound or secondary emulsification in an -carrageenan dispersion (Guzey et al. 2004; Gu et al. 2005a, 2005b). Both techniques allowed creation of protein±polysaccharide multilayers at the oil droplet interface. Interestingly, these results led to the design of multilayered emulsion composed by the superimposition of several protein±polysaccharide layers, such as, for example, -lactoglobulin/carrageenan/gelatine, each layer being able to respond specifically to an environmental stress, such as, for example, pH change, ionic strength increase or temperature change (Gu et al. 2005b). This latest interfacial engineering might be useful for the development of functional capsules to be used in food applications, and will be discussed in the next section. We now turn to a review of the air/water interfacial and foaming properties of protein/polysaccharide complexes and coacervates.
16.5.3 Adsorption at air/water interface and foaming properties The ability of protein±polysaccharide electrostatic complexes to stabilize the air/ water interface was reported early for the whey protein/carboxymethylcellulose system (Hansen and Black 1972). Thus, these complexes exhibit air/water surface activity due to their amphiphilic nature, as was the case for oil/water interface stabilization. It was shown that complexes obtained between BSA and -carrageenan were able to lower the air/water interfacial tension, to the same extent as BSA alone, but at a reduced adsorption rate (Dickinson and Pawlowsky 1997). This delay in interfacial adsorption might be related to the reduced
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diffusion coefficient of the complexes compare to the protein alone. This hypothesis was confirmed for the -lg/pectin system (Ganzevles et al. 2006a). It was shown that the rate of surface pressure increase was lower for conditions where more protein±polysaccharide complexes were formed, i.e., for lower pH values or high protein±polysaccharide ratios. An interesting feature of this system was also that pectins with a lower degree of esterification (more negative charges) were delaying more importantly the interface adsorption because of the size of the complexes formed compared to highly esterified pectins (Ganzevles et al. 2006a). Similar observations of lower diffusion at the air/water interface were made upon adsorption of ovalbumin/pectin complexes at acidic pH (Kudryashova et al. 2007). Results on adsorption kinetics might be triggered by the molecular characteristics of the polysaccharide. Hence, complexes of -lg with the globular polysaccharide acacia gum were able to lower interfacial tension as fast as the protein alone (Schmitt et al. 2005a). This might be due to the more compact structure of these complexes compared to pectin-based ones, pectin molecules being more linear biopolymers than acacia gum. This hypothesis was tested recently on a system composed by -lg interacting with pullulan grafted with carboxyl group leading to charge densities ranging from 0.1 to 0.61 (Ganzevles et al. 2007b). It was shown that the adsorption kinetics were lowered for the -lg/pullulan complexes having the highest charge densities, which in turn led to the soluble complexes with the largest hydrodynamic radius. The interfacial properties of the protein±polysaccharide films were shown to be strongly dependent on the way complexes were formed. Complex formation at the interface led to dilational modulus up to two times higher than for complexes adsorbed from the bulk (Ganzevles et al. 2006b). The authors explained that, in the first case, pectin molecules were able to reinforce the existing adsorbed protein layer, whereas in the second case, protein adsorbed less easily at the interface due to its interaction with the pectin chains. Interestingly, it was shown that the surface shear modulus was independent of the protein±polysaccharide ratio for the already adsorbed protein layer, but was maximized when complexes formed in the bulk reached electroneutrality. Neutral -lg/acacia gum complexes were also shown to increased interfacial dilational viscoelasticity compared to the protein alone (Schmitt et al. 2005a). This led also to thicker interfacial films with reduced gas permeability and increased lifetimes (Schmitt et al. 2005a; Liz et al. 2006). Schmitt et al. (2005b) reported the importance of possible rearrangements of -lg/acacia gum complexes at the interface on the stability of the resulting air/bubble and foams. Adsorption of neutral complexes led to thick viscoelastic hydrated films with low gas permeability, whereas already formed coacervates led to thin elastic films with lower water content and higher gas permeability. Figure 16.6 clearly shows the formation of a thick interfacial layer composed of -lg/acacia gum complexes having rearranged into a coacervate phase at the interface of air bubble within foam at the EEP. Interestingly, flowing isolated coacervates could be seen in the Plateau border (due to gravitational drainage), but were not
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Fig. 16.6 Confocal scanning laser micrograph of aqueous foam stabilized by fluorescently labelled -lg/acacia gum complexes and coacervates at a total biopolymer concentration of 1 wt% at pH 4.2 and a protein to polysaccharide ratio of 2 : 1. Arrows indicate interfacial layer stabilized by fused coacervates and free coacervates draining in the thin-film. Scale bar is 50 m. Copyright Nestle Research Center, Lausanne.
integrated into the interface. Ganzevles et al. (2007b) also reported that varying the charge density of the pullulan (by grafting of carboxylic groups) upon complex formation with -lg led to various interfacial shear moduli at the air/ water interface. In this case, the polysaccharide was controlling the protein conformation at the interface through its charge density by the formation of more or less compact complexes. Several studies reported the improved foam stabilization properties of protein±polysaccharide complexes and related the improvement compared to the single protein mainly to a combined effect of interfacial properties but also from the local increase of the bulk viscosity in the thin-films due to the formation of the complexes (Poole 1989; Ahmed and Dickinson 1991; Glaser et al. 2007). As was shown above, protein±polysaccharide complexes exhibit a variety of functional properties enabling their use in several food and non-food applications (Samant et al. 1993; Schmitt et al. 1998; Cooper et al. 2005), and these are now discussed in Section 16.6 and 16.7, respectively.
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16.6 Food applications of protein±polysaccharide complexes and coacervates Some food and non-food applications of protein±polysaccharide complexes and coacervates will overlap (protein separation, microencapsulation). Nevertheless, in order to give a clearer overview of both fields, these applications will be considered separately. In this section, we consider the use of complex and coacervate formation for protein purification, microencapsulation, production of fat substitutes and texturization in food products. In the last subsection, some other food applications will be reviewed. 16.6.1 Protein purification The differences in the IEP of the various food proteins allow recovering them selectively by complex formation with polysaccharides upon proper adjustment of the environmental conditions such as pH, ionic strength and protein to polysaccharide ratio. This purification method offers the advantage of not using organic solvents. In addition, it enables the re-use of the polysaccharide and allows large-scale purification processes. The main drawback is that it is mainly a batch process, unless the polysaccharide can be immobilized on a support. Several studies reported on the extraction of the major whey protein fractions upon complex formation with carboxymethylcellulose (CMC) in acidic conditions (Hansen et al. 1971; Hill and Zadow 1974). For example, it was shown that BSA and -lg could be specifically removed from whey at pH 4.0 using CMC. Further decrease of the pH to 3.2 enabled extraction of -la, the remaining soluble fraction being composed mainly of proteose peptones (Hidalgo and Hansen 1971). Similar results could be obtained using apple pectin that allowed recovery of 90% of all the whey proteins at pH 3.4, but with no selectivity (Serov et al. 1985). Whey protein recovery could also be achieved in basic pH conditions when chitosan was used for complex formation. For example, up to 90% of -lg could be removed from cheese whey upon complex formation with chitosan at pH ranging from 8 to 10 and ionic strengths ranging from 0.08 to 0.3 M (Montilla et al. 2007). Interestingly, a variety of other proteins (e.g., soy or potato proteins) could be fractionated using the same principle (Smith et al. 1962; Vikelouda and Kiosseoglou 2004). For example, an efficient and simple technique for isolation of immunoglobulins Y (IgY) from egg yolk by anionic polysaccharides was developed (Chang et al. 2000). These proteins can be used as immunological supplements in infant formulae and other foods. Isolation conditions of IgY in egg yolk were optimized by the addition of various levels of Na-alginate, -carrageenan, Na-CMC, and pectin to diluted egg yolk. It was found that the pH value of the polysaccharide±yolk mixture affected both the quantity of polysaccharide-lipoprotein precipitates and the immunoactivity recovery of IgY. The IgY recovery was determined to be 33±74% by means of single radial immunodiffusion method when IgY was isolated under optimal conditions. Among the polysaccharides tested in this
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study, the use of 0.15% pectin exhibited the best immunoactivity recovery of IgY at pH 5.0.
16.6.2 Microencapsulation In food applications, microencapsulation can be used in order to protect food bioactive molecules that are sensitive to external stresses such as pH, oxygen content or temperature (Table 16.2). For example, Oliveira et al. (2007) evaluated the resistance of microcapsules containing Bifidobacterium lactis and Lactobacillus acidophilus, produced by complex coacervation using a casein/ pectin system, to the spray drying process, a shelf-life of 120 days at 7±37 ëC and the in vitro tolerance after being submitted to acid pH (pH 1.0 and 3.0) solutions. The process used and the wall material were efficient in protecting the micro-organisms; however, microencapsulated Bifidobacterium lactis lost its viability before the end of the storage time. Other important uses could be to provide a controlled release of aroma or flavours before or upon consumption of the food or even to mask the bad taste of some biologically active material (Gouin 2004). Any type of triggers can be used to prompt the release of the encapsulated ingredients, such as pH changes, mechanical stress, temperature, enzymatic activity, time, osmotic force, etc. In the case of probiotics, the use of microencapsulation for controlled-release applications is a promising alternative to solving the major problems of these organisms that are faced by food industries (Anal and Singh 2007). The probiotics are protected from bacteriophage and harsh environments, such as freezing and gastric solutions and their encapsulated form can be used in many fermented dairy products, such as yoghurt, cheese, cultured cream and frozen dairy desserts, and for biomass production (Anal and Singh 2007). Yeo et al. (2005) used temperature as a trigger to controlled release of flavour oil to improve the appeal of frozen baked foods upon heating. They have encapsulated flavour oil in complex coacervate microcapsules using gelatine and gum arabic. The oil remained encapsulated for 4 weeks of storage at 4 and ÿ20 ëC (freezing and thawing) but was released by exposure to 100 mM NaCl at room temperature. When particles were cooled after releasing their oil content, the oil was re-encapsulated. It is worth mentioning that two methods of encapsulation can be followed for food applications. Interfacial complex coacervation or layer-by-layer deposition around an oil phase can be used. This technique generally leads to microcapsules having diameters ranging from 1 to 50 m (Lamprecht et al. 2001; Weinbreck et al. 2004a). Alternatively, complexes can be formed at the surface of a protein or polysaccharide matrix, leading to sub-micrometer particles (Chen and Subirade 2005; Hong and McClements 2007). In both cases, the oil phase and the matrix contain the molecule(s) of interest. In the case of oil droplet encapsulation, besides the fact that pH, ionic strength, protein to polysaccharide ratio or total biopolymer concentration must be optimized, an important subsequent processing step is to stabilize the
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Table 16.2 Examples of protein±polysaccharide systems used for microencapsulation purposes in food and non-food applications Systems Food applications Whey protein isolate/ xanthan gum, /fenugreek gum Whey protein isolate/ xanthan gum, /galactomannans 2-gliadin/, pea globulin/ acacia gum Gelatine/acacia gum
Whey protein/gum arabic Gelatine/gum arabic
Technique/Encapsulated material
References
Oil in water in oil emulsion/ Medium chain trigycerides/ triacetin, flumethrinÕ Water in oil in water emulsion/ Medium chain triglycerides, vitamin B1 Oil in water emulsion/Medium chain triglycerides Oil in water emulsion/ Eicosapentaenoic acid ethyl ester (EPA) Oil in water emulsion/Lemon and orange oil flavour Oil in water emulsion/10% natural oils from anise, oregano, garlic, black pepper in 90% vegetable oil
Benichou et al. (2007a)
Non-food applications Gelatine/acacia gum Oil in water or water in oil emulsion using microchannels/Soybean oil Gelatine/gum arabic Oil in water emulsion/Paraffin oil Alginate/poly-L-lysine Aqueous coacervation and CaCl2 reticulation/Erythropoietin-secreting cells Gelatine/acacia gum Oil in water emulsion/ Methoxybutropate Gelatine/sodium Oil in water emulsion/Dalarelin alginate Gelatine/acacia gum Oil in water or water in oil emulsion/Clove oil, sulphamethoxazole Gelatine/acacia gum/ Oil in water emulsion/Capsaicin tannins Gelatine/acacia gum Oil in water microemulsion/ Cypermethrin
Benichou et al. (2007b)
Ducel et al. (2004) Lamprecht et al. (2001)
Weinbreck et al. (2004a) Yeo et al. (2005)
Nakagawa et al. (2004) Mayya et al. (2003) Murua et al. (2007)
Palmieri et al. (1999) Ryszka et al. (2002) Thimma and Tammishetti (2003) Xing et al. (2003) Zhu et al. (2005)
interface against pH and ionic strength changes to avoid capsule disruption. Hence, it has been clearly shown that unstabilized microcapsules were much more sensitive to leakage or oxidation than crosslinked ones (Weinbreck et al. 2004a). If this can be achieved by formation of covalent bonds between protein and polysaccharide by use of glutaraldehyde for non-food applications, other biocompatible means of crosslinking have been developed for the food industry.
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Some authors described the formation of multilayers around the oil droplets, each layer being sensitive to different environmental stresses (Harnsilawat et al. 2006b). Another approach could be to use food grade solvents (ethanol) or spray drying to dehydrate the interfacial coacervates as has been shown for gelatine/ acacia gum capsules loaded with -3 unsaturated fatty acids (Lamprecht et al. 2001). Interestingly, it was recently shown that the use of glycerol to crosslink gelatine/acacia gum capsules loaded with sesame oil extract led to similar results to capsules crosslinked with formaldehyde (Huang et al. 2007). An alternative solution is to use an oxidizing agent such a dihydroascorbic acid or even `foodgrade' crosslinking enzymes (Weinbreck et al. 2003a; Strauss and Gibson, 2004; Littoz and McClements 2008). The obtained microcapsules can thereafter be introduced in a food matrix and release aroma and bioactives, as has been shown for limonene-loaded whey protein/acacia gum capsules in Gouda cheese (Weinbreck et al. 2004a). When a protein or a polysaccharide matrix is used, stabilization can be achieved by subsequent heat treatment of the system. This promotes strong hydrophobic interactions in addition to the initial electrostatic ones as has been shown for -lactoglobulin/chitosan complexes (Chen and Subirade 2005; Hong and McClements 2007). Nevertheless, it was also shown that non-heat-stabilized chiosan/ -lg core/shell nanoparticles loaded with brilliant blue were less prone to acid and pepsin degradation in simulated gastric conditions, probably because of the different reactivity of native and denatured forms of -lg (Chen and Subirade 2005).
16.6.3 Food products texturization Protein±polysaccharide complexes have been shown to be able to control gellike product texture or replace fat or meat in several food applications (Sanchez and Paquin 1997; Schmitt et al. 1998; Norton and Frith 2001). Owing to the rheological and heat sensitivity of several globular proteins and fibrillar polysaccharides, appropriate processing conditions (heat treatment, hom*ogenization) allowed the production of mixed microparticles matching the textural attributes of fat (spherical aggregates in the range of 1±10 m) or meat (fibrillar aggregates in the range 1 to 50 m). For example, Chen et al. (1989) reported the use of microfragmented milk and/or egg proteins/xanthan gum complexes obtained upon interaction at pH 3.7 to 4.2 as a fat substitute. The formed complexes had a diameter of around 15 m, which is close to values reported for electrostatic complexes between whey proteins and xanthan gum (Laneuville et al. 2000). These fibrillar complexes were thereafter heat treated to increase their stability by exposure of hydrophobic regions of the protein and microfragmented under high pressure (200±800 bars) and shearing, leading to microparticles of about 2 to 10 m. Such whey proteins/xanthan gum complexes were shown to efficiently allow reduction of fat in cake frostings or sandwich cookie fillings, provided that the amount of complexes was adapted to the water activity of the sample (Laneuville et al. 2005a). Recently, it was shown that
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thermogels with a very wide range of texture could be obtained upon complex formation of -lg or -la with carboxymethylcellulose (Capitani et al. 2007). The production of meat substitutes was achieved in a very similar way, but in this case, alginate and pectins were used to complex milk and soy proteins (Tolstoguzov et al. 1974). Interestingly, it was also shown that texturization properties could be obtained without requiring a heat treatment of the complexes as was the case for chitosan/chicken salt-soluble proteins (Kachenechai et al. 2007) or amidated pectins and sodium caseinate (Matia-Merino et al. 2004). In this respect, polysaccharides played the function of electrostatic crosslinking agents. Recently, similar modulation of protein gel texture was reported upon insitu acidification of -lg/xanthan gum (Laneuville et al. 2006) or skimmed milk/ exopolysaccharide mixtures using GDL (Girard and Schaffer-Lequart 2007). In these studies, the importance of the molecular weight of the xanthan gum or the molecular structure of the exopolysaccharides in the rheological properties of the obtained gels was clearly shown.
16.6.4 Interface stabilization Protein±polysaccharide complexes can be used to stabilize interfaces in several food products. For example, creaming stability of a 30 wt% soya oil emulsion stabilized by whey protein isolate/-carrageenan complexes (Singh et al. 2003) and 40 wt% rapeseed oil stabilized by whey protein isolate/chitosan complexes (Speiciene et al. 2007) could be efficiently improved in acidic conditions. This was due mainly to the formation of a weak electrostatic network between the oil droplets, as it has been previously described for cold-protein gels. Use of spray dried complexes made of whey protein and carboxymethylcellulose (CMC) was tested in order to stabilize 10 or 20 wt% corn oil in comparison to pure whey protein isolate (Girard et al. 2002b). It was concluded that the complexes were clearly improving the stability of the oil droplets against coalescence, but were promoting some flocculation due to the presence of a minor fraction of uncomplexed CMC. Similar freeze-dried complexes at a concentration of solids of 4 wt% were able to produce very dense and thick foams that were very close to those obtained with egg white (Hansen and Black 1972). Poole (1989) reported the very high foaming and foam stabilizing properties of whey protein/chitosan complexes in a model sucrose/corn oil emulsion, likely because complexes were encapsulating the fat, preventing its detrimental spreading at the air/water interface. More recently, the use of whey protein isolate/acacia gum complexes was shown to improve significantly the heat shock stability of air bubbles within a complex acidic ice cream matrix (Kolodziejczyk and Schmitt 2004). Figure 16.7 shows whey protein isolate/ acacia gum complexes formed instantaneously in the ice cream mix during whipping/freezing and adsorbed at the interface of the air bubbles. A coacervate layer was formed, conferring very low gas permeability to the bubbles upon several freeze/thawing cycles.
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Fig. 16.7 Confocal scanning laser micrograph of a strawberry ice-cream stabilized by whey protein isolate/acacia gum complexes and coacervates at pH 4.2 after 7 days of storage at ÿ40 ëC. The protein was coloured by the rhodamine 6G reagent. Arrows indicate the coacervates adsorbed at the interface of the air bubbles. The scale bar is 20 m. From Kolodziejczyk and Schmitt (2004). Copyright Nestle Research Center, Lausanne
16.6.5 Other food applications Some other food applications of protein±polysaccharide complexes might be the design of edible films as has been described for a system composed by soy protein/sodium alginate or /propylene glycol alginate (Shih 1994). Interestingly, it was recently shown that a combination of lysozyme and chitosan with an edible film was able to confer a very high potential for antimicrobial activity (Park et al. 2004). In this case an important parameter to control was the protein to polysaccharide ratio that was both controlling the antimicrobial activity, but also the physical properties of the film. Finally, it has recently been shown that protein±polysaccharide complexes were probably importantly involved in some in-mouth fat perception attributes as the flocculation of lysozyme-stabilized emulsion was due mainly to interaction with the glycated saliva mucin proteins (Silletti et al. 2007). Interestingly, such type of electrostatic complex formation has also been discovered between mucins and chitosan (Dedinaite et al. 2005).
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16.7 Non-food applications of protein±polysaccharide complexes and coacevates A variety of applications of protein±polysaccharide or protein±polyelectrolyte complexes can be described. They range from hydrogels for protein or enzyme release and entrapment, synthesis of biosensors, use as surfactants in cosmetic applications (T.X. Wang et al. 2000) to protein separation (Strege et al. 1990) as it was already the case for food applications. Recent examples of these applications can be found in a very detailed review by Cooper et al. (2005). In the next two subsections, we consider mainly uses for microencapsulation and synthesis of biomaterials. 16.7.1 Microencapsulation An important field for non-food applications of protein±polysaccharide complexes and coacervates is microencapsulation for the pharmaceutical industry (Burgess 1994; Table 16.2). Early on, Bungenberg de Jong (1949b) reported that is was possible to entrap carbon particles or ink directly into the dispersed gelatine/acacia gum coacervate phase. This technique was also used to encapsulate micronized Naproxen into gelatine A/gelatine B microcapsules (Burgess and Carless 1985). The drug was dispersed in glycerol and mixed with gelatine B dispersion before coacervation occurred. The yield of drug encapsulation was around 50% in the best conditions of protein to polysaccharide ratio and temperature. In the same vein, Ryszka et al. (2002) reported the preparation of microcapsules in the form of a biodegradable coacervate containing dalarelin (analogue of the gonadotrophin-releasing hormone), with a gelatine/alginate coating. The microcapsules were obtained with yield of 66:4 2:1%. It was found that the dalarelin in the form of microcapsules had better bioavailability and was active longer in rats when compared with the dalarelin solution injections. Ponsart and Burgess (1996) reported the encapsulation of the -glucuronidase enzyme into three different coacervating systems consisting in gelatine/acacia gum, gelatine/sodium alginate and BSA/acacia gum in the optimum coacervation conditions of pH, ionic strength and protein to polysaccharide ratio. Stabilization of the microparticles was achieved by spray drying, leading to diameters around 5 m. The enzyme encapsulation yield was shown to be low, 28.8%, but was, however, higher than for non-spray-dried microparticles (11.3%). Interestingly, it was shown that the activity of the enzyme could be maintained upon hydration of the microparticle in phosphate buffer over a period of 8 days, revealing the interesting controlled release properties of these systems. Recently, a similar approach was used to increase the oral delivery of low molecular weight heparin, tinzaparin, which is not highly negatively charged (Lamprecht et al. 2007). In this case, an encapsulation yield of more than 90% was obtained using gelatine A or B in combination with acacia gum. Oral delivery quantification showed that gelatine B particles lead to 4.2% oral
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absorption compared to no adsorption with the pure protein or in gelatine A/ acacia gum particles, showing the importance of the microparticle charge for promoting adsorption at a given pH. Xing et al. (2006) studied the antimicrobial activities of capsaicin microcapsules prepared by the complex coacervation of gelatine, acacia gum and tannins. The results showed that the optimum pH for the antimicrobial effect was about 5.0, which might be related to the strongest protein-precipitating ability of tannins at this pH value. Another type of sub-micronic pH sensitive particles was obtained upon complex formation between chitosan and ovalbumin (Yu et al. 2006). Worth noting was the fact that the hydrophobicity/hydrophilicity of these nanogels could be triggered upon pH adjustment as was demonstrated from fluorescence spectroscopy upon pyrene addition. It was shown that between pH 2 and 5, nanogels were hydrophobic, whereas they became more hydrophilic up to pH 6.8. The likely explanation was the change in the intensity of the electrostatic interactions between the two biopolymers upon pH increase, leading to several molecular conformations of the chitosan chains within the gels. Another common way of producing microcapsules using complex coacervation is to achieve interfacial coacervation around an oil droplet as already described for food applications. Such technique allows encapsulating hydrophobic molecules that cannot be easily dispersed in the coacervate phase. This was done for example for the anti-inflammatory drug, ketoprofen, using gelatine/acacia gum microcapsules (Palmieri et al. 1996). Recently, Lamprecht et al. (2000) gave a very interesting insight into the structure of the microcapsules made of gelatine and acacia gum around oil droplets. Using confocal scanning laser microscopy, they were able to localize specifically the two biopolymers into the shell of the microcapsule and demonstrated that the two biopolymers were evenly distributed. Remarkably, the authors showed that if a ternary labelled hydrophobic protein was used (casein in this case), it had the tendency to adsorb preferentially at the oil/water interface, enabling an additional control of the release of the encapsulated material. The same system was used by Mayya et al. (2003) to encapsulate paraffin oil in the presence of sodium dodecyl sulfate. They reported that encapsulation yield increases from 35% to 70% in the presence of small quantities of the oppositely charged surfactant.
16.7.2 Biomaterials Another important field of use of protein±polysaccharide complexes is the synthesis of biomaterials. When used as biomaterials and matrices in tissue engineering, biopolymers offer important options to structure, morphology and chemistry as reasonable substitutes or mimics of extracellular matrix systems (ECM). These features also allow controlling material functions such as mechanical properties in gel, fibre and porous scaffold formats (Velema and Kaplan 2006). Hence, as these materials are supposed to be in direct contact with living organisms, replacing the extracellular matrix, they should be
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composed with biopolymers that can be found in the organisms or be as close as possible in terms of structure (Ellis and Yannas 1996). In this respect, chitosan was shown to be relatively close to the structure of glycosaminoglycans (GAGs) occurring in living cells. It was therefore extensively used as a complex with collagen or with its hydrolysate product, gelatine. Taravel and Domard (1996) studied the interaction of collagen with chitosan and reported on the mechanical and structural properties of the films that were obtained. Such structural properties are very important in order to control the assimilation of the biomaterial by the living body, as, for example, the porosity of the protein±polysaccharide-based ECM was shown to control the speed development and colonization of the graft by the fibroblasts. Porosity could, for example, be modulated by controlling the freezing rate upon drying of collagen/ GAG complexes (O'Brien et al. 2004). Interestingly, protein±polysaccharide complexes could also serve as a carrier for bioactive molecules in gene therapy as has been shown for the delivery of plasmid DNA to articular chondrocytes using a collagen/GAG matrix (Samuel et al. 2002). Here, it was shown that DNA incorporated in the scaffold at pH 7.5 was better released over 28 days upon incubation in Tris-EDTA buffer compared to pH 2.5. In addition, DNA kept a more `natural' coiled structure in neutral pH conditions. Erbacher et al. (1998) studied pharmacological approaches to gene therapy based on non-viral DNA vectors using chitosan to form polyelectrolyte complexes. They found that chitosan presents some characteristics favourable for gene delivery, such as the ability to condense DNA and to form hom*ogeneous population of complexes (diameter: 50±100 nm). The characterization of physico-chemical properties of this system was carried out by Mao et al. (2001). They also confirmed the offered protection for encapsulated DNA from nuclease attack. Recently, the in vitro and in vivo transfection efficiency of chitosan-pDNA nanoparticles obtained by complex coacervation were studied using quaternized chitosan ± 60% trimethylated chitosan oligomer (TMCO60%) ± and two other chitosan molecules differing in molecular weight (Fang et al. 2007). It was found that TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two molecules, with especially most prominent delivery in the gastric and upper intestinal mucosa. In the same vein, it was recently shown that 5±10 m chitosan/gelatine microspheres could be used for controlled and site-specific release of fibroblasts growth factor (Liu et al. 2007). It was shown that the growth factor released could be extended for 14 days, with an accumulated release yield of around 72% leading to a significant development of fibroblasts compared to a control with no added micropheres.
16.8
Conclusions
In this chapter, we have attempted to give a concise overview of the field of protein±polysaccharide complexes and coacervates. Thanks to the development
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of new investigation techniques and the use of computer simulations, increasing knowledge has been acquired on these systems in the last few years. Hence, thermodynamic parameters of the complex formation/coacervation are now accessible, enabling the probing and refining of theories from the late 1950s. However, more refined thermodynamic binding models are needed to take into account the inherent complexity of thermograms obtained with protein± polysaccharide mixtures, especially the existence of both exothermic and endothermic signals. In addition, computer simulations on monodispersed model systems allow prediction of phase behaviour and complex formation in relatively simple real synthetic or semi-synthetic systems. Increasing complexity of the models with experimentally measurable parameters would be required for application to more polydispersed natural systems and for more complex biomolecular architectures. In this respect, one would also expect models enabling prediction of the phase separation kinetics in protein±polysaccharide systems to be developed. The use of mesoscopic and multi-scale modelling techniques can be anticipated in the next few years. An important contribution to the understanding of the complex and coacervate structure was permitted by the use of novel scattering and microscopy techniques, such as, for example, confocal scanning laser microscopy (CSLM) coupled with fluorescence recovery after photobleaching (FRAP) or particle tracking. Such experiments clearly demonstrated the dynamics of the coacervate phase that can be compared to a heterogeneous electrostatic network of polymers exhibiting capability to diffuse along the polyelectrolyte chains; the diffusion being controlled by the strength of the electrostatic interactions between the protein and the polysaccharide (depending on pH, ionic strength, mixing ratio and other biopolymer intrinsic properties). In the last few years, emphasis has been given to extend the study of the functional properties of these complexes and coacervates, enabling identification of several interesting fields of applications in food and non-food products. Thus, the rheological and interfacial properties of protein±polysaccharide complexes might be advantageously used for designing food products with various textures, stability and delivery properties. The same conclusions apply to non-food applications, where an increased number of studies reported on the formulation of novel delivery systems sensitive to environmental stresses, enabling specific controlled release of bioactive molecules in the right organs or tissues. In order to expand the different applications, especially at the industrial scale, it would be necessary to better understand the effects of processing conditions such as temperature, shear stresses and pressure on the structure, stability and functional properties of protein±polysaccharide complexes and coacervates. Also, since the formation of protein±polysaccharide complexes and coacervates is mostly totally reversible upon pH changes, stabilization of these systems using physical parameters or by using specific enzymes or chelating biological molecules is expected to be developed in the future.
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16.9
Acknowledgements
The authors would like to thank Dr Eric Kolodziejczyk from Nestle Research Center for providing Figs 16.6 and 16.7.
16.10
References
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(1996) Factor determining the character of biopolymer-biopolymer interactions in multicomponent aqueous solutions modelling food systems. In: N. Parris, A. Kato, L.K. Creamer and J. Pearce (eds), Macromolecular Interactions in Food Colloids and Food Technology, ACS Symposium Series 650, Washington DC, pp. 37±49. SEROV, A.V., ANTONOV, Y.A. and TOLSTOGUZOV, V.B. (1985) Isolation of lactic whey proteins in form of complexes with apple pectin. Nahrung 1, 19±30. SEYREK, E., DUBIN, P.L., TRIBET, C. and GAMBLE, E.A. (2003) Ionic strength dependence of protein±polyelectrolyte interactions. Biomacromolecules 4, 273±282. SHALOVA, I.N., ASRYANTS, R.A., SHOLUKH, M.V., SASO, L., KURGANOV, B.I., MURONETZ, V.I. and IZUMRUDOV, V.A. (2005) Interaction of polyanions with basic proteins, 2: Influence of complexing polyanions on the thermoaggregation of oligomeric enzymes. Macromol. Biosci. 5, 1184±1192. SHIEH, J.Y. and GLATZ, C.E. (1994) Precipitation of proteins with polyelectrolytes: role of the polymer molecular weight. In: P.L. Dubin, J. Bock, R. Davis, D.N. Schulz and C. Thies (eds), Macromolecular Complexes in Chemistry and Biology, Springer Verlag, Berlin Heidelberg. SHIH, F.F. (1994) Interaction of soy isolate with polysaccharide and its effect on film properties. JAOCS 71, 1281±1285. SILLETTI, E., VINGERHOEDS, M.H., NORDE, W. and VAN ACKEN, G.A. (2007) Complex formation in mixtures of lysozyme-stabilized emulsions and human saliva. J. Coll. Interf. Sci. 313, 485±493. SINGH, H., TAMEHANA, M., HEMAR, Y. and MUNRO, P.A. (2003) Interfacial compositions, microstructure and stability of oil-in-water emulsions formed with mixtures of milk proteins and -carrageenan: 2. Whey protein isolate (WPI). Food Hydrocoll. 17, 549±561. SINGH, O. N. and BURGESS, D. J. (1989) Characterisation of albumin-alginic acid complex coacervation. J. Pharm. Pharmacol. 41, 670±673. SINGH, S. S., SIDDHANTA, A.K., MEENA, R., PRASAD, K., BANDYOPADHYAY, S. and BOHIDAR, H.B. (2007) Intermolecular complexation and phase separation in aqueous solutions of oppositely charged biopolymers. Int. J. Biol. Macromol. 41, 185±192. È , M. and LINSE, P. (2003) Complexation, phase separation, and redissolution in SKEPO polyelectrolyte-macroion solutions. Macromolecules 36, 508±519. SMITH, A.K., NASH, A.M., ELDRIDGE, A.C. and WOLF, W.J. (1962) Recovery of soybean whey protein with edible gums and detergents. Agric. Food Chem. 10, 302±304. SPEICIENE, V., GUILMINEAU, F., KULOZIK, U. and LESKAUSKAITE, D. (2007) The effect of chitosan on the properties of emulsions stabilized by whey proteins. Food Chem. 102, 1048±1054. STRAUSS, G. and GIBSON, S.M. (2004) Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients. Food Hydrocoll. 18, 81±89. STREGE, M.A., DUBIN, P.L., WEST, J.S. and FLINTA, C.D. (1990) Protein separation via polyelectrolyte complexation. In: M. Ladisch, R.C. Wilson, C.C. Painton and S.E. Builder (eds), Protein Purification: From Molecular Mechanisms to Large Scale Processes, ACS Symposium Series 427, Washington DC, pp. 66±79. SYRBE, A., FERNANDES, P.B., DANNENBERG, F., BAUER, W. and KLOSTERMEYER, H. (1995) Whey protein + polysaccharide mixtures: polymer incompatibility and its application. In: E. Dickinson and D. Lorient (eds), Food Macromolecules and Colloids, Royal Society of Chemistry, Cambridge, pp. 328±339. TAINAKA, K.-I. (1979) Study of complex coacervation in low concentration by virial sem*nOVA, M.G.
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expansion method. I. Salt free systems. J. Phys. Soc. Jpn 46, 1899±1906. and DOMARD, A. (1994) Relation between the physicochemical characteristics of collagen and its interaction with chitosan. I. Biomaterials 14, 930±938. TARAVEL, M.N. and DOMARD, A. (1996) Collagen and its interaction with chitosan. III. Some biological and mechanical properties. Biomaterials 17, 865±871. THIMMA, R.T. and TAMMISHETTI, S. (2003) Study of complex coacervation of gelatin with sodium carboxymethyl guar gum: microencapsulation of clove oil and sulphamethoxazole. J. Microencapsulation 20, 203±210. TIRKKONEN, S., TURAKKA, L. and PARONEN, P. (1994) Microencapsulation of indomethacin by gelatin-acacia complex coacervation in presence of surfactants. J. Microencapsulation 11, 615±626. TOLSTOGUZOV, V.B. (1986) Functional properties of protein±polysaccharide mixtures. In: J.R. Mitchell and D.A. Ledward (eds), Functional Properties of Macromolecules, Elsevier Applied Science, London, pp. 385±415. TOLSTOGUZOV, V.B. (1991) Functional properties of food proteins and role of protein± polysaccharide interaction. Food Hydrocoll. 4, 429±468. TOLSTOGUZOV, V.B. (1993) Functional properties of food proteins. Role of interactions in protein systems. In: K.D. Schwenke and R. Mothes (eds), Food Proteins, Structure and Functionality, VCH, Weinheim, pp. 203±209. TOLSTOGUZOV, V.B. (1997) Protein±polysaccharide interactions. In: S. Damodaran and A. Paraf (eds), Food Proteins and Their Applications, Marcel Dekker, New York, pp. 171±198. TOLSTOGUZOV, V.B. (2000) Compositions and phase diagrams for aqueous systems based on proteins and polysaccharides. Int. Rev. Cytol. 192, 3±31. TOLSTOGUZOV, V.B. (2003) Review. Some thermodynamic considerations in food formulation. Food Hydrocoll. 17, 1±23. TOLSTOGUZOV, V.B., IZJUMOV, D.B., GRINBERG, V.Y., MARUSOVA, A.N. and CHEKHOVSKAYA, V.T. (1974) Method of making protein-containing foodstuffs resembling mincedmeat. United States patent application US3829387. TUINIER, R., ROLIN, C. and DE KRUIF, C.G. (2002) Electrosorption of pectin onto casein micelles. Biomacromolecules 3, 632±638. TURGEON, S.L., BEAULIEU, M., SCHMITT, C. and SANCHEZ, C. (2003) Protein±polysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects. Curr. Opin. Coll. Interf. Sci. 8, 401±414. TURGEON, S.L., SCHMITT, C. and SANCHEZ, C. (2007) Protein±polysaccharide complexes and coacervates. Curr. Opin. Coll. Interf. Sci. 12, 166±178. ULRICH, S., SEIJO, M. and STOLL, S. (2006) The many facets of polyelectrolytes and oppositely charged macroions complex formation. Curr. Opin. Coll. Interf. Sci. 11, 268±272. VEIS, A. and ARANYI, C. (1960) Phase separation in polyelectrolyte systems. I. Complex coacervates of gelatin. J. Phys. Chem. 64, 1203±1210. VEIS, A., BODOR, E. and MUSSEL, S. (1967) Molecular weight fractionation and the selfsuppression of complex coacervation. Biopolymers 5, 37±59. VELEMA, J. and KAPLAN, D. (2006). Biopolymer-based biomaterials as scaffolds for tissue engineering. Adv. Biochem. Eng./Biotechnol. 102, 187±238. VIKELOUDA, M. and KIOSSEOGLOU, V. (2004) The use of carboxymethylcellulose to recover potato proteins and control their functional properties. Food Hydrocoll. 18, 21±27. WANG, T.X., DIGIROLAMO, D.M.V. and RUSS, J.G. (2000) Cosmetic compositions containing TARAVEL, M.N.
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polysaccharide/protein complexes. PCT patent application WO00/23038 from Revlon Consumer Products Corporation. WANG, X., LEE, J., WANG, Y.-W. and HUANG, Q. (2007a) Composition and rheological properties of -lactoglobulin/pectin coacervates: effects of salt concentration and initial protein/polysaccharide ratio. Biomacromolecules 8, 992±997. WANG, X., LI, Y., WANG, Y.-W., LAL, J. and HUANG, Q. (2007b) Microstructure of lactoglobulin/pectin coacervates studied by small-angle neutron scattering. J. Phys. Chem B 111, 515±520. WANG, X., RUENGRUGLIKIT, C., WANG, Y.-W. and HUANG, Q. (2007c) Interfacial interactions of pectin with bovine serum albumin studied by quartz crystal microbalance with dissipation monitoring: effect of ionic strength. J. Agric. Food Chem. 55, 10425± 10431. WANG, Y., KIMURA, K., DUBIN, P.L. and JAEGER, W. (2000) Polyelectrolyte-micelle coacervation: effect of micelle surface charge density, polymer molecular weight, and polymer/surfactant ratio. Macromolecules 33, 3324±3331. WEINBRECK, F. and DE KRUIF, C.G. (2003) Complex coacervation of globular proteins and gum Arabic. In: E. Dickinson and T. van Vliet (eds), Food Colloids: Biopolymers and Materials, Royal Society of Chemistry, Cambridge, pp. 337±344. WEINBRECK, F., DE KRUIF, C.G. and SCHROOYEN, P. (2003a) Complex coacervates containing whey proteins. PCT patent application WO03/106014 from Nizo Food Research B.V. WEINBRECK, F., DE VRIES, R., SCHROOYEN, P. and DE KRUIF, C.G. (2003b) Complex coacervation of whey proteins and gum Arabic. Biomacromolecules 4, 293±303. WEINBRECK, F., NIEUWENHUIJSE, H., ROBIJN, GW. and DE KRUIF, C.G. (2003c) Complex formation of whey proteins-exocellular polysaccharide EPS B40. Langmuir 19, 9404±9410. WEINBRECK, F., MINOR, M. and DE KRUIF, C.G. (2004a) Microencapsulation of oils using whey protein/gum Arabic coacervates. J. Microencapsulation 21, 667±679. WEINBRECK, F., NIEUWENHUIJSE, H., ROBIJN, G.W. and DE KRUIF, C.G. (2004b) Complexation of whey proteins with carrageenan. J. Agric. Food Chem. 52, 3550±3555. WEINBRECK, F., ROLLEMA, H.S., TROMP, R.H. and DE KRUIF, C.G. (2004c) Diffusivity of whey protein and gum Arabic in their coacervates. Langmuir 20, 6389±6395. WEINBRECK, F., TROMP, R.H. and DE KRUIF, C.G. (2004d) Composition and structure of whey protein/gum Arabic coacervates. Biomacromolecules 5, 1437±1445. WEINBRECK, F., WIENTJES, R.H., NIEUWENHUIJSE, H., ROBIJN, G.W. and DE KRUIF, C.G. (2004e) Rheological properties of whey protein/gum Arabic coacervates. J. Rheol. 48, 1215±1228. XIA, J. and DUBIN, P.L. (1994) Protein±polyelectrolyte complexes. In: P.L. Dubin, J. Bock, R. Davis, D.N. Schulz and C. Thies (eds), Macromolecular Complexes in Chemistry and Biology, Springer Verlag, Berlin, Heidelberg, pp. 248±271. XIA, J., DUBIN, P.L. and KOKUFUTA, E. (1993) Dynamic and electrophoretic light scattering of a water soluble complex formed between pepsin and poly(ethylene glycol). Macromolecules 26, 6688±6690. XING, F., CHENG, G., YANG, B. and MA, L. (2003) Microencapsulation of capsaicin by complex coacervation of gelatin, acacia and tannins. J. Appl. Polym. Sci. 91, 2669± 2675. XING, F., CHENG, G. and YI, K. (2006) Study on the antimicrobial activities of the capsaicin microcapsules. J. Appl. Polym. Sci. 102, 1318±1321. YE, A., FLANAGAN, J. and SINGH, H. (2006) Formation of stable nanoparticles via
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electrostatic complexation between sodium caseinate and gum Arabic. Biopolymers 82, 121±133. YEO, Y., BELLAS, E., FIRESTONE, W., LANGER, R. and KOHANE, D.S. (2005) Complex coacervates for thermally sensitive controlled release of flavor compounds. J. Agric. Food Chem. 53, 7518±7525. YU, S., HU, J., PAN, X., YAO, P. and JIANG, M. (2006) Stable and pH-sensitive nanogels prepared by self-assembly of chitosan and ovalbumin. Langmuir 22, 2754±2759. ZAITZEV, V.S., IZUMRUDOV, V.A. and ZEZIN, A.B. (1992) A new family of water-soluble protein±polyelectrolyte complexes. Polym. Sci. USSR 34, 54±55. ZHANG, H., SAIANI, A., GUENET, J.M. and CURTIS, R. (2007) Effect of stereoregular polyelectrolyte on protein thermal stability. Macromol. Symp. 251, 25±32. ZHU, Y. Y., ZHANG, G. Y., HONG, X.L., DONG, J.F., ZHANG, X.G. and ZENG, H. (2005) Synthesis of nanocapsules by complex coacervation in microemulsion. Acta Chim. Sinica 63, 1505±1509. ZIEGLER, A. and SEELIG, J. (2004) Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters. Biophys. J. 86, 254±263.
17 Gum ghatti S. Al-Assaf, G. O. Phillips and V. Amar, Glyndwr University, UK
Abstract: This chapter describes the natural tree exudate gum from India. The exudate gum has a glassy appearance and the colour can vary from dark red to translucent white depending on the shape which can be either as a nodule or spiro. The chapter then reviews the structure and botanical source of gum ghatti. Technical information relating to its solubility, sugar composition, protein content, molecular weight, viscosity and emulsification performance are directly compared with gum arabic. Gum ghatti's current regulatory status together with various food and other applications are also described. Key words: gum ghatti regulatory status, ghatti molecular weight, ghatti in beverage emulsion, ghatti applications, GATIFOLIA.
17.1 Introduction Gum ghatti is a natural gum from India. It is exuded from the tree species Anogeissus Latifolia which is native to India and Sri Lanka. These trees constitute one of the largest forest coverages in India and are found mostly in dry deciduous forests. This tree can survive in harsh conditions and does not need a lot of water to survive, although if proper nutrients are provided this tree can grow very healthy and large. Gum exudation occurs during times of stress for the tree. The exudation process occurs very slowly over a period of days and depending on the size or age of the tree. The exuded gum nodules can range from 5 g to 50 g in weight. The shape of the exuded nodule depends on environmental conditions such as wind, sunlight, heat and other factors like the mass of the nodule, the pressure within the tree and the shape/size of the fissure from where the gum is exuding. There is no particular shape for a ghatti nodule as found for gum arabic variety Acacia senegal. Gum ghatti has a glassy appearance and the
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Fig. 17.1
(left) Gum ghatti nodule known in the industry as `SPIRO'; (right) gum ghatti nodule in a rounded tear shape.
colour of the nodule can vary from dark red to translucent white. The gum is collected in much the same way as most other natural tree exudate gums such as gum arabic and gum karaya. The local community in the forest region (Tribals), who constitute the main population of the forest areas, collect the exudate manually. These Tribals have extensive knowledge about the character of the trees in their local habitats, and provide a very good source of knowledge about the natural gum exudation and tree identification. Figure 17.1 shows two types of exudates known as a Spiro exudation which is clear and tubular in form and a rounded tear shaped nodule from trees in the Central Indian Forest. The tree grows on a well-drained slope and clay loam soil, and attains considerable height towards maturity in about 70±80 years. On dry rocky slopes, however, it tends to be stunted. It has a peculiar bark, smooth and greenish white in colour, and about 4±5 mm thick. The leaves are shaped somewhat like those of guava. Varying between 5 and 10 cm in length and 3 and 8 cm in width, these are alternate or sub-opposite in arrangement. These are thick and somewhat shining. Old leaves are shed between October and December, when these tend to get a beautiful reddish-brown hue. New leaves appear during February±March. Inflorescence starts appearing during May and stays on up to the end of June. The small flowers are off-white. Fruits start to show in July. When about to mature, these 8±10 mm 5±6 mm drupes are peculiarly compressed, showing a two-winged form. The ghatti tree grows best in the sunny aspect of peninsula ranges, Shivaliks and outer Himalayas at altitudes ranging from 200 m to 1250 m, and experiencing annual rainfall of 100±200 mm and temperatures varying from 5 to 40 ëC. However, it is quite capable of surviving in adverse climatic and geological conditions like dry rocky slopes. This tree has a moderate rate of growth. It has seven annual rings of growth in an inch of the cross section of its stem. Thus weighing about 28 to 32 kg to a cubic foot, it is one of the best hardwoods on the Indian sub-continent. Though quite difficult to season, it tends to split if left unattended in the sun. The splitting is less if the logs are kept in shade for about six months and sawn pieces are also kept in shade for a long period.
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The gum is exuded from the bark of the tree and can be found all over the tree irrespective of the location such as a branch or the trunk, etc. The gum is exuded from the tree in extreme climatic conditions and acts as the buffer stock of nutrients required by the tree for survival. There is no need to make wounds in the bark of this tree to exude the gum as is the common practice with some other natural tree exudate gums. The gum exudation from this species is mostly due to the pressures created within the bark due to extreme natural conditions. The gum comes naturally from the bark and can be collected by pulling the soft gel-like exude which collects on the tree bark at certain locations. If the gum is not plucked/picked from that location, fresh gum will not exude from the same position again. In order to increase the yield the common practice is to pick the old gum and make way for fresh gum to flow. The gum when freshly exuded has high moisture content and is very soft. When left in the open for days, the gum nodule loses moisture and hardens giving the nodule a rounded tear-like shape which is very hard and does not change. The gum has a faint sweetness to taste and a very faint odour which can only be recognised when the gum is kept in large volumes. Within the Genus Anogeissus several species have been identified: A. acuminata (A. pendula), A. bentii, A. dhofarica, A. latifolia, A. leiocarpus (A. leiocarpa), A. rotundifolia, A. Schimperi and A. sericea. The specific gum which is being described here is the species Latifolia with the following botanical taxonomy: Kingdom: Plantae Phylum: Angiosperms Class: Magnoliatae Subclass: Rosidae Order: Myrtales Family: Combretaceae Genus: Anogeissus Anogeissus is a genus of trees native to South Asia, the Arabian Peninsula, and Africa, belonging to the family Combretaceae. The genus has eight species, five native to South Asia, two endemic to the southern Arabian Peninsula, and one native to Africa. Anogeissus latifolia is one of the most useful trees in India. A. pendula is common in the Kathiarbar-Gir dry deciduous forests of western India, where it often forms pure stands in the rocky ridges of the Aravalli Range. A. leiocarpus is found in Africa from northeastern Ethiopia to Senegal, and its bark is used to produce Anogelline, a substance used in cosmetics. A. dhofarica and A. bentii are endemic to the woodlands of the southern Arabian Peninsula. Gum ghatti is known by many names throughout India and the rest of the world. It is commonly known as Bakli, Tirman, Davedi, Dindal, Dindiga, Dhanta, Dhao, Dhawra, Dhokra, Dho, Kardhai in different parts of India. Elsewhere in the world it is referred to as Indian gum, Gummi indicum, Gummi indici and the tree is known as axlewood in the United States and Australia.
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17.2
Manufacture
The gum is usually supplied from the original producer in India to the global industry in lump form without any particular manufacturing process being applied. It is, nevertheless, distinguished on the basis of the amount of extraneous contamination present in the raw material. The main factors that contribute to purity are the colour and extraneous material. White, yellow and red grades are distinguished. There could be further grading according to the quality of the gum in which all the impurities such as bark, wood, sand, and nonghatti products, etc., have been removed. In the past this mixture of different types was an inherent weakness, since the gum reaching the market in terms of colour, gel content and solubility, was very variable. Now, due to the establishment of a plantation, selection and cleaning before exporting, the quality has greatly improved. Due to this more recent improvement, it has been possible to produce a gum quality by special processing and spray drying which is almost completely soluble and of consistent colour. This special product has been termed GATIFOLIA.
17.3
Structure
Gum ghatti has an extremely complex structure composed of sugars such as Larabinose, D-galactose, D-mannose, D-xylose, and D-glucuronic acid in a 48 : 29 : 10 : 5 : 10 molar ratio.1 The gum contains alternating 4-O-subsituted and 2-O-subsituted -D-mannopyranose units and chains of 1 ! 6 linked -Dgalactopyrannose units with side chains which are most frequently single Larabinofuranose residues. Using 13C NMR spectroscopy suggested that ~6% of rhamnose are linked as side chain to the galactose backbone as -Rhap-(1 ! 4) -galactopyrannose in common with the linkage found in gum arabic.2 Most of the information that we have about the structure of the polysaccharide components of gum ghatti has come from the classical work of that doyen of polysaccharide chemistry, Professor Edmund Hirst together with his colleague Gerald Aspinall and their co-workers over the period 1955 to 1969 at the University of Edinburgh.1,3±5 They showed that the polysaccharide of gum ghatti has an extremely complex structure. Complex arrays of neutral sugar units (Galp, Araf, Arap) and GluA are attached to a molecular core of alternating -D-GlucA and D-Man residues, the former linked though O-4 and the latter though O-2. The base structural units were identified using classical carbohydrate techniques. The aldobiouronic acid: -GlcA(1-2) D-Manp was first isolated as a product of the partial hydrolysis of damson and cherry gums and has subsequently been found to be a structural fragment of many plant gum polysaccharides. This unit is frequently found in the interior chains of these complex polysaccharides. It was found by partial hydrolysis of gum ghatti along
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with another aldobiuronic acid: 6-O-( -D-glucopyranosyluronic acid)-Dgalactose. In this study by Aspinall3 the longest oligosacchalride sequences found were: -D-Galp-{(1-6)- -D-Galp-}n(1-6)-D-Gal -D-Galp-{(1-6)- -D-Galp-}n(1-3)-L-Ara Smith degradation led to the isolation of a degraded gum from which 3-O-/3-D-galactopyranosyl-D-galactose, 6-O-/3-D-galactopyranosyl-Dgalactose (n = 0), 3-O-/3-D-galacto-pyranosyl-L-arabinose (n = 0), and 3-O-L-arabinopyranosyl-D-mannose were obtained on partial hydrolysis. These results imply that D-mannosyl residues are located in the interior of the molecular structure, and the following partial structure was proposed, in which this structural unit was found: ±(1!6)- -D-Galp-(1!6)- -D-Galp-(1!3)-L-Arap-(1!3)-D-Manp-l The chains of (1!6)-linked -D-galactopyranosyl residues are joined through an L-arabinopyranosyl `link' unit to a D-mannosyl residue in the basal chain. Subsequently, an acidic oligosaccharide (at first erroneously reported to be a trisaccharide) was characterised as the tetrasaccharide: O- -D-glucopyranosyluronic acid-(1!2)-O-D-mannopyranosyl-(1!4)-O -D-glucopyranosyluronic acid-(1!2)-D-mannose, and this provided evidence for the following sequence of the sugar residues in the inner chains. -D-GpA-(1!2)-D-Manp-(1!4)- -D-GpA-(1!2)-D-Man Based on these results the overall nature of the assembly was proposed.6 R R " " 6 6 ! 4)- -D-GlcA-(1!2)-D-Manp-(1!4)- -D-GlcA-(1!2)-D-Manp-(1 3 3 " " 1 1 L-Arap L-Arap 3 3 " " R0 R0 R = ÿ L-Araf- or L-Araf-(1!2, 3 or 5)-L-Araf-(1
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# -D-GlcpA- " or (1 ! 6)-D-Galp -(1 3 n -D-Galp" R
The periodicity of the acidic groups in the main chain is an important feature and there are others scattered though the periphery. Also (1-3)- and (1-6)-linked Gal units make up associated side chains. This structure is further complicated by D-Man residues being present as double branch points. It is this molecular complexity that accounts for the quite spectacular rheological and solution properties of gum ghatti. It is the overall molecular shape arising from the periodicity of the chains which gives it a rod-like shape and accounts also for the chain entanglements which are a feature of systems of this gum in water.4 More recently further investigations of this gum using methylation and three successive, controlled Smith degradations confirmed the previous findings of Aspinall, and also showed that the side chains contained mainly 2-O- and 3-Osubstituted Araf units.2 A second Smith degradation eliminated the remaining Araf units, and their anomers became evident. The proportion of Galp units gradually increased in the form of non-reducing end and Galp units, although 3,6-di-O- and 3,4,6-tri-O-substituted Galp units diminished. After three degradations groups with consecutive 3-O-substituted -Galp units were formed. The proportion of periodate-resistant 3-O- and 2,3-di-O-substituted Manp units was maintained. These observations support the core nature of the Manp units and the linked 1-6 Gal chains. Rhamnose residues were located, which, although present in smaller amounts than the other sugars, are clearly observed in NMR studies. The study observed free -Araf-(1!2)-Araf and Araf-[ -Araf]n-Ara with n 4 and 7, which corresponds with 2-O- and 3-Osubstituted Araf side-chain structures in the polysaccharide, along with: -Rhap-(1 ! 4)-GlcpA, -Rhap-(1 ! 4)- -GlcpA-(1 ! 6)-Gal, and -Rhap-(1 ! 4)- -GlcpA-(1 ! 6)- -Galp-(1 ! 6)-Gal. On three successive, controlled Smith degradations, a product was formed with consecutive (1 ! 3)-linked -Galp units. One of the observations in NMR studies is of relevance to the difference in physical forms and heterogeneity which is a feature of this gum as collected from the forests in India. The variations in physical form and colour are not related to a basic difference in chemical composition or specific bonding since the spectra are essentially similar. Nevertheless, it is evident that much more is yet to be learnt about the structure of this remarkable gum.
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17.4 Technical data 17.4.1 Solubility Gum ghatti occurs in nature as a calcium-magnesium salt and contains soluble and insoluble fractions which partially dissolve in boiling water. Gel fractions in ghatti have been reported which can be mostly dissolved by maceration; the gel fractions have a higher proportion of calcium ions.5 The gel is not primarily due to the presence of calcium ions and forms intermolecular linkages as a result.6 The viscosity of the gel dispersion was found to be ca. 10±30 times that of the soluble fraction and the gel fraction in commercial batches can vary from 8±23%.5±7 17.4.2 Physico-chemical parameters The quality of gum ghatti, like gum arabic, is also influenced by factors such as location, geographic climate and the form of the exudates (i.e. spiro, white or yellow nodules). The physico-chemical parameters for gum arabic and ghatti are compared in Table 17.1.8 The sample selected in Table 17.1 is a currently available gum ghatti called Shimla. The percentage loss on drying obtained for gum ghatti is similar to that of gum arabic in the lump gum form. While gum arabic is highly soluble in water, gum ghatti contains varying proportions of Table 17.1 Physico-chemical and molecular weight parameters of gum ghatti compared with gum arabic
% loss on drying % insoluble matter Specific optical rotation (deg dmÿ1 cm3 gÿ1) % galactose % arabinose % rhamonse % uronic acid % mannose % xylose % nitrogen % protein* Intrinsic viscosity ( cm3/g) in 1 M NaCl Intrinsic viscosity ( cm3/g) in 0.2 M NaCl Mw (whole gum) 105 Rg/nm (whole gum) Polydispersity (Mw/Mn) whole gum Mw (1st peak) 106 % mass 1st peak Rg/nm 1st peak Mw (2nd peak) 105 % mass (2nd peak) Rg/nm (2nd peak)
Gum arabic
Gum ghatti (shimla)
6.3 0 ÿ30 32.3 30.2 13.3 24.2 ÿ ÿ 0.314 2.1 16.8 18.3 6.22 29 2.36 2.54 10.6 41 3.96 89.4 ÿ
11.9 2.1 ÿ56.5 27.6 55.7 ÿ 11.7 3.3 1.6 0.518 3.4 52.7 64.4 8.7 35 1.35 1.87 21.2 45 5.9 79 22
*nitrogen conversion factor of 6.6 was used to calculate the protein %.
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Table 17.2 Amino acid composition (mol%) of gum arabic and gum ghatti
Ala Arg Asp Cys Glu Gly His Hyp Ile Leu Lys Met Phe Pro Ser Thr Tyr Val
Gum arabic
Gum ghatti
2.6 0.9 5.1 Ð 3.5 5.6 5.8 26.9 1.3 8.0 2.9 0.1 3.6 7.5 14.1 7.5 0.8 3.8
7.6 2.3 17.2 Ð 7.9 13.8 3.3 nd 3.9 5.4 9.2 0.3 2.3 6.9 4.7 5.1 2.4 7.7
insoluble gel. The ghatti sample used in this study was specifically supplied as highly soluble gum as demonstrated by the small percentage obtained for the insoluble matter (Table 17.1). One of the major differences between the two gums is the specific optical rotation (gum ghatti ÿ56.5ë, gum arabic ÿ30ë) and it is directly related to the difference in sugar composition. The presence of xylose and mannose are often used as a means of detecting its presence in formulations. For acacia gums it has been possible to relate the specific optical rotation to the vectorial contributions made by the individual sugars present.9,10 The second notable difference between the two gums is the protein content. The protein content of gum arabic is ~2% whereas gum ghatti has higher percentage at 3.4%. Gum ghatti contains a considerably higher percentage of Asp, Gly, Lys with lower percentages of Phe, Pro and Ser compared to gum arabic (Table 17.2). The intrinsic viscosity is a measure of hydrodynamic volume of macromolecules and the value obtained for gum ghatti is at least three times higher than that of gum arabic. This indicates that ghatti molecules are either highly solvated or asymmetric, or both when compared to gum arabic. Both gums contain uronic acid which confers a polyelectrolyte character as demonstrated by the increase in the overall size, due to electrostatic charge repulsion, while decreasing the ionic strength of the solvent (Table 17.1).
17.4.3 Molecular weight The structure and molecular weight of gum arabic have already been described11 and are summarised here in order to compare with gum ghatti. Hydrophobic fractionation of gum arabic has shown that it is a highly heterogeneous complex
Gum ghatti
485
polysaccharide which consists of three main fractions. These fractions are: arabinogalactan protein complex (AGP), arabinogalactan (AG) and glycoprotein (GP). Each fraction contains an average of different molecular weight components with different protein content. One of the proposed models for the AGP fraction suggests that it is composed of hydrophilic carbohydrate blocks linked to a protein chain and has been reported to have a wattle blossom-type structure due to being readily degraded by proteolytic enzyme.12,13 Other models to describe the structure of the AGP fraction have been recently reviewed.14 Gel permeation chromatography coupled on line to a multi-angle laser scattering, refractive index and UV detectors (GPC-MALLS) is now routinely used to identify the components in gum arabic, as labelled in Fig. 17.2(a).8,15,16 The same method was applied for the fractionation gum ghatti and the elution profile is shown in Fig. 17.2(b). The light scattering response shows the presence of two peaks with partial separation of the second peak, which appears as a shoulder. The RI response also shows the presence of these two peaks and the first peak coincided with the light scattering response, whereas the second peak
Fig. 17.2 Elution profile of (a) gum arabic and (b) gum ghatti following fractionation by gel permeation chromatography and detection by light scattering, refractive index and UV detectors.
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was lower than the first peak and contained the majority of the gum. The UV response showed that there is protein associated with the high molecular weight materials and the second peak also appears as shoulder and follows the same RI response. The UV response shows a third low molecular weight peak which elutes at ~15±18.5 ml before the total volume of the column (~20 ml). The molecular weight parameters of the whole gum and when processed as two peaks are given in Table 17.1 and compared with gum arabic. The weight average molecular weight (Mw) and root mean square radius of gyration (Rg) of gum ghatti is higher than that of standard gum arabic but is less polydisperse. The Mw and Rg of the first peak in gum ghatti, known as the AGP fraction in gum arabic, is comparable but in ghatti gum with double the concentration. The molecular weight parameters of the second peak are also higher but with slightly lower amounts compared to gum arabic. A third proteinaceous peak with high UV absorption was also identified in gum ghatti (Fig. 17.2(b)) but was difficult to accurately determine its molecular weight due to very low light scattering response. However, integration of the area under the peak for a range of ghatti samples revealed that the proportion of this peak is ~1±2.2% of the whole gum. The proportion of the third for the sample shown in Fig. 17.2(b) was 1.38% of the total injected mass. The total mass recovery for gum ghatti was 102% which indicates a suitability of column used for the fractionation and in agreement with the solubility determined independently using the weight of the materials retained on the filter (Table 17.1). 17.4.4 Rheology The viscosity of gum ghatti, like other hydrocolloids, increases exponentially with increasing concentration. Gum ghatti makes a viscous solution at 30±35 wt% (Fig. 17.3). Rheological measurements showing the shear flow viscosity of gum arabic and gum ghatti as a function of shear rate are shown in Fig. 17.3 (inset). At relatively diluted concentration (5% ghatti and 10% gum arabic) both show evidence of shear thinning behaviour at low shear rates and were Newtonian at higher shear rates. However, the viscosity of gum ghatti is double that of gum arabic as demonstrated by the almost exact viscosity match between the two gums. With increasing the concentration (45% gum arabic, 30% gum ghatti), both gums show a clear shear thinning behaviour albeit at different shear rates. The viscosity difference between the two gums is largely explained by the higher intrinsic viscosity and proportion of high molecular weight present in gum ghatti compared to gum arabic (Table 17.1 and Fig. 17.2). 17.4.5 Emulsification The emulsification performance of gum ghatti is greatly influenced by the presence of insoluble components. These insoluble components are usually filtered and removed during the manufacture of beverage emulsion. Gum ghatti, like other hydrocolloid emulsifiers, such as gum arabic and sugar beet pectin, contain a small amount of protein linked to the carbohydrate unit. Enzyme
Gum ghatti
487
Fig. 17.3 Viscosity of gum ghatti as a function of concentration. (Inset) Shear flow viscosity plotted as a function of shear rate for (ú) 10% w/w, ( ) 45% w/w gum arabic and (n) 5%, (l) 30% w/w gum ghatti. Solutions were dissolved in distilled water containing 0.005% w/v NaN3. All measurements were carried out at 25 ëC.
digestion with protease does not greatly reduce the molecular weight. However, when the gum is subjected to processing, such as spray drying, the effect of enzyme digestion can be clearly seen as results of changes in the protein conformation which then become more accessible. Further evidence of protein± carbohydrate linkage is supported by the interaction with Yariv's reagent and hence can be classified as arabinogalactan-protein complex. The emulsification mechanism in gum ghatti is similar to that of gum arabic where the protein is responsible for the surface activity by acting as the strong anchor point at the oil±water interface with the hydrophilic polysaccharide chains providing the protective layer.17 This is why gum ghatti can emulsify 20% medium chain triglyceride (MCT) oil at much lower concentration (5 wt%) compared to gum arabic as shown in Fig. 17.4. In Fig. 17.4 the emulsification performance and stability of gum ghatti, which is completely soluble, is compared with standard gum arabic at a range of concentrations. The interfacial rheology of gum ghatti is superior to that of gum arabic at the same concentration due to the high protein content and possibly the conformation in solution. Additionally, gum ghatti is more resistant to heat and this is clearly reflected by the emulsion stability following acceleration at 60 ëC for 3 days. Detailed evaluation of emulsification performance of gum ghatti has recently been reported.18
17.5 Uses and applications Gum ghatti has an age-old reputation in India as being a very good medicinal product as well as having very good qualities for food products. This gum has been mentioned in ancient medicinal scriptures like the Ayurveda (Indian system
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Fig. 17.4 Volume weighted mean (d[4,3]) for initial and accelerated emulsions of gum arabic (5±20 wt%) and gum ghatti at 5 wt%. Oil phase 20% MCT, 0.12% citric acid, 0.13% sodium benzoate.
of medicine) and the Unani (Greek system of medicine). In some parts of India, the clean nodules of a variety of ghatti called Spiro, are eaten as a luxury product and are considered a sign of wealth and prosperity. It has been used throughout households and industry as a good adhesive. Other uses include hair gel, print industry in India particularly screen printing, confectionary and by the petroleum industry as a drilling mud conditioner. The newly processed gum ghatti (GATIFOLIA SD) gives a solution of moderate viscosity which falls between gum arabic and gum karaya. By utilising the excellent emulsifying properties and the complete solubility of GATIFOLIA SD it has been possible to show that it can be used in many emulsified products as an emulsifier in formulations which are difficult to stabilise using gum arabic. Examples of various formulations are given below.
17.5.1
Beverage emulsion
Formulation 17.1 MCT Ingredients Oil (MCT) Gum ghatti Citric acid Benzoate Ion exchange water
% 20 5 0.12 0.13 to 100
Gum ghatti
489
Preparation The emulsion is prepared using Nanomiser (Laboratory scale high pressure hom*ogeniser, Yoshida Kikai Co., Ltd) at 50 MPa, 2 passes. Formulation 17.2 Orange oil±ester gum Ingredients Oil Orange oil Ester gum Gum ghatti (GHATIFOLIA) Citric acid Benzoate Ion exchange water
% 14 7 7 5 0.2 0.1 to 100
Preparation 1. The oil phase is prepared by mixing ester gum and orange oil at 1:1 ratio. 2. The oil phase is then added to the aqueous phase, containing citric acid and sodium benzoate. 3. The emulsion is prepared using Nanomiser (Laboratory scale high pressure hom*ogeniser, Yoshida Kikai Co., Ltd) at 50 MPa, 2 passes. Formulation 17.3 Orange oil, ester gum, -carotene, MCT Ingredients Oil Orange oil Ester gum MCT -carotene Gum ghatti (GHATIFOLIA SD) Citric acid Benzoate Ion exchange water
% 14 4 4 4 2 5 0.2 0.1 to 100
Preparation 1. The oil phase is prepared by mixing ester gum and orange oil at 1:1 ratio. 2. The oil phase is then added to the aqueous phase, containing citric acid and sodium benzoate, followed by the addition of MCT. 3. The emulsion is hom*ogenised using Nanomiser (Laboratory scale high pressure hom*ogeniser, Yoshida Kikai Co., Ltd) at 50 MPa, 2 passes.
17.5.2
Mayonnaise type dressing (70% fat)
Formulation 17.4 Ingredients 1. Corn oil 2. Vinegar
% 70 4.5
490 3. 4. 5. 6. 7. 8. 9. 10.
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Apple vinegar Lemon fruit juice Gum ghatti (GHATIFOLIA SD) SAN ACE Sugar Salt Sodium glutaminate Ion exchange water
7.0 2.0 2.0 0.15 1.0 2.5 0.1 to 100
Note: typical emulsifier used in the above formulation is egg white at 10%. Preparation 1. Slowly add ingredients 5±9 into water while stirring. 2. Add ingredients 2±4 and continue stirring the blend. 3. Add egg yolk at the end of this step. 4. Slowly add oil and stir blend. 5. Emulsify using a colloid mill.
17.5.3
Dressings (30% fat)
Formulation 17.5 Ingredients 1. 2. 3. 4. 5. 6. 7. 8.
Corn oil Vinegar Gum ghatti (GHATIFOLIA SD) SAN ACE Sugar Salt Sodium glutaminate Ion exchange water
% 30 5.3 0.1 0.2 3.0 5.0 0.4 to 100
Note: typical emulsifier used in the above formulation is egg white at 0.5%. Preparation 1. Slowly add ingredients 5±7 into water while stirring. 2. Add ingredients 2±4 and stir blend. 3. Slowly add oil and stir blend. 4. Emulsify using a colloid mill or hom*o-mixer.
17.5.4
Butter cream
Formulation 17.6 Ingredients
%
Shortening Margarine Sugar syrup GATIFOLIA SD
30 25 20 0.2
Gum ghatti Milk flavour No. 71005 Carotene base No. 80
491
0.2 0.02
Preparation 1. Slowly add GATIFOLIA SD into sugar syrup while stirring the blend. 2. Add flavour and carotene into the syrup and stir blend. 3. Add the syrup into shortening/margarine mixture and stir blend.
17.6 Regulatory status South America Argentina, Bolivia, Chile and Peru: Not permitted Brazil: Permitted as an emulsifier to aid hom*ogenising flavours or to incorporate them in food products. No maximum level specified. Colombia: Permitted as a natural emulsifier in foodstuffs. Ecuador: Permitted as a natural emulsifier, stabiliser and thickener. Guatemala: Permitted in food as emulsifier, stabiliser and thickener. Maximum level in soft drink and non-alcoholic beverages specified at 0.2%. Mexico: Permitted in all foods as a natural emulsifier, stabiliser, thickener and gelling agent. Uruguay: Permitted as an emulsifier for flavours with no maximum level specified. Venezuela: Permitted as a natural emulsifier, stabiliser and thickener. No maximum level specified for carbonated beverages and sweets. Instant drink powder should not exceed 0.1%. Asia China: Permitted in food as an emulsifier for flavours. No maximum level specified (an appropriate amount as practically needed). India: Bureau of Indian Standards (BIS) ± IS 7239:1974 (Food Grade) Permitted in food as a natural emulsifier, stabiliser and thickener as detailed below: · Stabiliser and emulsifier in sugar-boiled confectionery, processed cheese spread with no maximum level set. · Emulsifier in chewing gum, bubble gum, malted milk food with no maximum level set. · Stabiliser in yoghurt, milk ices and milk lollies with maximum level set at 0.5%. · Emulsifier and/or stabiliser in ice candy, ice cream, kulfi, chocolate ice cream, ice lollies or edible ices with maximum level set at 0.5%. · Emulsifier, stabiliser and thickener in frozen desserts with no maximum level set. · Emulsifier, stabiliser and thickener in fat spread table margarine, bakery and industrial margarine, wafer biscuits and flavouring agent with no maximum level set.
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South Korea: Permitted in all foods (unless specifically prohibited for use in a compositional standard) as natural food additives with no specific function defined. Singapore: Permitted in all foods (unless specifically prohibited for use in a compositional standard and in unstandardised foods) as natural emulsifier and stabilizer with no specific function defined. United States Permitted in food as an emulsifier, emulsifier salt in · Beverages and beverage bases, non-alcoholic with a maximum level set at 0.2%. All other food categories at a maximum level of 0.1%. The following regulations are relevant to the use of gum ghatti in the United States. · Code of Federal Regulations(CFR), Food and Drug Administration, Department of Health and Human Services CFR ± Title 21, Code ± 184.1333 · The Flavor and Extract Manufacturers Association FEMA ± GRAS status, Limitation ± Stabilizer · Chemical Abstracts Service CAS ± 009000-28-6 · Priority Based Assessment of Food Additives PAFA ± 2149 · Registry of Toxic Effects of Chemical Substances RTECS ± LY8935000 Japan Permitted in all foods as natural food additives with no specific function defined and subject to the following specification: · Identification test 1: When 1 g is dispersed in 5 ml of water it forms a viscous, adhesive mucilage. · Identification test 2: To 5 ml of 1 in 100 solution of the sample (filter through diatomaceous earth if necessary) add 0.2 ml of dilute lead subacetate TS. A small or no precipitate is formed, but an opaque flocculent precipitate is produced upon the further addition of 0.5 ml of ammonia TS. · Identification test 3: A 1 in 50 solution of the sample filtered through diatomaceous earth is levorotatory. · Heavy metals: Not more than 4 ppm. · Lead: Not more than 10 ppm. · Arsenic (as As2O3): Not more than 4 ppm. · Loss on drying (105 ëC, 5 hours): Not more than 14%. · Ash content: Not more than 6%. · Acid insoluble ash: Not more than 1%. · Viable bacteria: Not more than 10,000/g. · Escherichia coli: Negative. Europe Not permitted.
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Russia Permitted as stabiliser, thickener and gelling agent. Not defined for particular oodstuff and no maximum level set. South Africa Permitted as an emulsifier in condiments and sauces, soft drinks, breakfast cereals, flour confectionery and cakes mixes with no maximum level set. Australia Gum ghatti falls under the list of approved herbs.
17.7 References 1.
ASPINALL GO, BHAVANADAN VP, CHRISTENSEN TB
(1965) `Gum ghatti (Indian gum)', J.
Chem. Soc., 2677±2684. 2.
3. 4. 5. 6. 7. 8.
9.
10.
11. 12.
13.
14.
et al. (2002) `New structural features of the polysaccharide from gum ghatti (Anogeissus latifola)', Carbohydrate Research, 337: 2205±2210. ASPINALL GO (1980) `Chemistry of cell wall polysaccharides', in The Biochemistry of Plants (vol 3), Preiss J (ed.), Academic Press, New York, pp. 473±500. ELWORTHY PH, GEORGE TM (1963) `The molecular properties of ghatti gum: A naturally occuring polyelectrolyte', J Pharm. Pharmacol., 15: 781±793. JEFFERIES M, PASS G, PHILLIPS GO (1977) `Viscosity of aqueous solutions of gum ghatti', J. Sci. Food Agric., 28: 173±179. JEFFERIES M, PASS G, PHILLIPS GO, ZAKARIA MB (1978) `Effect of metal-ion content on viscosity of gum ghatti', J. Sci. Food Agric., 29: 193±200. JEFFERIES M, KONADU EY, PASS G (1982) `Cation effects on the viscosity of gum ghatti', J. Sci. Food Agric., 33: 1152±1159. AL-ASSAF S, PHILLIPS GO, WILLIAMS PA (2005) `Studies on acacia exudate gums. Part I: the molecular weight of Acacia senegal gum exudate', Food Hydrocolloids, 19: 647±660. BISWAS B, BISWAS S, PHILLIPS GO (2000) `The relationship of specific optical rotation to structural composition for Acacia and related gums', Food Hydrocolloids, 14: 601±608. BISWAS B, PHILLIPS GO (2003) `Computation of specific optical rotation from carbohydrate composition of exudate gums Acacia senegal and Acacia seyal', Food Hydrocolloids, 17: 177±189. WILLIAMS PA, PHILLIPS GO (2000) `Gum arabic', in Handbook of Hydrocolloids, Phillips GO, Williams PA (eds), CRC Press, Cambridge, pp. 155±168. OSMAN ME, MENZIES AR, WILLIAMS PA, PHILLIPS GO, BALDWIN TC (1993) `The molecular characterization of the polysaccharide gum from Acacia senegal', Carbohydrate Research, 246: 303±318. CONNOLLY S, FENYO JC, VANDEVELDE MC (1987) `The effect of pronase on the aminoacid composition of gum arabic', Comptes Rendus des Seances de la SocieÂte de Biologie et de ses Filiales, 181: 683±687. WILLIAMS PA, PHILLIPS GO, STEPHEN AM, CHURMS SC (2006) `Gums and Mucilages', in TISCHER CA, IACOMINI M, WAGNER R,
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15.
16.
17. 18.
Handbook of hydrocolloids Foood Polysaccharides and Their Complexes, 2nd edn, Stephen AM, Williams PA (eds), Taylor & Francis, London, pp. 455±495. AL-ASSAF S, PHILLIPS GO, AOKI H, SASAKI Y (2007) `Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM (TM)): Part 1 ± Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier', Food Hydrocolloids, 21: 319±328. AOKI H, AL-ASSAF S, KATAYAMA T, PHILLIPS GO (2007) `Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUM (TM)): Part 2 ± Mechanism of the maturation process', Food Hydrocolloids, 21: 329±337. DICKINSON E (2003) `Hydrocolloids at interfaces and the influence on the properties of dispersed systems', Food Hydrocolloids, 17: 25±39. AL-ASSAF S, AMAR V, PHILLIPS GO (2008) Characterisation of gum ghatti and comparison with gum arabic, in Gums and Stabilisers for the Food Industry 14, Williams PA, Phillips GO (eds), Wrexham, Royal Society of Chemistry, pp. 280± 290.
18 Other exudates: tragancanth, karaya, mesquite gum and larchwood arabinogalactan Y. LoÂpez-Franco and I. Higuera-Ciapara, Centro de InvestigacioÂn en AlimentacioÂn y Desarrollo, Mexico, F. M. Goycoolea, Universidad de Santiago de Compostela, Spain and Centro de InvestigacioÂn en AlimentacioÂn y Desarrollo, Mexico and W. Wang, Andi-Johnson Konjac C. Ltd., China
Abstract: The collection, processing and trading of plant exudate gums, other than gum arabic, and the production of arabinogalactan from the heartwood of Western larch tree represent an important economic activity in many regions of the world. What this family of materials shares in common is that they are comprised of highly branched heteropolysaccharide structures. This chapter addresses the manufacture, chemical structure, functional properties, main applications and regulatory issues for three wellestablished hydrocolloids, namely gum tragacanth, gum karaya and larchwood arabinogalactan along with those of mesquite gum, whose full potential utilization is still to be exploited in several fields of application. Key words: gum tragacanth, gum karaya, mesquite gum, larchwood arabinogalactan, exudates.
18.1 Introduction Although gum arabic is by far the most important plant exudate hydrocolloid, there are other related gums that have retained their economic and technological importance for centuries despite the availability of several new alternative industrial hydrocolloids. In fact, natural plant gums are the most widely used and traded non-wood forest products other than items consumed directly as food, fodder and medicine (Upadhayay, 2006). Their collection by hand still
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represents a source of income for millions of people, dwelling in rural areas mostly in Africa, India, Iran, Turkey, and to a less extent, in Mexico. In northern USA, the extraction of arabinogalactan from larch trees also represents an important economic activity. Gums are exuded by the bark of trees in the form of tear-like, striated nodules or amorphous lumps, which are vitrified upon drying, thus forming hard, glassy lumps (gum karaya and mesquite gum) or tough opaque thin ribbons (gum tragacanth) of different colours, ranging from red-amber for mesquite gum, pale gray to dark brown for karaya gum, and white to dark brown for tragacanth. In general, the gums are produced by the stem under conditions of heat and drought stress, partly as a natural phenomenon (as part of the normal metabolism of plants) and partly as a result of injury to the bark or stem (due to fungal or bacterial attack) by a process known as gummosis. The other type of polysaccharide gum addressed in this chapter is not strictly an exudate like the others, but it is extracted from the vacuoles of the heartwood of the Western larch tree and related species. Chemically, these materials are known to be comprised to varying extents either by arabinogalactan (AG) heteropolysaccharides (e.g., larchwood arabinogalactan) or occur as complex mixtures of other acetylated polysaccharides such as rhamnogalacturonan (e.g., gum karaya); mixtures of galacturonan regions and type II AG as gum tragacanth (Verbeken et al., 2003) or macromolecular complexes of type II AG and proteoglycans (arabinogalactan-protein, AGP) comprising ca. 4% of protein such as mesquite gum (Goycoolea et al., 2000). As a consequence of this chemical structural diversity, these polysaccharides exhibit very different functional properties and thus they have found applications in various fields. The individual properties of gum tragacanth, gum karaya, mesquite gum and larch arabinogalactan are discussed throughout the various sections of this chapter.
18.2
Manufacture
18.2.1 Gum tragacanth Gum tragacanth was first described by Theophrastus several centuries before Christ. The name tragacanth, from the Greek tragos (goat) and akantha (horn), probably refers to the curved shape of the ribbons, the best grade of commercial gum. The gum is obtained from small shrubs of the Astragalus genus, comprising up to 2000 species indigenous to mountain areas of south west Asia from Pakistan to Greece (Whistler, 1993). A. gummifer was considered to be the main tragacanth yielding species, but a field survey established that A. microcephalus was the principal source of the gum (Dogan et al., 1985); A. kurdicus and A. gossypinus have also been documented as botanical sources. The plants are small, low bushy perennial shrubs having a large tap root along with branches. The root and lower stem are tapped for gum.
Other exudates
497
The main areas of commercial production are the arid and mountainous regions of Iran (accounting for ~70% of the supplies) and the Anatolia region in Turkey (Anderson, 1989), and in lesser amounts in Afghanistan and Syria. In the past, several thousand tonnes of tragacanth were used in food, pharmaceutical and technical applications. However, as a result of very high costs, erratic supply and strong competition from xanthan gum, demand for the gum fell dramatically from several thousand to 200±300 tonnes per year (Anderson, 1989). Iran's recovery in the gum tragacanth export market suggests that, with a correct understanding of the world market and supply of premium product, there is a vast prospect for a bigger and better market for this gum. Trade sources in London have quote prices (mid-1995) at around US$22/kg free on board (FOB) for the top grade (Ribbon no. 1), US$16/kg for Ribbon no. 4 and falling to US$3±4/kg for the lowest grades. Current quoted price for gum from Azerbaijan is US$30/kg. 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 exudate is produced spontaneously on the bark of the shrub, but both the yield and quality are often increased by making incisions in the tap root and lower stem. Abundant rainfall prior to the tapping season, and dry conditions during the harvesting season, constitute optimum climate conditions for gum production. Tapping is carried out in May or June with subsequent collection in August and September (after 6 weeks) for ribbon grades and August to November for Flake grades (Wareing, 1997). The gum is obtained in two basic physical forms, namely ribbons (superior quality) and flakes (inferior quality). These two forms are obtained from different sub-species of the shrub. Both types of shrubs normally do not grow in the same locality (Robbins, 1988). After collection, the gum is sorted by hand by the natives and carried to sorting centres where it is graded into several grades of ribbons and flakes and exported. The Iranian grading system is more clearly defined than that of Turkey and comprises nine different grades. The most commonly used Iranian qualities are ribbons 1 and 4, mixed ribbon and flakes 27, 28 and 55, while in Turkey there are four grades, namely, Fior Extra and Fior for ribbons and Bianca and Pianto for flakes. The best qualities are regarded as those with higher viscosity, good solution colour and low microbiological limits. Blending is necessary to ensure the desired properties. Processors in the US and Europe purchase material following approval of pre-delivery samples. Quality control inspections of each incoming batch are necessary to ensure powder blends meet well-defined specifications for powder and solution colour and viscosity. Food applications for sauces, dressings, icings, and confectionery normally use mixed ribbon or flake grades. Lower qualities are used where solution colour is less important and where thermal processing, pH and/or the soluble solids level are sufficient to prevent microbial proliferation in the final product. Limited mechanical treatment to remove foreign matter may be carried out in the exporting countries but no further processing is undertaken. Importers in the US and Western Europe, primarily in the UK and Germany, ensure consistent
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quality standards are maintained for the powdered material after milling. The best ribbon grades have low total viable counts of bacteria comprising mainly resistant spores from the soil and airbone contamination. These problems were previously controlled through fumigation with ethylene oxide (ETO). This process was forbidden around 1987 in the treatment of gum destined for food uses, because of carcinogenicity of ETO. The alternative methods of bringing down the microbial counts also cause chemical changes in the gum and accordingly are not acceptable (Anderson and Weiping, 1994). In the US propylene oxide is allowed but its efficacy is limited and permission for its use may be revoked.
18.2.2 Gum karaya Karaya gum, also known as sterculia gum, is the dry exudate of Sterculia urens (Roxburgh), a large and bushy tree. The majority of commercial material is obtained from wild S. urens trees, indigenous to central and northern India and more than half of the gum is produced in the state of Andhra Pradesh. Other significant sources are from S. setigera, in Senegal and Mali, and minor supplies from S. villosa in Sudan, India and Pakistan. The history of gum karaya trading, in contrast to tragacanth, is quite recent. It goes back to the 1920s when the gum used to be sold as an adulterant to tragacanth. World production and usage is currently 1500 tonnes per year. The major users of gum karaya are the US, France and the UK. Minor quantities are imported into Japan, Belgium, Germany and other European countries (Robbins, 1988). The export of Indian gum karaya declined from 4000 tonnes in 1982 to 1000 tonnes in 1992 and has remained roughly constant up to 2002, mostly due to a sharp decrease in the number of trees available for tapping due to unsustainable harvesting methods (Upadhayay, 2006). Over the past two decades, the prices in India have risen as a consequence of the increase in demand and shortage in production. In turn, exports from Senegal and other countries increased their production in the late 1980s to 1500 tonnes per annum and this has resulted in more competition and more stable prices. Indicative FOB prices quoted by importers in London for Indian karaya (mid-1995) are in the range US$2250± 6000/tonne according to grade. Fair average quality (FAQ) gum is about US$3000/tonne. For production, the trees are incised or tapped and exudation begins immediately and continues for several days forming irregular lumps (or tears) which may weigh more than 1 kg, and large trees can produce up to 4.5 kg (Whistler, 1993). The exudate is allowed to dry on the tree and is later collected, broken, cleaned and sorted. The highest quality of raw gum collected is during the hot months of April, May and June. In September, the gum is again picked. This autumn crop has a greyish colour and is normally less viscous. The gum is cleaned to remove bark and foreign matter (BFM) before sorting. Commercially available quality grades are hand-picked selected (HPS), superior no. 1, no. 2 and no. 3 (FAQ), and siftings (Verbeken et al., 2003). BFM can be found in
Other exudates
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white to very light tan HPS and superior no. 1 grades in proportions of 0±0.5% and 1.0±2.0%, respectively; 1.5±3.5% in very light tan superior no. 2; 2.5±4.0% in tan FAQ gum and 5.0±7.0% in the brown colour siftings (Wareing, 1997). Gum karaya is processed to remove impurities such as bark, stones, fibres and sand. It is then milled, blended and classified according to mesh, viscosity and purity. The gum is offered as granules or in powder form. The granule size ranges from 4±8 mesh and 8±14 mesh and powder size is 160 mesh with viscosity ranging from 500±1200 cps. The powder is light to pinkish gray and has a slight acetic taste and odour. The microbial quality of this gum is similar to that of other exudates, and its use in sauces and dressings is safe, as the low pH of these products and the heat treatments they are regularly subjected to are sufficient to ensure safety.
18.2.3 Mesquite gum Mesquite trees are leguminous plant trees that are widespread in arid and semiarid regions of the world and account for one of the major plant species in such places. In fact, the genus Prosopis comprises about 44 different species that grow mostly in North and South America, and also in Australia, Africa, and eastern Asia. In Mexico, around ten species are found, of which the most abundant is P. juliflora (Vernon-Carter et al., 2000), which has also been suggested to correspond with P. laevigata. This species grows from the coastal areas of the Pacific Ocean in the Mexican state of Sinaloa to Panama, in the centre and south of Mexico, reaching all the way to the south-eastern United States under environmental conditions that range from subhumid to areas with an average rainfall of up to 1500 mm. It is well documented that the bark of Prosopis spp. produces an exudate known as mesquite gum as a response to insect attack, wounding or physiological stresses such as severe water and heat. The gum could be defined as `the dried gummy exudation obtained from the stems and branches of Prosopis species'. By contrast with commercial gum arabic, karaya and tragacanth gums, mesquite exudate is not an established hydrocolloid in the world market. However, the gum was widely used by the Indian cultures of the Mexican Northwest (Seri and Yaqui) and southwestern United States (Papago and Pima) (Felger, 1977), mainly as a sweet and as a medicinal aid to prepare eye drops (Felger and Moser, 1974). Presently, mesquite gum, known in Sonora as chuÂcata, is used in few household applications. However, in the past, mesquite gum has been used extensively in food applications and has been traditionally considered as a `substitute or adulterant of gum arabic, of inferior quality due to its darker colour' (Smith and Montgomery, 1959). In Mexico, there are two main regions where mesquite gum is produced, namely, in the desert plains of the northwestern state of Sonora, where the predominant source is P. velutina and in the lowlands of the Northeastern state of San Luis Potosi, where the gum is sourced mostly from P. laevigata. The structural and functional properties of mesquite gum have been studied extensively mostly by two independent
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Mexican research groups, namely, the group led by Dr J. Vernon-Carter, at Universidad AutoÂnoma Metropolitana, that has worked with the gum from San Luis Potosi, and our group that has worked with the material from Sonora. The production season of mesquite gum in Sonora begins during the late spring and early summer months (May±July), ending with the summer rains at late July/early August. At present, the collection of mesquite gum is not properly organized and there is not a quality grading system to sort it. Nevertheless, as with other gum exudates, the nodules can be classified by size, colour and by the contents of bark and foreign matter. In some cases dark gum nodules are eliminated by their high tannin content depending on their intended use (OrozcoVillafuerte et al., 2000). Ultrafiltration studies in mesquite gum from Sonora showed that this technology was feasible to reduce the contents of naturally occurring tannin compounds (Goycoolea et al., 1998). Quantitative analysis of the removed tannins indicated that up to ~62% of the original tannin contents can be removed using a hollow fibre membrane of 10 kDa molecular weight cutoff without compromising the emulsification capacity of mesquite gum. The only information available on production of mesquite gum from wild plantations comes from a few field studies that have tried to estimate the availability of the gum in the two collection regions in Mexico. In San Luis Potosi, it has been estimated that the potential production in an area of 600 km2 is ~2000 tonnes p.a. (Vernon-Carter et al., 2000), while in Sonora the estimated total annual production was nearly half as much, at ~800 tonnes (Goycoolea et al., 2000). These figures allow us to conclude that the potential production of mesquite gum from wild mesquite forests could fulfil the 2004 demand for gum arabic which was in the order of ~1417 tonnes (SecretarõÂa de EconomõÂa, 2004). Unfortunately, to date mesquite gum is neither produced on a large scale, nor are there commercial plantations, extraction methods or efficient collection systems. Besides, the price at which gum arabic is currently imported to Mexico at ~$US3200/tonne (SecretarõÂa de EconomõÂa, 2005) renders it economically unfeasible to collect mesquite gum from the wild areas. In light of the above, alternative production methods have been investigated. In vitro studies for culturing of P. laevigata and laboratory conditions for the gum production by stem segments (Orozco-Villafuerte et al., 2000) have demonstrated that application of combined environmental conditions (temperature increase) and biotic elicitors, can be utilized for increasing mesquite gum production with similar characteristics to those produced in situ by wild trees (Orozco-Villafuerte et al., 2005).
18.2.4 Larchwood arabinogalactan Arabinogalactan is particularly abundant in larchwood (genus Larix) and especially in Western larch (Larix occidentalis) from whose heartwood AG can be extracted in high yield (Stephen, 1983). Extraction of water-soluble arabinogalactan from shavings of the butt of the Western larch tree was first described in 1898 (Trimble, 1898), though no quantitative data was reported.
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During the twentieth century this material continued to receive economic and scientific interest and later it was found that arabinose and galactose were its main constituents (Wise and Peterson, 1930; Nikitin and Soloviev, 1935). Subsequent efforts at large-scale commercialization were hampered by the economics of extraction and purification (Anderson, 1967). However, technological improvements were made and presently larchwood arabinogalactan is produced on an industrial scale and its market developed as food fibre and for biomedical and healthcare applications (Gallez et al., 1994). The most practical method of extraction consists of hot water treatment using countercurrent flow of the drilled or chipped heartwood (Adams and Ettling, 1973) from Dahurian larch (Larix dadurica), Siberian larch (Larix siberica), Eastern larch (Larix laricina), European larch (Larix deciduas), Japanese larch (Larix leptolepsis) and Western larch tree (Larix occidentalis), which contains quantities up to 35% of the arabinogalactan in vacuoles and it is the most abundant and available source (Stephen and Churms, 1995). Arabinogalactan is available commercially in ultrafiltered (AG±UF) and in food grades (AG±FG) (Christian et al., 1998). Conditions used to isolate arabinogalactan from L. occidentalis include extraction at 70 ëC for several days and use of magnesium oxide (Adams and Ettling, 1973). The Swiss company Lonza Inc. (which has recently acquired Larex Inc.), is presently the major manufacturer of larch arabinogalactan for commercial applications in the world, including medicinal and food supplements. The company owns patents on composition and extraction processes for a range of AG products of varying qualities, depending on the application fields they are intended for. Their industrial facility has a production capacity for 3.7 million metric tonnes (dry weight) of arabinogalactan. The amount of arabinogalactan that could be obtained from 1% of larch trees each year in the United States is 4.6 million metric tonnes. The intellectual property of the processes to produce this gum from larch trees is covered under various patents (DeWitt, 1989; Adams and Knudson, 1990; Price et al., 1995).
18.3 Structure 18.3.1 Gum tragacanth The structures of polysaccharides of gum tragacanth were investigated in detail by James and Smith (1945a, 1945b) followed by Aspinall and Baillie (1963a, 1963b). The gum is a slightly acidic salt occurring naturally with calcium, magnesium and sodium cations (Whistler, 1993). Gum tragacanth has a molecular weight of about 840 kDa, calculated by Svedberg's method and formula and an elongated shape of 450 nm by 1.9 nm, providing a high viscosity. Astragalus species (A. gummifer, A. microcephalus and A. kurdicus) have 1± 3.6% of protein with the proportions of the major amino acid constituents (Asp, Hyp, Ser, Pro and Val) also varying (Whistler, 1993; Stephen and Churms, 1995).
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Gum tragacanth is composed of two major components: tragacanthic acid and a small amount of a water soluble arabinogalactan and the bassorin fraction which is insoluble but swells in water to form a gel. The water soluble tragacanthin, accounts for 30±40% of the gum and is reported as a neutral, highly branched arabinogalactan (of type II) comprising (1!6)- and (1!3)linked core chain containing galactose and arabinose (both in furanose and pyranose forms) and side groups of (1!2)-, (1!3)- and (1!5)-linked arabinose units occurring as monosccharide or oligosaccharides (Stephen and Churms, 1995; Tischer et al., 2002). Acid hydrolysis revealed that tragacanthin (Astralagus gummifer) contains neutral monosaccharides such as L-fucose (LFuc), L-(L-Ara), D-xylose (D-Xyl), D-mannose (D-Man), D-galactose (D-Gal) and D-glucose (D-Glc) in a 3:52:29:6:5:5 molar ratio and the arabinogalactan contained L-rhamnose (L-Rha), L-Fuc, L-Ara, D-Xyl, D-Man, D-Gal and D-Glc in a 1:1:68:2:5:22:1 molar ratio (Tisher et al., 2002). This polysaccharide component is soluble in a mixture of ethanol-water (7:3). Recently, intrinsic viscosity [], molecular weight MW, and radius of gyration hS 2 iz1=2 of tragacanthin from Astragalus gossypinus were calculated to be, [] 9.077 10±3 MW0.87 (mL g±1), hS 2 iz1=2 0:021 MW0.59 (nm) in the range of MW from 1.8 105 to 1.6 106. The conformational parameter of tragacanthin were 1111 g mol for molar mass per unit contour length (ML), 26 nm for persistence length (q) and 1.87 ratio of RGRH (Mohammadifar et al., 2006). Bassorin, a pectic component (Fig. 18.1), has a chain of (1!4)-linked -Dgalacturonic acid units some of which are substituted at O-3 with -Dxylopyranosyl units and some of these being terminated with D-Gal or L-Fuc. Bassorin appears to contain some methyl groups. It was reported that for most species of Astragalus, the insoluble part has less methoxyl and galacturonic acid than the soluble part. Pectic component is dissolved partly in dilute aqueous sodium hydroxide. Grade precipitation of alkali soluble material gave fractions similar to those isolated from the water-soluble proportion of the gum. Bassorin and tragacanthin have quite different rheological properties: while 1% bassorin solution at 25 ëC shows a high viscosity gel-like structure, tragacanthin solution behaves like semi-dilute to concentrated solution of entangled, random coil polymers (Mohammadifar et al., 2006).
Fig. 18.1
Structure of gum tragacanth pectic component (Astragalus spp) (from Stephen and Churms, 1995).
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18.3.2 Gum karaya Chemically, gum karaya is a partially acetylated polysaccharide of the substituted rhamnogalacturonoglycan type. The exudate occurs in the salt form containing calcium and magnesium ions. It has a branched structure and a very high molecular weight (ranging from 9,000 to 16,000 kDa) (Stephen, 1990; Whistler, 1993; Stephen and Churms, 1995). It contains about 37% uronic acid residues and approximately 8% acetyl groups. Due to these acetyl groups gum karaya is insoluble and only swells in water. The native acetylated gum assumes a rather compact and branched conformation in aqueous solution. In contrast, the fully deacetylated karaya gum assumes a more expanded conformation and behaves as a random coil (Le Cerf et al., 1990). After acid hydrolysis gum karaya produces D-galacturonic acid (D-GalA), DGal, L-Rha and small proportions of D-glucuronic acid (D-GlcA). The sugar composition of gum karaya has been given as (in wt%): 37.6% uronic acids; 26.3% D-Gal and 29.2% L-Rha (Aspinall et al., 1986). However, the sugar composition of the gum is dependent on the botanical sources and age of the tree and there is also more than average variability in the proportions of amino acids in the proteinaceous components. It is worth pointing out that gum karaya has a much higher rhamnose content than other commercial exudate gums. More detailed structural studies after partial acid hydrolysis, acetolysis, methylation analysis and Smith degradation, suggest that the polysaccharide component of karaya corresponds with that shown in Fig. 18.2 (Stephen and Churms, 1995).
Fig. 18.2
Structure of gum karaya (Sterculia urens) (from Stephen and Churms, 1995).
18.3.3 Mesquite gum Mesquite gum is the neutral salt of a complex acidic branched polysaccharide formed by a core of -D-Gal residues comprising a (1!-3)-linked backbone with (1!6)-linked branches, bearing L-Ara (pyranose and furanose rings form), D-glucuronic acid and 4-O-methyl- -D-glucuronic acid (Fig. 18.3) (White, 1946, 1947a, 1947b, 1948; Cuneen and Smith, 1948a, 1948b; Akher et al., 1952; Aspinall and Whitehead, 1970a, 1970b). On acid hydrolysis mesquite gum from P. velutina yields L-Ara and D-Gal as main carbohydrate residues with Ara/Gal ratio between 7.32 and 10.61, and traces of D-Glc, D-Man and D-Xyl were also detected (LoÂpez-Franco et al., 2008). 1H NMR spectroscopy studies have recently been used to analyse the structure of gum from P. velutina (Rinaudo et
Fig. 18.3 Primary structure for the carbohydrate component of mesquite gum (from Aspinall and Whitehead, 1970a, 1970b); R Ara-(1!2)-Ara-(1!2)-Ara-(1!2)-Ara-(1!2)-Ara-(1!4)-Ara-(1!3)-Ara-(1 y Ara-(1!6)-Gal-(1!3)-Ara-(1!3)-Ara-(1.
Other exudates
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Fig. 18.4 Twisted hairy rope structure proposed to AGPs from gum arabic (A. senegal) (from Qi and Lamport, 1991; reproduced with permission of American Society of Plant Biologists).
al., 2008), and it was confirmed that L-Rha is not present in mesquite gum, in contrast with gum arabic whose spectrum shows the corresponding signal for this residue at ~1.32 ppm. By contrast, in the material from P. laevigata, a small concentration of L-Rha of ~1.3 mol% has been reported (Orozco-Villafuerte et al., 2003). In addition to the polysaccharide component, mesquite gum contains a small amount of protein (2±4%) (Fincher et al., 1983; Goycoolea et al., 1998; OrozcoVillafuerte et al., 2003; LoÂpez-Franco et al., 2004) which plays an important role in its emulsification properties (Vernon-Carter et al., 1996b, 1998; Goycoolea et al., 1995). The adequacy of models that explain the tertiary structure of mesquite gum has not yet been assessed experimentally. However, light scattering studies have shown that mesquite gum (P. velutina) with molecular weight of 386,000 g/mol and radius of gyration (RG) of 50.47 nm and hydrodynamic radius (RH) of 9.48 nm (LoÂpez-Franco et al., 2004), resembles a polydisperse macrocoil in agreement with the `twisted hairy rope' proposal AGP for gum arabic (Fig. 18.4) (Qi and Lamport, 1991). The intrinsic viscosity of mesquite gum has been recently given as [] 1.47 10±2 MW0.50 (mL g±1) (Rinaudo et al., 2008). From the absolute values of the constants of the Mark±Houwink relation, it follows that a very low intrinsic viscosity is obtained in consideration of the molecular weight; this is directly related to the highly branched structure.
18.3.4 Larchwood arabinogalactan Arabinogalactan from larchwood is known to be composed of two main fractions, the more abundant fraction (70±95%) being the high molecular weight
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(MW ~ 37±100 kDa), AG-A, and a proportionally less abundant (5±30%) low molecular weight fraction (MW ~ 7.5±18 kDa), AG-B (Swenson et al., 1969; Clarke et al., 1979). It is unclear whether a typical A/B ratio exists and differences in reported ratios have been attributed to analytical methodology (Jones and Reid, 1963). The principal material available commercially is the ultrafiltered product, which is known to correspond with AG-A (Ponder and Richards, 1997a). By contrast with other plant arabinogalactans, AG-A is protein free (Clarke et al., 1979; Prescot et al., 1995). Chemically, arabinogalactans from larchwood have a general structure given by a backbone of (1!3)-linked -D-galactopyranosyl units that account for about one-third of the molecule, each of which contain a side chain at position C6. Most of these side chains are galactobiosyl units containing a (1!6)- -Dlinkage. Another side chain type that occurs is a single L-Ara unit or 3-O-( -Larabinopyranosyl)--L-arabinofuranosyl units. Less frequent is a single -DGalp or -L-Araf or a dimer -L-Arap-(1!3)--L-Araf (Ponder and Richards, 1997b). The side group distribution is not uniform and the overall ratio of Lgalactose to L-arabinose is ~6:1. Traces of uronic acid units have also been reported as part of the structure of AG-A (Ponder and Richards, 1997a). The representative chemical structure of larchwood AG is shown in Fig. 18.5. Purified AG-A has been suggested to occur naturally as ordered assemblies of molecules that can be disrupted by alkali to form individual, unassociated molecules, i.e., disordered AG (DAG) (Ponder and Richards, 1997b). This order± disorder transition can be reversed by drying or freezing. Parallel studies have shown that when AG-A (37 kDa) is treated with sodium hydroxide solutions of 0.5 M or greater and 0.1 M sodium borohydride, the average molecular weight of the resulting arabinogalactan falls approximately four-fold to yield fractions of AG (~9 kDa) (Prescot et al., 1995). Based on this evidence it has been proposed that larch AG consists of a series of subunits joined through au unknown type of linkage which is susceptible to cleavage at low alkali concentrations and moderate temperatures. 13C-NMR spectra of AG-A (37 kDa) and AG (9 kDa) are identical except that broader spectral lines are observed in the AG-A spectrum due to its greater molecular weight. Whilst in vitro comparison of both materials using isolated asialoglycoprotein receptor shows equivalent bioactivity (Prescot et al., 1995), it has been proposed that the low molecular weight material in the crude AG extract is possibly a biological precursor of the predominant, larger molecular weight form of AG in the extract (Prescot et al., 1997). More recent Xray fibre diffraction data supports a model for a curdlan-type triple helical structure for the ordered structure of arabinogalactan (Chandrasekaran and Janaswamy, 2002), whereby a galactan triple helix can accommodate disaccharide D-Gal-(1!6)-D-Gal substituents at C6 of every D-Gal unit in the main chain. This side group attachment is not unique and it can be done in several ways while preserving the helix symmetry. Under the proposed model, the arabinogalactan molecule resembles a bottle brush. AG-B is the form of AG that exists naturally as discrete molecules. It constitutes some 5% or less of a typical AG sample and its average L-Ara
Fig. 18.5
Major structural features of a typical larchwood arabinogalactan molecule (from Ponder and Richards, 1997b).
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content is about 38 mol%. It is distinguished from DAG by GPC, having a longer retention time, and its Mw is about 7,000 to 10,000 (Simson et al., 1968; Swenson et al., 1969). Neither drying nor freezing causes it to assume a multimolecular structure, and it contained no uronic acid residues.
18.4
Technical data
18.4.1 Gum tragacanth Gum powder made from ribbon is white to light yellow in colour, 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 high grade commercial gum tragacanth powder is: Appearance: Off white to creamy coloured fine powder Loss on drying: 12% Ash: 3.0% Acid insoluble ash: 0.3% Viscosity 1% in water: 800 150 cPs Particle size: 90% through BSS 150 mesh Specifications of lower grade gum diverge from top quality gum mostly in that the colour tends to cream and yellow and viscosity values may be as low as ~280 cPs. Minimum quality and safety standards for gum tragacanth to be used in food and pharmaceutical products have been defined in the United States Pharmacopeia USP31 (USPC, 2007): Arsenic: 3 ppm Heavy metals (as Pb): 20 ppm Microbiology: Salmonella/E. coli ± absent The main inherent functional properties of tragacanth exudates are briefly discussed next. Solubility Gum tragacanth swells rapidly in either cold or hot water to form a viscous colloidal solution, which acts as a protective colloid and stabilizer. 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
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suspending agent, stabilizer or emulsifier. The viscosity of 1% solutions may range from about 100±3,500 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 3,500 cPs (1.0%, 25 ëC, 24 h, 20 rpm by Brookfield viscometer). Tragacanth highly viscous colloidal sols or semi-gels can serve as protective colloids and stabilizing agents. The high viscosity of tragacanth solutions results from the molecular characteristics of the gum, and these depend on the grade and physical form, and the manner in which it is taken up in water. For example, the same concentration of solution prepared from whole gum is more viscous than one prepared from powdered gum. Unlike many other gums, solutions of tragacanth have a very long shelf-life without loss of viscosity. The solution viscosity reaches a maximum in 24 h at 25 ëC, 8 h at 40 ëC and 2 h at 50 ëC. Fine powdered gum has shorter hydration time than coarse powder and good dispersion is needed to avoid the formation of aggregates. The maximum initial viscosity of tragacanth solutions is at pH 8, but maximum stable viscosity is at about pH ~ 5 (Stauffer, 1980). The viscosity is quite stable over a wide pH range from 2±10, particularly for the flake type of the gum (Wareing, 1997). 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 The apparent viscosity of tragacanth solutions decreases as the shear rate increases and is reversible, with the original viscosity returning upon the reduction of the shear rate. Such 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 pH 4±8, but with very good stability at both the higher pH and at the lower end of pH 2. 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, they 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% (Glicksman, 1982a). 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 and Andon (1975) reported that 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.
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Emulsification ability Gum tragacanth, regarded as a bifunctional emulsifier, is a most efficient natural emulsifier for acidic oil-in-water emulsions. It thickens the aqueous phase and also lowers the interfacial tension between oil and water. It has a reported hydrophilic lipophilic balance (HLB) value of 11.9 (Griffin and Lynch, 1972), but it is believed HLB values run from 11±13.9 depending on the grade of the gum because flake types have lower interfacial tension between oil and water than ribbon types (Anderson and Andon, 1988). 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 that has been attributed to the molecular association between both gum species (Rabbani et al., 1995). Although the precise mechanism for this interaction is still unclear, it is exploited commercially to produce superior, thin, pourable, smooth emulsions with fish and citrus oils, which also have a long shelf-life. 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 94 mL/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 pH 6. A combination 0.17% methyl and 0.03% propyl parahydroxybenzoate is effective at pH 3±9. Benzoic acid esters are also effective for maintaining solution properties throughout product preparation and shelf-life (Wareing, 1997). 18.4.2 Gum karaya Gum karaya has a slightly acetous odour and taste. The 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 the loss on drying is higher than this due to the presence of volatile substances. A typical specification for top quality commercial gum karaya is shown below (from Importers Service Corporation, NJ, USA): Appearance: Off white to buff fine powder Odour: Light acetic acid
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Taste: None Loss on drying: 20% Total ash: 5.0% Acid insoluble ash: 1.0% Particle size: 99.9% through USS 80 mesh; 98% through USS 140 mesh Viscosity 1% in water: 400 mPas Viscosity 2% in water: 8000 mPas pH 1% solution: 4.3±5.0 Salmonella: Negative E. coli: Negative Specifications for gum karaya from other manufacturers (Arthur Branwell & Co Ltd.) include maximal heavy metal contents, namely: Heavy metals (as Pb): 20 ppm Pb: 5 ppm As: 3 ppm Solubility Gum karaya is the least soluble of commercial gums and forms true solutions only at very low concentrations ( G00 moduli and no frequency dependence, thus indicating that a gel network is formed. The presence of acetyl groups in both gums seems to stabilize the gel. In turn, a separate study has shown that karaya gum forms true gels (i.e. G0 =G00 > 3) only at concentrations greater than 4% and that the addition of NaCl decreases the gel strength (Brito et al., 2005). Karaya gels studied by the latter group did not present any sharp variation in G0 or G00 with increasing temperature. pH stability The pH of a 1% solution of gum karaya 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 (Glicksman, 1982b). Above pH 8, alkali irreversibly transforms the characteristic short-bodied solution into a ropy, 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
Other exudates
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quite well and resist hydrolysis in 10% hydrochloric acid solution at room temperature for at least 8 h (Whistler, 1993). Heat stability Heating karaya dispersions changes the polymer conformation and increases the solubility, but results in a permanent viscosity loss. Maximum concentrations of 4±5% can be prepared by cold water hydration, but when heating under pressure, smooth, hom*ogeneous, translucent and colloidal solutions at concentrations as high as 18±20% can be obtained (Glicksman, 1982b). Water-binding properties Gum karaya has a strong water-binding ability. It can absorb water and swell to more than 60 times its original volume. Film-forming properties Gum karaya forms smooth films when plasticized with compounds such as glycols. Adhesive properties At high concentrations of 20±50% gum karaya in water gives heavy pastes with strong wet-adhesive properties. This enables karaya gels and pastes to resist loss of strength when diluted (Glicksman, 1982b). These are used in dental adhesives and colostomy bag sealing rings (Wareing, 1997). 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 (Le Cerf and Muller, 1994). However, karaya gels are incompatible with pyrilamine maleate, a strong hydrotrope and antihistaminic agent. Strong electrolytes or excessive acid cause a drop in viscosity, while alkalis make karaya solutions very ropy (Meer, 1980). 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.
18.4.3 Mesquite gum Unprocessed mesquite gum is available as vitrified nodules of varying size and shape and has red amber to tan colour. Dry mesquite gum is dissolved in water to form solutions which are dextrorotatory (ca. +60ë). The Mexican Ministry of Health has proposed specifications for gum intended for use in foods (SecretarõÂa de Salud, 1996). These along with the main physico-chemical characteristics
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Table 18.1 Analytical parameters for Prosopis velutina and specifications for P. laevigata gum
Appearance Color Loss on drying (%) Ash (total, %) Ash (acid insoluble (%)) Arsenic (as As) Heavy metal (as Pb) Lead Tannin (%) Starch or dextrin Insoluble matter (%) Specific rotation []D20 Total nitrogen (%) Protein (%)b Viscosity 20% (cps)c Microbiology pH Acid equivalent weight (g mol) Glucuronic acid (mol%) Arabinose (mol%) Galactose (mol%) Rhamnose (mol%)
P. velutina (hand sorted)
P. laevigataa
Vitrous nodules Red amber 9.7 0.1 2.6 0.01 NA NA NA NA 0.46 0.03 NA 0.6 0.1 + 66.7 5.3 0.7 0.1 4.6 0.6 25 Coliform negative 4.5 1282 3 71 26 ND
Vitrous nodules Red amber 15 4.0 0.5 3 ppm 40 ppm 10 ppm 2.0 Passes test 1.0 % + 77.0 0.4 0.07 2.6 0.06 NA NA NA NA 16.2 1.3d 40.4 2.04d 43.3 1.4d 1.3 0.2d
a
Maximum values are taken from specifications from Ministry of Health of Mexico, SecretarõÂa de Salad (1996), the rest of the values have been measured experimentally. b Protein = N 6.53. c At 20 ëC in 0.1 M NaCl. d From Orozco-Villafuerte et al. (2003). NA = Not available; ND= not detected
derived from various studies on the gums from P. velutina and P. laevigata are compiled in Table 18.1. Solubility Mesquite gum has extremely high solubility in aqueous medium, which can yield solutions above 50% (w/w) concentration (Goycoolea et al., 1995). It is also soluble in aqueous ethanol up to 70% ethanol, and has limited solubility in glycerol and ethylene glycol but is insoluble in organic solvents and oils (Vernon-Carter et al., 2000). Prosopis gum solutions present colours that vary from slight yellow or amber to a dark brown colour depending on the concentrations and botanical origin. Viscosity The viscosity of mesquite gum solutions even at high concentrations is very low when compared with that of other polysaccharide gums (Vernon-Carter and
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Sherman, 1980). At concentrations below 15% (w/v) the solutions have been reported as being `shear thickening' as the shear rate increased beyond 100 sÿ1, and attributed this effect to either a change in molecular shape at high shear rates or to an experimental artifact caused by turbulent flow in the coaxial cylinder geometry used. The viscosity of 20% (w/w) solutions in 0.1 M NaCl at 25 ëC was ~25±30 mPas and presented a Newtonian behaviour (Goycoolea et al., 1995), though shear thinning occurred at greater concentrations. At concentrations of 50% (w/v) mesquite gum solutions exhibited a clear nonNewtonian behaviour (Rinaudo et al., 2008). Effect of pH The functional properties of mesquite gum are affected by pH. The relative viscosity ( rel) of mesquite gum solutions increases as pH increases from 4.0 to 7.0, due to the substitution of the H+ ions by Na+ ions with a greater degree of dissociation, leading to the macroion unfolding and hence to the increase in viscosity. As the pH increases from 7.0 to 9.0 the macroion cannot expand further due to steric constraints, and as the amount of Na+ counterions increases, they shield the macroion charges and cause it to fold and hence the solution viscosity to decrease (Vernon-Carter et al., 2000). In turn, it has been observed that the effective electrical surface charge (given by the zeta potential) of orange oil-in-water emulsions stabilized with mesquite gum, increases with pH reaching an approximately constant value at pH ~7.0. As the concentration of added NaCl increases from 10ÿ3 to 10ÿ2 M, the compression of the electrical double layer, due to charge shielding, leads to a comparatively lower zeta potential values at all pHs (Acedo-Carrillo et al., 2006). Surface activity Mesquite gum solutions reduce the interfacial tension as a function of the concentration and time (Vernon-Carter and Sherman, 1981). As the gum concentration increases in the range 0.5±25%, the interfacial tension decreases faster. The solution pH also influences the lowering of the interfacial tension with time, an effect that has been directly related to the mesquite gum conformation in solution. The more compact mesquite gum molecular species are, the faster and lower is the decrease in interfacial tension. This has been attributed to the diffusion and/or the conformation of the gum species at the interface. In parallel studies, the absorption of water and oil by mesquite gum at temperatures in the range of 23±45 ëC were greater than those of gum arabic. The activation energy values obtained for water and oil absorption for gum arabic were 21.98 and 39.57 kJ molÿ1, respectively, compared to those of mesquite gum with values of 15.79 and 46.16 kJ molÿ1, respectively (Beristain et al., 1996). In separate studies, changes in the surface tension of an orange oil±water interface, as probed by a Wilhemly plate, were measured. These measurements showed that the adsorbed surface per molecule for gum arabic was an order of magnitude greater than that of gum mesquite (23.0 and 2.2 nm2, respectively)
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(Goycoolea et al., 2000), revealing that the structural microheterogeneity plays a key role in the functional behavior of these materials. In turn, mesquite gum and its major fractions, separated by hydrophobic affinity chromatography, of varying protein contents (7.18±38.60%) and macromolecular dimensions (Mw ~ 3.89 105±8.06 105 g mol-1, Rg ~ 48.83±71.11 nm, Rh ~ 9.61±24.06 nm), have been studied in Langmuir monolayers spread at an air±water interface and compared with whole gum arabic and its corresponding fractions (LoÂpez-Franco et al., 2004). The most active species at the interface were those containing greater amounts of protein. These results have been related with the fine structural differences between the constituent macromolecular species comprising the gum (LoÂpez-Franco et al., 2004). Emulsification ability Like gum arabic, mesquite gum also forms and stabilizes oil-in-water emulsions and has the ability to encapsulate orange citrus oil during spray drying (Goycoolea et al., 1997; Vernon-Carter et al., 1996b; Beristain et al., 1996). Mesquite gum solutions of 15 w/w% are able to form emulsions with n-decane, n-dodecane, n-tetradecane and n-hexadecane with mean droplet diameters of 4± 4.5 m; whereas with orange oil the average droplet diameter was found to vary in the range 2.5±3.0 m (Valdez et al., 2006; Acedo-Carrillo et al., 2006). Moreover, the particle size of emulsions of orange oil-in-water stabilized with 1% mesquite gum remained unchanged for up to 100 h. By contrast, in emulsions with D-limonene and n-decane, phase separation starts within the first 24 h (Acedo-Carrillo et al., 2006). This behaviour of mesquite gum on the orange oil emulsions to stop or control Ostwald ripening is attributed, among other causes, to the fact that orange oil is less water soluble than D-limonene. In addition, mesquite gum-stabilized emulsions of orange oil showed the ability to form a gel structure with time, in contrast with similar emulsions stabilized with gum arabic, with those obtained with alkane oils and with D-limonene. These results seem to indicate that the nature of the oil used is a key factor for gel formation and for the prolonged stability of the emulsions formed with mesquite gum (Valdez et al., 2006; Acedo-Carrillo et al., 2006; Rinaudo et al., 2008). In other studies, it has been found that mesquite gum with a nitrogen content of 0.49% had better emulsifying capacity for chilli oleoresin than gum arabic with nitrogen contents of 0.35% (Vernon-Carter et al., 1996b). The mesquite gum stabilized emulsions had similar initial particle size and exhibited monodisperse particle size distribuition over 8 days, while gum arabic emulsions had a larger initial particle size and polydisperse particle size distribution that broadened with ageing time up to 8 days. Encapsulation ability Several materials are commercially available for encapsulation of essential oils, flavours, colorants and vitamins by spray-drying. The most widely used encapsulation agents are gum arabic and modified or hydrolysed starches. Mesquite gum has been reported as having the ability to encapsulate orange peel oil
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(Goycoolea et al., 1997) (80.5% of the starting oil) (Beristain and VernonCarter, 1994). A blend of 60:40% gum arabic to mesquite gum was able to encapsulate the same amount of orange peel oil as pure gum arabic (Beristain and Vernon-Carter, 1995), whereas a 3:2 ratio of maltodextrin 10 DE to mesquite gum, retained 84.6% of the starting orange peel oil, thus providing a better encapsulating capacity. Film forming Mesquite gum-based films have become an important research topic mainly due to their ability to regulate moisture, lipid and gas migration. Such films can be used to extend the shelf-life of foodstuffs. Emulsion films using mesquite gum as structural agent and a blend of candelilla wax with white mineral oil as the lipid phase, prolong the shelf-life of treated guava fruit (Psidium guajava L.) by retarding ethylene emission and enhancing the texture of the fruits (TomaÂs et al., 2005a). On the other hand, blends of mesquite gum (Prosopis spp) with whey protein concentrate, have been reported to form edible films with poor moisture barrier properties (TomaÂs et al., 2005b). A complex of mesquite gum and chitosan complex has also been used to form edible films again with low water vapour permeability (RuõÂz-Ramos et al., 2006). Compatibility Mesquite gum has been used successfully for various purposes in combination with other gums (e.g., gum arabic), maltodextrins, lipids (candelilla wax), vegetal and animal proteins (e.g., corn zein, soy, whey, peanut proteins, gelatin, casein and milk whey proteins) and with other polysaccharides such as chitosan (RuõÂzRamos et al., 2006; PeÂrez-Orozco et al., 2004), sodium alginate and carrageenan (TomaÂs et al., 2004). Blends of gum arabic and mesquite gum exhibited a synergistic effect that provided greater long-term stability against drop coalescence than either component on its own; however, mesquite gum provided better stability against drop coalescence and deterred pigment degradation better than gum arabic and its blends (Vernon-Carter et al., 1996b). When mixed with gelatin, mesquite gum forms complex coacervates at a 1:1 mass ratio. Microcapsules based on this complex coacervate system have been exploited in encapsulation of corn and orange oil (Vernon-Carter et al., 2000). Preservatives Mesquite gum solutions can be preserved with benzoic acid, formic acid and phydroxybenzoic acid or a combination of sodium benzoate, potassium sorbate and citric acid (Vernon-Carter et al., 2000). 18.4.4 Larchwood arabinogalactan Commercial larch arabinogalactan (LAG) is a dry slightly yellow free-flowing powder with a very slight pine-like odour and sweetish taste (Kelly, 1999). Food grade LAG (98% purity) is free of phenolic, terpenoid or other extraneous chemical and is completely colourless, odourless and tasteless. This material is
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used for clinical applications. Low purity arabinogalactan has significant levels of polyphenolic lignin impurities which impart a light yellow colour and strong odour. Typical specifications for food grade LAG are given below (from Lonza Ltd): Appearance: Fine, off-white to white free-flowing powder Carbohydrates > 90% Physical state: Texture ± free-flowing powder; flavor ± minimal; odor ± minimal Color (CWF): L > 85; a ± Record; b 60 Dissolution: Sink ± wet pass; lumps ± None Heavy metals < 5 ppm Lead HPC > PVP and PVA. Other scientific approaches can be found in the literature calculating adhesion energy of different formulations attached to biological membranes.198 It is interesting to note that although LBG-xanthan systems were evaluated as mucosal glues and found unsatisfactory, they are nevertheless used because of their good mouthfeel and for health safety reasons.256 Biological hydrocolloid glues for the vagin*l area and eyes, and in sprays for the nose and other mucosal areas, have been developed.200,256 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.204 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 of hydrocolloids such as gum karaya, gum arabic, cellulose derivatives, polyox, and others for dentistry.
28.16 Structure-function and hydrogel-adherend relationships Hydrocolloids can be classified into adhesive and non-adhesive. Upon hydration, adhesive hydrocolloids are generally sticky, whereas non-adhesive hydrocolloids form a `slippery' mucilage.258,259 It is well known that the linear structure of many polysaccharides (e.g., pectin, CMC, etc.) hampers their ability to form water-based adhesives. In contrast, branched molecules (e.g., gum arabic, larch arabinogalactan, synthetic polymers) are inherently tacky.260 Many of these branched molecules are gum exudates and among them, numerous form adhesive gels with PSA properties. The branched structure of Sterculia urens polysaccharide is a prerequisite for wet adhesion. Nevertheless its uniqueness lies mainly in its inherently high molecular weight and high degree of acetylation, which are fine starting points for the production of a wide range of depolymerized and deacetylated products.260 Adhesive hydrogels formulated from PVA were evaluated and despite its commercial availability in a wide range of molecular weights and degrees of hydrolysis, they were found to be inferior to the S. urens-based hydrogels as a model, due mainly to the complex and time-consuming preparation involved.261
Hydrocolloids for coatings and adhesives
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The unique chemical structure of the S. urens polysaccharide is a gift from nature as it would most likely be impossible, from a practical point of view, to synthesize such a complex heteropolysaccharide. Its outstanding adhesive features and unique structure have not only raised it to commercial success, but also make it an exceptional tool for studying the adhesive mechanism of hydrogels in a reliable model system.261 Acetyl-free S. urens polysaccharide-based hydrogels (in typical composition including water and polyols) have a more compact macromolecular structure than systems in which acetyl is present. This is characterized by multiple molecular self-associations, a considerable decrease in molecular mobility and reduced bound water. Pressure-sensitive tack behavior of acetyl-free hydrogels follows the Dahlquist criterion developed for the loss of pressure-sensitive tack in PSAs (tack ceases completely when creep compliance approaches 10ÿ6 Paÿ1).262 In contrast, native S. urens polysaccharide-based hydrogels containing acetyl showed evidence of an expanded conformation, increased molecular mobility, increased bound water and improved bonding-debonding characteristics. Acetylation was assumed to influence the interactions between the polysaccharide chains by simple steric hindrance, preventing the close molecular associations that would allow the formation of hydrogen bonds, or by altering the hydrophilicity of the polysaccharide, thus affecting its bonding with water molecules (hydrophobic hydration). As a result, bonding and debonding were, respectively, characterized by increased wettability and elongation, and hence a higher measured tack energy. An excess increase of water in the hydrogel accounted for the network's low rigidity, which in turn increased wettability on the one hand, but reduced the energy dissipated during debonding on the other, resulting in a decrease in tack energy.262 Likewise, relative to acetyl-free hydrogels, any change in the degree of acetylation or in the proportion of acetyl and water, or the addition of a plasticizing auxiliary, could account for changes in adhesion performance presumably owing to conformational shifts. Relations between hydrogels and adherends were considered in terms of some basic adhesion theories (see also Section 28.13). The effect of ionic (electrostatic) primary bonds on adhesion using versatile chitosan probes and hydrogels, which varied in their degrees of cationic and anionic charge, was studied. It was clearly shown that the quantity of carboxylate groups has a direct effect on the adhesion and the interface between the hydrogels and the protonated chitosan probes. The presence of polyols was found to increase the free carboxyl, presumably mainly by increased molecular pseudoplasticity of the polysaccharide-polyol aqueous mixture. It turned out that this ionic bonding is a time-dependent kinetic process which relies, concurrently, on the extent of anionic-cationic charges, rheological characteristics of the hydrogel and possible interdiffusion.262 The surface energy of adherends has a prominent effect on dissipation energy (during debonding), compared with only a modest rheological effect of the hydrogel. The bonding process of typical S. urens polysaccharidebased hydrogels to low- and high-energy surfaces (18.5 and 39.2 mJ mÿ2,
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respectively) is on the order of 10ÿ4 s, typical of industrial PSAs and a prerequisite for providing instantaneous tackiness. To complete bond formation, a value of ~100 s is likely to be a common rule for many hydrogels, regardless of their rheology or the surface energy of the probe.262 Ra (arithmetic mean of the absolute values of the roughness profile's deviation from the centerline within the evaluation length) values in the range of 0 to 0.5 m could potentially serve as a general tack criterion for viscoelastic materials, particularly in terms of achieving improved hydrogel adhesion. Increasing Ra values in the range of 0.5 to 25 m had a negative effect on tack, because roughness induces a delay in the establishment of intimate contact between the adhesive and the probe. Adhesion of polysaccharide-based hydrogels is derived from the structure of their threedimensional network, and from their physico-chemical properties, dictated by the extent of hydration and solubility. Possible influences of degree of acetylation on hydration or of molecular weight on solubility could be important factors affecting hydrocolloid adhesion. In addition, hydrogel-adherend interfacial phenomena are liable to exist via polyanion-polycation electrostatic interactions, intrinsic surface energy, adherend surface roughness, contact pressure and contact dwell time.262
28.17
Hydrocolloid adhesion tests
A relatively large number of tests have been proposed to evaluate adhesivebonding strength. They include peeling at 90ë (Fig. 28.3(a)), and tensile-bond and lap-shear tests. In tensile tests, tension is used to examine adhesive joints, bonds, or design via the application of perpendicular forces on the adhesive layer. Different modes of failure have been recognized: failure within the adhesive layer ± cohesive failure; failure at the interface between the adherent and the adhesive ± adhesive failure, and failure of the adherent itself. After testing, the ratio between adhesive and cohesive failure should be estimated. Samples in which the failure occurs within the substrates should be discarded, since they do not constitute a test of the adhesive material.263,264 Peel tests are used for quality control or for comparing different adhesives, and are important in quantifying stripping or peeling forces. In peel tests, a flexible adherend that is adhesively bonded to another rigid or flexible adherent is stripped. During this operation, the distribution of the stress within the peel joints is complex and influenced by the properties of the adherent and the joint geometry. The width of the peeled adherend is a major factor in calculating the peel strength, usually in pounds per inch. The average load needed to maintain the peeling after initiation is used to estimate the peeling force.1 In shear tests, the structural adhesive within bonded structures is usually designed so that it will sustain shear loads most of the time, the reason being the strength of adhesives in shear, as compared to that under peel or tensile loads. Applied loads act in the plane of the adhesive layer when the adhesively bonded structure is considered under shear. As a result of the loads, the adherents can slide, which in turn causes shearing or
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Fig. 28.3 (a) Specimen mounted on the Instron Universal Testing Machine (UTM) during the 90ë peel test. (b) Specimen mounted on the Instron UTM prior to application of the lap-shear test. (c) A modified probe-tack test in which a special device is capable of detecting the first contact between the probe and a pressure-sensitive adhesive (PSA) and determining this contact as the initial dwell time.
sliding of the adhesive. A certain length of overlap between adherents is typical when lap-shear tests are applied (Fig. 28.3(b)). The layer between the overlapping area consists of the structural adhesive, and tension-to-failure is used during the lap-shear test. The critical shear strength is calculated by dividing the load at failure by the area of the overlap.263 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,202 adhesive-layer thickness, 203 water-holding capacity of a specimen, 194,208 length of contact,194,203 the effect of molecular weight203 and the type of adhesive.194 Recently, a modified probe-tack test (Fig. 28.3(c)) was described in which a special device is capable of detecting the first contact between the probe and a PSA and of determining this contact as the initial dwell time.265 In tests using viscoelastic PSA materials (hydrogels), this modified device gives results which differ significantly from those obtained with conventional probe-tack testers. In particular, when tack values are plotted against dwell time, the modified tester reveals a regime of sigmoid low-energy tack and then a transition to the normal power-law high-energy tack. This transition behavior was studied as a function of crosshead velocity, compression pressure, adhesive rheology, and adherend surface roughness. The practical implication is that far more information can be obtained from this modified device than from conventional probe-tack testers. The modified machine and methodology should prove particularly valuable as an experimental tool and for quality control tests in the manufacture of PSA products, particularly soft and tacky viscoelastic substances.265
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28.18
Hydrocolloids as wet glues
Twenty-six 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 properties at predetermined gum concentrations ranging from 10 to 75% by weight. The hydrocolloids were: gum talha, gum ghatti, gum arabinogalactan (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. 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 color 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 min later had risen by 0:97 0:14 ëC. 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 utilization of bioadhesive materials. Studies have shown108 that gum karaya (pH ~ 3.6) may cause allergic reactions such as hives and angioneurotic edema. Paste color may be a factor in choosing an ointment or bioadhesive for a particular application. A variety of different colors could be found among the wet glues, ranging from off-white to yellowish or darkbrown.266 Once in very thick paste form, the 13 hydrocolloids were smeared hom*ogeneously onto two different substrates: (a) cellulose-acetate film, normally used for dialysis, and (b) a skin-surface model (SSM), proposed266 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.266 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.266 Typical curves for the 90ë peel, tensile load and lapshear tests are presented in Fig. 28.4. 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
Hydrocolloids for coatings and adhesives
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Fig. 28.4 Typical curves for the 90ë peel test: (a) pectin, 25%; (b) gum talha; (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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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 well-defined maxima and minima, and the distance between two minima or maxima is independent of testing rate.267 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.267 Tensile load (Fig. 28.4(c)) and lap shear (Fig. 28.4(d)) were applied and plotted as load (g cmÿ2 ) vs. displacement (cm) curves.266 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, respectively268). 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 D1002, ASTM D-3528). Data were collected continuously as the specimens were stretched uniaxially to rupture. The maximal force was recorded in both cases.266 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.266 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. 90ë peel 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 peel bond 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.
28.19
Future trends
An adhesive is basically defined by its function, which is to hold two surfaces together. To fulfill this function, the properties of an adhesive must include easy positioning at the interface, rapid and complete bond formation and subsequent hardening, and a bond strength that is adapted to the specific application
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(structural, permanent, removable; rigid, or soft).269 A variety of solutions exist in practice, and their application requires an understanding of both polymer chemistry and materials science.269 Conventional PSAs are soft viscoelastic polymers that degrade, leave residue behind, self-adhere, and attach accidentally to inappropriate surfaces. Engineered adhesive nanostructures inspired by geckos (geckos attach and detach their adhesive toes in milliseconds while running with reckless abandon on nearly any surface and the adhesive on gecko toes differs dramatically from that of conventional adhesives) may become the glue of the future but it is too early and therefore difficult to estimate whether hydrocolloids will be part of this development.270 On the other hand, future trends and research directions for PSAs, from the point of view of end-use properties alone, are moving towards heterogeneous polymer structures and the incorporation of additional functions, such as thermal or electrical conductivity or controlled drug release, as in nicotine patches, into a PSA matrix without altering its self-adhesive properties.271 It is also evident that the nano revolution, as was discussed for edible coatings, will also be a part of the adhesives' future. Evidence for this can be found in a study that included nano-organo-clays within PSAs. It was observed that during drug release, the initial burst is reduced and can be controlled. Moreover, by optimizing the level of the organo-silicate additive to the polymer matrix, superior control over drug-release kinetics and simultaneous improvements in adhesive properties could be attained for a transdermal PSA formulation.272 Another future research direction may be related to cell adhesion and changing surface modifications,273,274 or the involvement of adhesive hydrocolloids, or those that change adhesiveness, in tissue engineering and regenerative medicine.275
28.20 1. 2. 3. 4. 5. 6. 7. 8.
References
`Hydrocolloid coating of foods: a review' The Leatherhead Food Industry J, 1998 1 (3) 174±88. DAVIDSON, R. L. Handbook of Water-Soluble Gums and Resins, New York, McGraw-Hill Book Company, 1980. NUSSINOVITCH, A. Hydrocolloid Applications. Gum Technology in the Food and Other Industries, London, Blackie Academic and Professional, 1997. NUSSINOVITCH, A. Water-Soluble Polymer Applications in Foods, Oxford, Blackwell Science Ltd., 2003. KAMPF, N. and NUSSINOVITCH, A. `Hydrocolloid coatings of cheeses' Food Hydrocolloids, 2000 14 531±7. CUQ, B., GONTARD, N. and GUILBERT, S. `Packaging materials based on natural polymers' Industries Alimentaires et Agricoles, 1997 114 110±16. DEBEAUFORT, F., QUEADA-GALLO, J. A. and VOIILLEY, A. (1998) `Edible films and coatings: tomorrow's packaging: a review' Crit Rev Food Sci, 1998 38 299±313. MCHUGH, T. H. `Effects of macromolecular interactions on the permeability of composite edible films' in Macromolecular Interactions in Food Technology, Washington DC, American Chemical Society, 1996, pp. 134±44. NUSSINOVITCH, A.
794 9.
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29 Alginates K. I. Draget, Norwegian University of Science and Technology, Norway
Abstract: Being essentially independent from temperature with respect to their gelling properties, alginates from brown marine algae differ from most other gelling polysaccharides and proteins. The present chapter focuses mainly on the major structure/function correlations in alginates and how they can be used to control gel properties. Furthermore, their application limitations linked to their inherent stability and solubility properties are also discussed. Finally, some aspects of food and nutrition, as well as their regulatory status, are also addressed. Key words: structure/function, gels, gelling technologies, stability, food and nutrition.
29.1 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. The sources for industrial production of alginate may be regarded as unlimited even for a steadily growing industry since macroalgae may also be cultivated and since production by fermentation is technically possible (although not economically feasible at the moment). The biological function of alginate in brown algae is as a structure-forming component. The intercellular alginate gel matrix gives the plants both mechanical strength and flexibility (Andresen et al., 1977). This relation between structure and function is reflected in the compositional difference of
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alginates in different algae or even between different tissues from the same plant (see Section 29.3.2). In Laminaria hyperborea, an algae growing in very exposed coastal areas, the stipe and holdfast have a very high content of guluronic acid, giving high mechanical rigidity (see Section 29.5.1). 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. In bacteria, like Azotobacter vinelandii (Sadoff, 1975) and Pseudomonades (Linker and Jones, 1966), the biological function is not fully understood but in the case of A. vinelandii it has been shown that alginate production is required for dormant cyst formation. Pharmaceutical, food and technical applications (such as in printing paste for the textile industry) are the quantitative main market areas for alginates, but 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 (Smidsrùd and SkjaÊkBrñk, 1990; Soon-Shiong et al., 1992). 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 research has made alginate one of the best characterised and well understood gelling polysaccharides. The focus in this chapter will be 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 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.
29.2
Manufacture
Alginate was first described by the British chemist E.C.C. Stanford in 1881, 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 (Haug, 1964). Alginate is widely used in industry because of its ability to retain water, and its gelling, viscosifying and stabilising properties. Several bacteria also produce alginate exocellularly (Linker and Jones, 1966; Gorin and Spencer, 1966; Sutherland, 1977), and Azotobacter vinelandii has been evaluated as a source for industrial production. But at present, all commercial alginates stem from algal sources. The extraction of alginate from algal material is schematically illustrated in Fig. 29.1. 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 treating 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
Alginates 809
Fig. 29.1
Principal scheme for the isolation of alginate from seaweeds.
carbonate or sodium hydroxide to form the water-soluble sodium alginate. After extensive separation procedures such as sifting, flotation, 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 required and finally dried and milled. In addition to 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; see also Section 29.4.1). Following the increased popularity of alginate as an immobilisation matrix, FMC BioPolymer/Novamatrix A/S commercially manufactures ultrapure alginates highly compatible with mammalian biological systems. These qualities are low in pyrogens, and a low content of aggregates facilitate sterilisation of the alginate solution by filtration.
29.3 Structure and physical properties 29.3.1 Chemical composition and sequence Alginate is a family of linear binary copolymers of (1!4)-linked -Dmannuronic acid (M) and -L-guluronic acid (G) residues (see Fig. 29.2(a) and (b)) of widely varying composition and sequence. The first information about the sequential structure of alginates came from the work by Haug and co-
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workers (Haug, 1964; Haug and Smidsrùd, 1965; Haug and Larsen, 1966; Haug et al., 1966, 1967a) where alginate was separated into three fractions of widely differing composition. Two fractions contained almost hom*opolymeric molecules of guluronic and mannuronic acid, whereas a third fraction consisted of nearly equal proportions of both monomers containing a large number of MG dimer residues. It was concluded that alginate was a true block copolymer composed of hom*opolymeric regions of M and G, termed M- and G-blocks, respectively, interspersed with regions of alternating structure (MG-blocks; see Fig. 29.2(c)). In a series of papers (Painter et al., 1968; Larsen et al., 1970; Smidsrùd and Whittington, 1969) it was shown that alginates have no regular repeating unit and, furthermore, that 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. Modelling of the alginate molecule suggests that a second order Markov approach 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 spectroscopy (Grasdalen, 1983; Grasdalen et al., 1977, 1979; Penman and Sanderson, 1972) in the sequential analysis of alginate. NMR has made it possible to determine the monad frequencies FM and FG, the four nearest neighbouring (diad) frequencies
Fig. 29.2 Structural characteristics of alginates: (a) alginate monomers, (b) chain conformation, (c) block distribution.
Alginates 811 FGG, FMG, FGM, FMM, and the eight next nearest neighbouring (triad) frequencies. Knowledge of these frequencies enables calculation of the average G-block length larger than 1: NG>1 (FG ÿ FMGM)/FGGM, which has shown to correlate well with gelling properties (see Section 29.5.1). It is important to realize that in a population of alginate molecules, neither the composition nor the sequence of each chain will be alike. This results in a compositional distribution of a certain width.
29.3.2 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 29.1 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 (Haug, 1964; Indergaard and SkjaÊk-Brñk, 1987). 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 gel strength. Alginates with more extreme compositions can be isolated from bacteria (SkjaÊk-Brñk et al., 1986) 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 29.1), by chemical fractionation (Haug and Smidsrùd, 1965; SkjaÊk-Brñk et al., 1986) or by enzymatic modification in vitro using mannuronan C-5 epimerases from A. vinelandii (ErtesvaÊg et al., 1999). 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 Table 29.1
Composition and some sequential parameters of algal alginates
Source 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
FG
FM
FGG
FMM
FGM,MG
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
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epimerases from A. vinelandii have been cloned and expressed, and they represent at present a powerful new tool for the tailoring of alginates. Commercial alginates with less molecular heterogeneity, with respect to chemical composition and sequence, can also be obtained by a treatment with one of the C-5 epimerases.
29.3.3 Molecular weight Alginates, like polysaccharides in general, are polydisperse with respect to molecular weight. This may arise for two different reasons: (i) polysaccharides are not directly coded for by the DNA, but are synthesised by polymerase enzymes, and (ii) a depolymerisation occurs during extraction. Due to this polydispersity, the `molecular weight' of an alginate becomes an average over the total distribution of molecular weights. There are several methods for averaging the molecular weight, the two most n (which weighs the polymer molecules common are the number-average, M according to the number of molecules in a population having a specific w (which weighs the polymer molecular weight), and the weight-average, M molecules in a population according to the weight of molecules having a specific n is called the polydispersity index (PI). A w =M molecular weight). The fraction M PI 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 PI of more than 2.0 indicates a wider distribution, and suggests that blending of different batches with different molecular weights to obtain a sample of a certain average molecular weight (viscosity) or that a non-random degradation of the polymer has taken place. The molecular weight distribution can have implications for the uses of alginates, as low molecular 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 may cause problems (Stokke et al., 1991; Otterlei et al., 1991).
29.3.4 Selective binding of ions The ion-binding characteristics 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 (Haug, 1964). These characteristic affinities are exclusive to polyguluronate; polymannuronate is almost without selectivity. The affinity of alginates for alkaline earth metals increases in the order Mg 1). An important feature in the diffusion setting method is that the final gel can exhibit an inhom*ogeneous distribution of alginate, with the highest concentration at the surface and gradually decreasing towards the centre of the gel. Extreme alginate distributions have been reported (SkjaÊk-Brñk et al., 1989a) with a five-fold 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. It is important to know that the degree of hom*ogeneity can be controlled, and how different parameters can govern the final alginate distribution. Maximum inhom*ogeneity is obtained by letting a low molecular weight alginate gel at low concentration of the gelling ion and in the absence of non-gelling ions. Maximum hom*ogeneity is reached by gelling a high molecular weight alginate in the presence of high concentrations of both gelling and non-gelling ions (SkjaÊk-Brñk et al. 1989a). Non-gelling ions are
Alginates 821 also important with respect to the 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 increasing FM (Martinsen et al., 1989). Internal setting As outlined earlier, this technique is based on 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 (Draget et al., 1991). The main difference between internal and diffusion setting is the gelling kinetics. Internal setting allows for the tailoring of an alginate gelling systems towards a given manufacturing process. For example, in the alginate/CaCO3/Dglucono--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 (Draget et al., 1991). 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 hom*ogeneous gels, except where a low viscosity solution allows particle sedimentation. The gel strength of internally set alginate gels depends more on molecular weight compared to gels set by diffusion (Draget et al., 1993). Whereas gels made by the latter method describe almost a step function where gel strength becomes independent of molecular weight at around 100 kDa (Smidsrùd, 1974) (weight average degree of polymerisation, DPw ~ 500), the internally set gels still depend on molecular weight even at 300 kDa (DPw ~ 1,500). This is, at least partly, due to the fact that the internally set gels are calcium limited compared to the gels made by diffusion, implying 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 syneresis 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 (Draget et al., 1991). There is no detailed understanding of this difference at the moment, but part of the explanation is certainly the different modes of gelation. Diffusion setting gives a gelling zone, moving towards the centre of the gelling
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body, where the alginate molecules become saturated with Ca2+ and their activity drops towards zero. Internal setting implies a process where gelling starts simultaneously at a large number of locations. This puts some topological strains on the alginate molecules, but their activity and translational mobility does not equal zero. One can therefore imagine that after the primary gel network has been formed, elastic segments with free G-blocks will still be present that could create new junction zones given the proximity of another free G-block and the presence of calcium ions. Thus, 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.
29.5.2 Alginic acid gels It has been known and utilised for several decades that alginates precipitate at pH below the pKa value (Haug, 1964). In fact, the discovery that alginates were block co-polymers originated from the discovery that the different types of blocks had different solubility at low pH. 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 (e.g. the anti-reflux agent Gaviscon), 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 an instantaneous precipitation rather than a gel. pH must therefore be lowered in a controlled fashion, and this is most conveniently carried out by the addition of slowly hydrolysing lactones like Dglucono--lactone (GDL). It has been shown (Draget et al., 1994) 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. The strength of the acid gels is more or less independent on 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. But in contrast to ionic gels, also poly-mannuronate sequences support acid gel formation. Poly-alternating sequences seem to perturb gel formation in both cases. The obvious demand for hom*opolymeric sequences in acid gel formation suggests cooperative processes to be involved just as in the case of ionic gels. A broad molecular weight dependence has been observed, and this dependence becomes more pronounced with increasing content of guluronic acid residues.
Alginates 823
29.6 Foods, nutrition and health 29.6.1 Applications in food products Some examples of the use of alginates in food products have already been given in the previous chapters. Their ability to improve, modify and stabilise the texture of foods represents the basis for applying alginates as food additives, e.g. as a viscosity enhancer, gel former and in the stabilisation of aqueous mixtures, dispersions and emulsions in general. Alginates are also used to control the melting behaviour of ice cream. Most applications are based on the physical properties of alginates themselves, but may also result from interactions with other components of the food product, e.g. proteins or fibres. For detailed descriptions and formulation, see Cottrell and Kovacs (1980), Sime (1990) and Littlecott (1982). Restructured food based on Ca-alginate gels is simple (gelling being independent of temperature), and it is a steadily growing alginate application. Such processes are based on binding together a flaked, sectioned, chunked or milled foodstuff to make it resemble the original. Examples of such products already on the market are meat products (both for human consumption and as pet-food and feed), onion rings, pimento olive fillings, crabsticks and co*cktail berries. The synergetic gelling between alginates high in guluronate and highly esterified pectins may be utilised for the use in jams, jellies, fruit fillings, etc. (Toft et al., 1986). These mixed alginate/pectin systems may give thermoreversible gels in contrast to the purely ionically crosslinked alginate gels. Such gels are also almost independent of sugar content, in contrast to pectin gels, and may therefore be used in low calorie products. As already mentioned, propylene glycol alginate is the only alginate derivative used in food products. The main advantage of PGA is that it may be used to stabilise liquids under acidic conditions where the unmodified alginate would precipitate. 29.6.2 Nutritional aspects and health benefits Alginates as such are regarded as essentially non-digestible in the human gastrointestinal (GI) tract and are classified as so-called poorly fermentable soluble (viscous) fibres (Brownlee et al., 2005). Hence, they can not act as a direct source of energy but may provide other health-related benefits. Such soluble dietary fibres do in general reduce the rate of small intestinal absorption of nutrients (Jenkins et al., 2000), which in turn may reduce the likelihood of cardiovascular diseases (by reducing the overall cholesterol levels) as well as the onset of diabetes type II (by reducing the glycaemic load). Specific to alginates, it has been shown (Sunderland et al., 2000) that, to a large extent, they can inhibit the activity of proteases. As outlined in Section 29.4.2 it has been shown that alginates high in mannuronate stimulate the production of cytokines in vitro. It has not been proven that an oral administration of such alginates would result in a general immuno-response in humans, but it has been suggested that they could enhance the repair of mucosal damage in the GI tract (Brownlee et al. 2005).
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The fact that alginate also may undergo a sol-gel transition may be exploited to reduce the overall energy intake by stimulating endogeneous satiety signalling (Pelkman et al., 2007) and that the products for this type of application can be formulated as a calcium-gelled fibre beverage. It has also been shown that microencapsulation (see Section 29.5.1) of probiotic bacteria has a profound effect on improved survival rates when such bacteria are exposed to acids and bile salts (Ding and Shah, 2007). Finally, it has also been proposed that the highly specific ion binding properties of the alginate molecule (see Section 29.3.4) could represent a way to reduce the damage of ingested radioactive strontium isotopes following a nuclear accident. One study (Beresford et al., 1999) shows that when 5% calcium alginate is incorporated into the diet of dairy goats, the transfer of radioactive Sr into the milk was reduced by approximately 50%.
29.7
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 39th meeting in 1992. An ADI `not specified' was allocated. JECFA allocated an ADI of 0±70 mg/kg bw to propylene glycol alginate at its 41st meeting in 1993. 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 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 under the quantum satis principle in the EU (with the exception of those cited in Annex II and those described in article II of the Directive). A general prohibition exists on the use of alginate (as well as against several other gelling biopolymers) in so-called jelly mini-cups (amendment of 2005). Propylene glycol alginate (PGA) is inscribed in Annex IV (`other permitted additives') of the directive with a maximum level ranging from 0.1 mg/l±10 g/l (1.2±8 g/kg) depending on the food product.
29.8
Future trends
Containing only the two monomer units M and G linked by the same 1,4 linkages, the alginate molecule may look very simple. This may have lead potential commercial users of alginate to treat alginate as a commodity in much the same way as many of the cellulose derivatives. But as already outlined,
Alginates 825 alginates exhibit a very high diversity with respect to chemical composition and monomer sequence resulting in a large variety of physical and biological properties. So, contrary to what may be expected by just looking at the monomer composition, the family of alginate molecules represents a challenge to the unskilled users of alginate, but an advantage for those end-users of alginate aiming at research-based, high value applications. The possibility of the tailoring of alginates to fit new and demanding applications is increased even further if epimerase-modified alginates are taken into consideration. It is therefore possible to foresee a future trend, which has already started, where the exploitation of alginate gradually shifts from low-tech commodity applications with increasing competition from low-cost alternatives, to more advanced knowledge-based applications in the food, pharmaceutical and biomedical areas.
29.9 References and sources of further reading and HEMMER PC, `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±381. BáKKEN é, SMIDSRéD O, DRAGET KI and JOHNSEN F, `Gelled feed products, means for making the product and method for manufacturing of said products', Patent WO/ 2001/001792, 2001. BERESFORD NA, MAYES RW, MACEACHERN PJ, DODD BA and LAMB S, `The effectiveness of alginates to reduce the transfer of radiostrontium to the milk of dairy goats', J Env Radioactivity, 1999 44 43±54. BROWNLEE IA, ALLEN A, PEARSON JP, DETTMAR, PW, HAVLER ME, AUTHERTON R and ONSéYEN E, `Alginate as a source of dietary fiber', Crit. Rev. Food Sci. Nutr., 2005 45 497± 510. COTTRELL IW and KOVACS P, `Alginates', in: Handbook of Water-soluble Gums and Resins (HB Crawford and J Williams, eds). Auckland: McGraw-Hill, 1980, pp. 21±43. DING WK and SHAH NP, `Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria', J Food Sci, 2007 72 M446±M450. DRAGET KI and SMIDSRéD O, `Modification of gelling kinetics and elastic properties by nano structuring of alginate gels exploiting the properties of polyguluronate'. Proceedings from the 13th Gums and Stabilisers Conference for the Food Industry (PA Williams and GO Phillips, eds), The Royal Society of Chemistry, Cambridge, 2006, pp. 227±233. Ê K-BRáK G and éSTGAARD K, `Regeneration, cultivation and DRAGET KI, MYHRE S, SKJA differentiation of plant protoplasts immobilized in Ca-alginate beads', J Plant Physiol, 1988 132 552±556. DRAGET KI, éSTGAARD K and SMIDSRéD O, `hom*ogeneous alginate gels: a technical approach', Carbohydr Polym, 1991 14 159±178. DRAGET KI, SIMENSEN MK, ONSéYEN E and SMIDSRéD O, `Gel strength of Ca-limited alginate gels made in situ', Hydrobiologia, 1993 260/261 563±565. Ê K-BRáK G and SMIDSRéD O, `Alginic acid gels: the effect of alginate DRAGET KI, SKJA chemical composition and molecular weight', Carbohydr Polym, 1994 25 31±38. Ê G K, ONSéYEN E and SMIDSRéD O, `Na- and K-alginate; effect on Ca2+DRAGET KI, STEINSVA ANDRESEN I-L, SKIPNES O, SMIDSRéD O, éSTGAARD K
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Ê G H, HéIDAL HK, SCHJERVEN H, SVANEM BIG ERTESVA
and VALLA S, `Mannuronan C5epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology', Metabolic Engineering, 1999 1 773±783. Ê K-BRáK G, `Application of alginate gels in biotechnology and ESPEVIK T and SKJA biomedicine', Carbohydr Eur, 1996 14 19±25. GACESA P, CASWELL RC and KILLE P, `Bacterial alginases; Pseudomonas aeruginosa infection', Antibiot Chemoter, Basel, Karger, 1989 42 67±71. GORIN PAJ and SPENCER JFT, `Exocellular alginic acid from Azotobacter vinelandii', Can J Chem, 1966 44 993±998. GRANT GT, MORRIS ER, REES DA, SMITH PJC and THOM D, `Biological interactions between polysaccharides and divalent cations: the egg-box model', FEBS Lett, 1973 32 195±198. 1 GRASDALEN H, `High-field H-nmr spectroscopy of alginate: sequential structure and linkage conformations', Carbohydr Res, 1983 118 255±260. 13 GRASDALEN H, LARSEN B and SMIDSRéD O, ` C-NMR studies of alginate', Carbohydr Res, 1977 56 C11±C15. GRASDALEN H, LARSEN B and SMIDSRéD O, `A PMR study of the composition and sequence of uronate residues in alginate', Carbohydr Res, 1979 68 23±31. HAUG A, `Ion exchange properties of alginate fractions', Acta Chem Scand, 1959 13 1250± 1251. HAUG A, `Composition and properties of alginates', Thesis, Norwegian Institute of Technology, Trondheim, 1964. HAUG A and LARSEN B, `The solubility of alginate at low pH', Acta Chem Scand, 1963 17 1653±1662. HAUG A and LARSEN B, `A study on the constitution of alginic acid by partial acid hydrolysis', Proc Int Seaweed Symp, 1966 5 271±277. HAUG A and SMIDSRéD O, `Fractionation of alginates by precipitation with calcium and magnesium ions', Acta Chem Scand, 1965 19 1221±1226. HAUG A, LARSEN B and SMIDSRéD O, `The degradation of alginates at different pH values', Acta Chem Scand, 1963 17 1466±1468. HAUG A, LARSEN B and SMIDSRéD O, `A study of the constitution of alginic acid by partial hydrolysis', Acta Chem Scand, 1966 20 183±190. HAUG A, LARSEN B and SMIDSRéD O, `Studies on the sequence of uronic acid residues in alginic acid', Acta Chem Scand, 1967a 21 691±704. HAUG A, LARSEN B and SMIDSRéD O, `Alkaline degradation of alginate', Acta Chem Scand, 1967b 21 2859±2870. HAUG A, MYKLESTAD S, LARSEN B and SMIDSRéD O, `Correlation between chemical structure and physical properties of alginate', Acta Chem Scand, 1967c 21 768± 778. Ê K-BRáK G, `Characteristics of alginate from Laminaria digitata INDERGAARD M and SKJA cultivated in a high phosphate environment', Hydrobiologia, 1987 151/152 541± 549. JENKINS DJA, AXELSEN M, KENDALL CWC, AUGUSTIN LSA, VUKSAN V and SMITH U, `Dietary fiber, lente carbohydrates, and the insulin-resistant diseases', Brit J Nutr, 2000 83 S157±S163. LARSEN B, SMIDSRéD O, PAINTER TJ 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±728.
Alginates 827 and MALONE DM, `Effects of sterilization treatments on some properties of alginate solution and gels', Biotechnol Prog, 1990 6 51±53. LINKER A and JONES RS, `A new polysaccharide resembling alginic acid isolated from Pseudomonas', J Biol Chem, 1966 241 3845±3851. LITTLECOTT GW, `Food gels ± the role of alginates', Food Technol. Aust., 1982 34 412± 418. 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±341. Ê K-BRáK G and SMIDSRéD O, `Alginate as immobilization material: I. MARTINSEN A, SKJA Correlation between chemical and physical properties of alginate gel beads', Biotechnol Bioeng, 1989 33 79±89. NEISER S, DRAGET K and SMIDSRéD O, `Gel formation in heat-treated bovine serum albumin±sodium alginate systems', Food Hydrocolloids, 1997 12 127±132. NEISER S, DRAGET KI and SMIDSRéD O, `Interactions in bovine serum albumin±calcium alginate gel systems', Food Hydrocolloids, 1998, 13, 445±458. ONSéYEN E, `Commercial applications of alginates', Carbohydr Eur, 1996 14 26±31. Ê K-BRáK G, SMIDSRéD O, SOON-SHIONG P and ESPEVIK T, OTTERLEI M, éSTGAARD K, SKJA `Induction of cytokine production from human monocytes stimulated with alginate', Int J Immunother, 1991 10 286±291. PAINTER TJ, SMIDSRéD O and HAUG A, `A computer study of the changes in compositiondistribution occurring during random depolymerisation of a binary linear heteropolysachharide', Acta Chem Scand, 1968 22 1637±1648. PARSONS BJ, PHILLIPS GO, THOMAS B, WEDLOCK DJ and CLARK-STURMAN AJ, `Depolymerization of xanthan by iron-catalysed free radical reactions', Int J Biol Macromol, 1985 7 187±192. PELKMAN CL, NAVIA JL, MILLER AE and POHLE RJ, `Novel inatake dietary clacium-gelled, alginate-pectin beverage reduced energy in nondieting overweight and obese women: interactions with restraint status', Am J Clin Nutr, 2007 86 1595±1602. PENMAN A and SANDERSON GR, `A method for the determination of uronic acid sequence in alginates', Carbohydr Res, 1972 25 273±282. SADOFF HL, `Encystments and germination in Azotobacter vinelandii', Bacteriol Rev, 1975 39 516±539. SIME W, `Alginates', in: Food Gels (Harris P, ed.), London: Elsevier, 1990, pp. 53±78. SIMENSEN MK, DRAGET KI, ONSéYEN E, SMIDSRéD O and BORE TF, `Use of G-block polysaccharides', Patent WO/1998/002488, 1998. Ê K-BRáK G, SMIDSRéD O and LARSEN B, `Tailoring of alginates by enzymatic SKJA modification in vitro', Int J Biol Macromol, 1986 8 330±336. Ê K-BRáK G, GRASDALEN H and SMIDSRéD O, `Inhom*ogeneous polysaccharide ionic SKJA gels', Carbohydr Polym, 1989a 10 31±54. Ê K-BRáK G, MURANO E and PAOLETTI S, `Alginate as immobilization material. II: SKJA Determination of polyphenol contaminants by fluorescence spectroscopy, and evaluation of methods for their removal', Biotechnol Bioeng, 1989b 33 90±94. SMIDSRéD O, `Some physical properties of alginates in solution and in the gel state', Thesis, Norwegian Institute of Technology, Trondheim, 1973. SMIDSRéD O, `Molecular basis for some physical properties of alginates in the gel state', J Chem Soc Faraday Trans, 1974 57 263±274. SMIDSRéD O, `Structure and properties of charged polysaccharides', Int Congr Pure Appl Chem, 1980 27 315±327. LEO WJ, MCLOUGHLIN AJ
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and SKJAÊK-BRáK G, `Alginate as immobilization matrix for cells', Trends Biotechnol, 1990 8 71±78. SMIDSRéD O and WHITTINGTON SG, `Monte Carlo investigation of chemical inhom*ogeneity in copolymers', Macromolecules, 1969 2 42±44. SMIDSRéD O, HAUG A and LARSEN B, `Degradation of alginate in the presence of reducing compounds', Acta Chem Scand, 1963 17 2628±2637. SMIDSRéD O, HAUG A and LARSEN B, `The influence of pH on the rate of hydrolysis of acidic polysaccharides', Acta Chem Scand, 1966 20 1026±1034. SMIDSRéD O, HAUG A and LARSEN B, `Oxidative-reductive depolymerization: a note on the comparison of degradation rates of different polymers by viscosity measurements', Carbohydr Res, 1967 5 482±485. Ê K-BRáK G, SMIDSRéD O, HEINTZ R, LANZA RP and ESPEVIK SOON-SHIONG P, OTTERLEI M, SKJA T, `An immunologic basis for the fibrotic reaction to implanted microcapsules', Transplant Proc, 1991 23 758±759. SMIDSRéD O
Ê K-BRáK G, ESPEVIK SOON-SHIONG P, FELDMAN E, NELSON R, KOMTEBEDDE J, SMIDSRéD O, SKJA T, HEINTZ R and LEE M, `Successful reversal of spontaneous diabetes in dogs by intraperitoneal microencapsulated islets', Transplantation, 1992 54 769±774. SOON-SHIONG P, FELDMAN E, NELSON R, HEINTS R, YAO Q, YAO T, ZHENG N, MERIDETH G, Ê K-BRáK G, ESPEVIK T, SMIDSRéD O and SANDFORD P, `Long-term reversal of SKJA
diabetes by the injection of immunoprotected islets', Proc Natl Acad Sci, 1993 90 5843±5847. STANFORD ECC, British patent #142, 1881. STEGINSKY CA, BEALE JM, FLOSS HG and MAYER RM, `Structural determination of alginic acid and the effects of calcium binding as determined by high-field NMR', Carbohydr Res, 1992 225 11±26. STEINER AB, `Manufacture of glycol alginates', U.S. Patent no. 2,426,215, 1947. STEINER AB and MCNEILLY WH, `High-stability glycol alginates and their manufacture', U.S. Patent no. 2,494,911, 1950. Ê K-BRáK G, `Distribution of uronate residues STOKKE BT, SMIDSRéD O, BRUHEIM P and SKJA in alginate chains in relation to alginate gelling properties', Macromolecules, 1991 24 4637±4645. Ê K-BRáK G, `Distribution of uronate STOKKE BT, SMIDSRéD O, ZANETTI F, STRAND W and SKJA residues in alginate chains in relation to gelling properties 2:Enrichment of -Dmannuronic acid and depletion of -L-guluronic acid in the sol fraction', Carbohydr Polym, 1993 21 39±46. STOKKE BT, DRAGET KI, SMIDSRéD O, YUGUCHI Y, URAKAWA H and KAJIWARA K, `Smallangle X-ray scattering and theological characterization of alginate gels. 1. Caalginate gels', Macromolecules, 2000 33 1853±1863. SUNDERLAND AM, DETTMAR PW and PEARSON JP, `Alginates inhibit pepsin activity in vitro; A justification for their use in gastro-oesophagal reflux disease (GORD)', Gastroenterology, 2000 118 347. SUTHERLAND IW, Surface carbohydrates of the prokaryotic cell, London, Academic Press, 1977, pp 22±96. TOFT K, GRASDALEN, H and SMIDSRéD O, `Synergistic gelation of alginates and pectins', ACS Symp Ser, 1986 310 117.
30 Inulin D. Meyer, Sensus, The Netherlands and J.-P. Blaauwhoed, Cosun Food Technology Centre, The Netherlands
Abstract: This chapter describes inulin and some of its application aspects in food. First the structure and industrial production are described followed by the technical properties relevant for food applications (e.g., solubility, viscosity, stability, interaction with hydrocolloids and gelling properties). Secondly, the nutritional and health benefits such as the low caloric value and the prebiotic properties are outlined, followed by some examples of the use of inulin in food applications. Emphasis is on those applications in which inulin is used as a fat and sugar replacer, to improve mouthfeel, etc. Finally, the regulatory status and some future developments are discussed. Key words: inulin, gelling properties, fat replacement, sugar replacement, interaction.
30.1 Introduction Inulin has been well known for a long time. In 1804 Rose isolated a substance from Inula helenium (elecampane) that was later (in 1811) called inulin by Thomas (see Suzuki, 1993). These carbohydrates are synthesised in at least ten families of higher plants. They can be found in many economically important plants such as chicory, Jerusalem artichoke, onion, garlic, barley, rye and wheat. The data compiled in Table 30.1 show that inulins are an intrinsic ingredient of many of our common foodstuffs and as such these ingredients have been consumed for longer than we can remember. Inulin-containing crops were and are consumed not only from the crops mentioned in Table 30.1, which belong to a western type diet, but also in other countries, such as yacon tubers in South America and Japan, and murnong by Australian aboriginals. Jerusalem artichoke was consumed in Europe in the sixteenth century and in the early twentieth
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Table 30.1 Inulin content of different crops Source Banana (Musa cavendishii) Barley (Hordeum vulgare) Chicory (Cichorium intybus) Garlic (Allium sativum) Globe artichoke (Cynara scolymus) Jerusalem artichoke (Helianthus tuberosus) Leek (Allium ampeloprasum) Onion (Allium cepa) Wheat (Triticum aestivum)
Inulin content (% of fresh weight)
Range of DP
0.3±0.7 0.5±1.5 15±20 9±16 2±3 3±10 16±20 1±8 1±4
2±5 No data 2±60 2±50 2±250 2±50 No data 2±12 2±8
Based on data of van Loo et al. (1995) and Sensus (unpublished). DP: degree of polymerisation (see Fig. 30.1)
century inulin was already known as a carbohydrate suitable for diabetics, as it did not give rise to a glycemic response. Inulin is built up of 2±60 fructose units with one terminal glucose molecule (Fig. 30.1). Inulin is the generic name covering all -(2,1) fructans. In most cases inulins are a polydisperse mixture of fructan chains with a chain length (DP: degree of polymerisation) distribution dependent on the source (Table 30.1) and moment of harvesting. The term oligofructose or fructooligoaccharides is used for -(2,1) fructans with a DP up to about 10, and an
Fig. 30.1 Basic chemical structure of inulin GFn with terminal glucose residue (G: glucose; F: fructose; DP n 2; with n 1±60 for inulin from chicory).
Inulin
831
average DP of about 4. The topic of this chapter is mainly (long-chain) inulin with an average DP of at least 20.
30.2 Production process of inulin In the early 1990s production of inulin as a food ingredient started in Europe. Industrial production of inulin almost exclusively uses chicory roots as raw material and is concentrated in the Netherlands and Belgium. Processing of chicory roots to produce inulin takes place under more or less the same conditions and with the same equipment as for the production of sucrose from sugar beet. As with sugar beet, processing also takes place in a campaign period from autumn to winter. After washing and slicing the roots, inulins are extracted with hot water. The so called diffusion juice can be transformed into a clear juice with liming and carbonation, similar to the purification of the diffusion juice in sugar beet processing. Alternatively, proteins and cell components can be precipitated and flocculated under slightly acidic conditions and separated off by filtration. Further refining by crystallisation at this stage as for white sugar production is not suitable for native inulin: the low solubility of long-chain inulin allows separation of this fraction by crystallisation, but the short-chain inulin fraction will remain in solution. For this reason separation techniques common for production of liquid sugars such as glucose are used in the refining of native inulin solutions. Removal of salts and most of the colour takes place by ion exchange. To prevent hydrolysis of inulin at low pH the whole process of ion exchange is carried out at low temperatures. After this step a slightly acidic, nearly fully demineralised inulin thin juice is obtained which is also low in colour. Finally the characteristic bitter taste of chicory is removed by activated carbon, which also removes any residual colour. The refined inulin juice is concentrated by evaporation and finally spray dried to obtain a powdered product. Spray drying takes place from completely dissolved inulin solutions resulting in amorphous powders with good solubility. Native inulin can be separated in short- and long-chain fractions by crystallisation. After crystallisation both long- and short-chain fractions can be spray dried to amorphous powders. This delivers types of inulin with a chain length distribution different from native inulin, which offer possibilities for different applications. The long-chain inulin with a DP ranging from about 10 to 60 is the main type of inulin discussed in this chapter.
30.3 Technical properties 30.3.1 Solubility Solubility in water is mostly dependent on the inulin chain length distribution. In Fig. 30.2, the maximum or direct solubility of native and long-chain inulin types is shown. As can be seen, the solubility increases with decreasing chain length.
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Fig. 30.2 Maximum solubility of native and long-chain inulin as a function of temperature. Data from Sensus (2003a).
Oligofructose is commercially available as syrup with a dry matter content of 75% which stays clear even at 5 ëC. Storage time and temperature are important factors for transparency of inulin solutions. Longer storage time might result in development of haze due to precipitation of long-chain inulins. In Table 30.2 the concentrations are given for obtaining a stable clear inulin solution in water for a long period of time. This is important, e.g., for the development of beverages which should stay clear for a longer period, or in products where precipitation may lead to a rough texture by the precipitated inulin particles. 30.3.2 Viscosity Solutions of native inulin in water are very low in viscosity, and also long-chain inulin exhibits only a slightly higher viscosity in solution compared to native inulin: e.g., a 20% solution in water has a viscosity of about 4.5 mPa s at 20 ëC. Table 30.2 Long-term solubility of inulin at different temperatures Type of inulin
Native inulin Long-chain inulin
Storage temperature 5 ëC
20 ëC
< 2.0% < 1.0%
< 2.5% < 1.5%
Inulin concentration in relation to inulin type and storage temperature resulting in a clear solution after 13 weeks of storage (Sensus, 2003a)
Inulin
833
30.3.3 Heat stability Inulin is stable to temperatures up to 140 ëC (when dissolved at near neutral pH) and can therefore be processed easily in most if not all food applications. It will break down at temperatures above 140 ëC. In powder form the heat stability is much higher.
30.3.4 Acid stability The -(2,1) glycosidic bond in inulin is susceptible to acid hydrolysis. The degree of hydrolysis depends on pH, food product composition (dry matter content) and time-temperature combination (during heat treatment or shelf-life). In general, hydrolysis will not occur at pH levels above 4.0. At pH levels below 4.0, hydrolysis may occur with the rate dependent on actual pH and temperature. For example, hydrolysis is not observed at temperature levels below 10 ëC at any pH between 3.0 and 7.5, but at pH 3 at 25 ëC about 10% hydrolysis takes place in 1 week. An example of inulin loss in acid solution with different inulin types is given in Fig. 30.3.
30.3.5 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 high molecular weight polysaccharides. 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
Fig. 30.3 Inulin hydrolysis of different inulin types at pH 3.5 and 20 ëC. Inulin or FOS solutions (1%) were prepared in buffer and stored at pH 3.5 and 20 ëC. The inulin content (defined as inulin with DP > 2) was determined using Shodex GPC measurements.
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high water binding effect, they can be used at low dosage levels. Proteins like gelatin also have the ability to form gels. 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. Inulin has different thickening and stabilising properties from hydrocolloids. The inulin molecule is much smaller and the water-binding ability is low compared to hydrocolloids. When concentrations exceed about 15%, inulin has the ability to form a gel or cream. Below this concentration, low viscous aqueous solutions are obtained, as mentioned above. Gel formation of inulin is also different compared to hydrocolloids. Inulin forms particle gels, similar to those formed by some starches (Harris and Day, 1993). Gel formation by hydrocolloids or the increase of viscosity with most hydrocolloids is due to interaction between the polymer chains. Due to the high number of hydroxyl groups present in inulin, it can be expected that inulin plays a role in hydrogen bonding in food systems, and that it may influence the solubility of other water-binding ingredients such as guar or xanthan gum, carrageenan, alginate, pectin, maltodextrin and starch. In other words, inulin takes part in the competition for water as solvent. Development of viscosity or gel formation will be changed as a result of this interference of inulin with the hydration of other hydrocolloids. This might result in a retardation of viscosity development, a higher viscosity, a more brittle gel (see Fig. 30.4 for carrageenan and inulin interaction) or a smoother product flow, and also syneresis can be reduced (Sensus, 2002). Research in model systems has shown that inulin in combination with hydrocolloids can influence the rheological behaviour of these ingredients and
Fig. 30.4 Effect of inulin on rheology of a carrageenan gel. Gel strength of a carrageenan gel with inulin or sucrose was measured in a Carrimed Rheometer. Based on data from Sensus (2002).
Inulin
Fig. 30.5
835
Effect of inulin on starch viscosity in relation with temperature.
additives. Inulin can provide a useful tool to affect rheology and textural perception of a food product. Although 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, inulin can optimise the rheological properties of products. The inulin product is highly polydisperse. It has significant influence (with its hydrogen bonding efficiency and consequent solubility (gel formation)) on various high water-binding 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. In Fig. 30.5, an example is shown how inulin may affect the viscosity of a starch solution at different temperatures. With high inulin concentrations viscosity is much lower than with lower concentrations. This may be due to the effect that the inulin network interferes with the starch network in such a way that viscosity is lowered. 30.3.6 Inulin as a gelling agent Preparation of an inulin gel is straightforward: inulin is dissolved in water and upon cooling the molecules start to precipitate and crystallise, and a gel is formed (Table 30.3). The rate of precipitation or crystallisation and consequently the size of the inulin particles depend 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
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Handbook of hydrocolloids Table 30.3 Typical procedure for making an inulin gel Preparation of a 25% (w/w) inulin gel 1. Dissolve 50 g inulin with DP about 25* in 150 g tap water. 2. Place the dispersion in a water bath at 72 ëC. 3. Heat the dispersion for 15 min while stirring. 4. Remove the solution from the water bath. 5. Place in a cold room, 5 ëC for at least 6 h. * E.g., FrutafitÕTEX as supplied by Sensus (the Netherlands)
the network of crystallised inulin particles, resulting in a gel structure. This gel has rheological properties very similar to those of fat and therefore inulin has been identified as an interesting ingredient for structuring in low- or zero-fat food products (Silva, 1996; Bot et al., 2004).
30.3.7 Parameters affecting gel characteristics The rheological features of an inulin gel can be influenced by the following parameters: · · · · ·
chain length distribution concentration preparation temperature amount of shear (Kim et al., 2001) use of seeding (Sensus, 2003b; Kim and Wang, 2001).
In Fig. 30.6, an example is given of the influence of concentration and inulin type on the firmness of a gel as measured with a Texture Analyser: long-chain inulins give a higher gel strength and for gel formation to occur with native inulin at least 20% of inulin is needed. With oligofructose no gels can be prepared at any concentration. 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 (DP > 10) participate in the gel structure and the smaller molecules remain dissolved (Hebette et al., 1998). It can be seen from Fig. 30.6, that inulin types with a higher average chain length, containing more long-chain inulin molecules, will form firmer inulin gels. An inulin gel belongs to the group of particle gels, and firmness increases with increasing concentration as shown in Fig. 30.6. 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. Seed material other than inulin, such as maltodextrin or -(2,6)-linked fructans are not effective (Sensus, unpublished observations). The degree of hydration depends on the inulin concentration and temperature. The application of a shear treatment immediately after pasteurisation or during cooling will have a positive effect on increasing the firmness of an inulin
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Fig. 30.6 Gel strength in relation to concentration of native inulin (DP 9), long-chain inulin (DP 25) from chicory roots and inulin (DP 36) from Cynara scolymus. Inulin gels were prepared at the concentrations given on the X-axis by dissolving inulin at 85 ëC and cooling down overnight (see Table 30.3 for the procedure). Gel strength was measured with a Stevens Texture Analyser (Sensus, 2003b).
gel (Fig. 30.7). 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 inulin seed crystals during cooling, in combination with a shear treatment will result in a firm inulin gel with perfect sensory properties (Fig. 30.7).
Fig. 30.7 Effect of shear treatment and seeding on firmness of inulin gels.
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Handbook of hydrocolloids
Nutritional and health benefits
As the glycosidic bonds between the fructosyl residues are resistant to gastric acid and since humans lack the digestive enzymes for splitting these bonds, inulins and fructo oligosaccharides (FOS) pass unharmed from mouth to small intestine. After reaching the colon they are completely fermented by the bacteria present in that organ. This behaviour explains why inulins are dietary fibres. The products from this fermentation process (short chain fatty acids, SCFA; acetic, propionic and butyric acid) will be absorbed by the host and subsequently metabolised. This salvages part of the energy, but only about a third of the caloric content compared with digestible carbohydrates is liberated by this process. This explains why inulins and oligofructose are low caloric food ingredients with a caloric value of about 1.5 kcal/g (Roberfroid, 1999). The fermentation in the large intestine leads to a change in the composition of the colonic microbiota: specifically bifidobacteria (and lactobacilli) are favoured in their growth which leads to an increase in the number of these genera. This effect is observed in children (Kim et al., 2007) and in adults (e.g., Kolida et al., 2007). Concomitantly, often a decrease in Clostridium and Bacteroides is found. This effect on the bacterial composition is called the prebiotic effect of inulins. Gibson and Roberfroid (1995) coined this term and described this effect as follows: a prebiotic modulates the colonic bacterial flora beneficially by stimulating the growth of health bacteria (Bifidobacterium and Lactobacillus) and decreasing the number of potentially harmful species (e.g. clostridia). This fermentation of inulins may lead to a whole range of other physiological effects, some of them related to the fibre functions and others more specifically to the increase of health associated bacteria as described. It is beyond the scope of this overview to discuss all effects in detail. It suffices here to describe the different effects briefly, with a focus on the results from studies with human volunteers. The fibre effects include the contribution to an improved defecation pattern (Kleessen et al., 1997; den Hond et al., 2000) and a stool bulking effect (den Hond et al., 2000), but the bifidogenic effect may also play a role here. The fibre properties (the non-digestibility) are also relevant for the low glycemic response elicited by inulins and FOS. The glycemic response (GR) of inulins is very low: the purest commercially available inulin has a GR of 5, and native inulin a GR of about 14 (all relative to glucose set at 100; Meyer, 2007). This makes inulin a suitable ingredient for food for diabetics, and for the development of low GR food products (Meyer, 2007). The fermentation of inulins may lead to a locally lowered pH thereby increasing the solubility of various salts. This improves their availability for absorption and it has been found that calcium and magnesium absorption in humans increases following inulin or FOS consumption (Meyer and StasseWolthuis, 2006). This may have a positive effect on bone density as has been found in various animal trials (e.g., Scholz-Ahrens et al., 2002) and in humans volunteers (Abrams et al., 2005).
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839
Another effect attributed to inulins is the lowering of serum lipids, thereby possibly contributing positively to heart health by lowering the risk for cardiovascular disease. Some studies showed a lowering of triglycerides (e.g., Causey et al., 2000) or cholesterol (e.g., Davidson et al., 1998), but others showed no effect (e.g., Pedersen et al., 1997). It appears that especially in dyslipidemic volunteers inulin consumption may lower serum lipids (Beylot, 2005). Recent data show that inulin can also have a positive effect on satiety feeling and energy intake. In people consuming inulin as a fat replacer in sausages, energy intake over the day is diminished (Archer et al., 2004) and similar data were reported by Cani et al. (2006) for volunteers consuming oligofructose. Inulins can also affect the immune system, possibly directly, but also through the increase in bifidobacteria and lactobacilli. Most of the effects described so far originate from experimental animal trials, and only a few examples from human studies have been published. These include a non-significant lowering of the incurrence of traveller's diarrhoea with FOS consumption (Cummings et al., 2001) and a reduced inflammation of the mucosa of the ileal reservoir in ileal pouch-anal anastomosis patients (Welters et al., 2002). There is also evidence to suggest that inulin may lower the risk for colon cancer. Especially in experimental animal trials with chemically induced colon cancer, positive results have been obtained showing that with inulin consumption, and especially in combination with probiotics, the occurrence of colon cancer can be prevented (e.g., Femia et al., 2002). It has also been reported that certain biomarkers for colon cancer risk in humans can be affected such that this risk may be lowered by such inulin-based synbiotics (Rafter et al., 2007).
30.5 Applications The rheological and sensory characteristics of inulin gels make them an excellent fat replacer in a wide range of foods (Silva, 1996). An overview is presented in Table 30.4, with emphasis on those applications for long-chain and native inulin in which the features discussed above play a role. For an overview of applications of all types of inulin, including oligofructose, we refer to Meyer et al. (2007). Generally speaking inulins are applied in the following markets: dairy, bakery, beverages, cereals and cereal bars, infant nutrition, confectionery, ice cream, but also in feed and pet food (see Verdonk et al., 2005 for a review of the latter applications). A positive influence is observed in the following applications by adding inulin. With thickeners (xanthan, guar gum and pectins) stabilised beverage systems become more hom*ogeneous 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.
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Table 30.4 Overview of applications of native and long-chain inulins Application
Chain Dosage Replacement lengtha (%) L/N/S Fat Sugar
Technological functionality
Dairy products Yoghurt Milk drink Breakfast drink Mousse Dairy spread
L/N N N L/N L
2±4 3±5 2±5 3±20 4±10
Mouthfeel Mouthfeel Mouthfeel Foam stabilisation, mouthfeel Texturiser, syneresis prevention
Bakery products Bread Hamburger bun Short dough biscuit
L/N L/N N/S
3±10 3±7 2±8
Maria biscuit
N/S
3±10
+ + + + +
+
+
+ +
Aerated cake filling N
5±15
+
Biscuit filling Microwave pizza Wafer
N L/N N
5±20 1±3 1±3
+
Miscellaneous Fat spread Extruded cereals Dressing Chocolate Meat products Tablets Pasta filling
L L/N L/N L/N L/N N L/N
4±20 3±20 2±10 3±20 2±7 5±40 2±7
+ + + + +
+
Flour replacement Flour replacement Crispiness, gluten softening, sweetness Crispiness, gluten softening, sweetness Texturiser, foam stabiliser, mouthfeel Bulking agent Crispiness enhancement Crispiness enhancement Texturiser Crispiness; bowl life Mouthfeel Bulking agent Texturiser, mouthfeel Direct compressible excipient Texturiser, mouthfeel
a Chain length: L: long-chain inulin (average DP 20±25); N: native inulin (average DP 9±11); S; oligofructose with average DP about 4. Based on data from Sensus (2005) and Franck (2000).
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 (Kip et al., 2006). 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. 30.5.1 Fat spreads and dairy spreads Addition of 7.5% native inulin to fat spreads with 20±40% fat (w/o emulsions) results in a product with a good structure and a perfect taste. The excellent
Inulin
841
spreadability and mouthfeel make it a perfect, low-fat substitute for sandwich margarines with 80% fat. An inulin gel based on 20% long-chain inulin 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. 30.5.2 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 flow properties is achieved. Furthermore, inulin is capable of masking the acidic/sour taste. 30.5.3 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. Research has shown that especially airiness in lowfat yoghurts is increased by the addition of inulin. The increase of this texture attribute contributes significantly to the increased creaminess (Fig. 30.8; Kip et al., 2006). A problem which occurs in other fat-free yoghurts is that the taste is more acidic. Inulin is capable of masking this effect.
30.5.4 Low-fat and low-sugar ice cream Fat in a premium quality ice cream contributes to sensory properties (such as mouthfeel, melting behaviour and creaminess) and physical properties (such as hardness and stability). The role of sugar in ice cream is very much related to
Fig. 30.8
Effect of long-chain inulin on creaminess of skimmed yoghurt. Data from Kip et al. (2003).
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texture, hardness and stability, as well as taste and sweetness. Removing fat and sugar from ice cream will thus affect taste, texture and mouthfeel of the final product. Inulin and oligofructose can return the desired sensory attributes to low-fat and low-sugar ice cream products (Schaller-Povolny and Smith, 1999) with inulin contributing to a creamy mouthfeel, hom*ogeneous melting and improved heat shock stability, and oligofructose providing a sweet taste, scoopability, and synergy with high intensity sweeteners, helping mask the aftertaste. Both ingredients help to maintain small ice cream crystals after heat shock, which is necessary for good mouthfeel and improves the shelf-life. Inulin and oligofructose offer excellent opportunities for sugar and fat replacement in ice creams. High quality products can be obtained in terms of the microstructure, sensory characteristics, melting properties and heat shock stability (McDevitt-Pugh and Peters, 2005).
30.5.5 Low-fat cake Partial replacement of the pastry margarine by an inulin gel results in a fatreduced cake with perfect sensory properties and a long shelf-life. Research has also shown that the combination of inulin with maltodextrin results in cakes with more volume.
30.5.6 Fillings The creamy structure of an inulin gel makes it a perfect base for fillings which allows biscuits to be made with a filling in which 25% of the fat was replaced.
30.5.7 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 crisper when 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. 30.9, curve 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. 30.9, curve 2). Inulin is able to bind water, so there will be less free water in the
Inulin
Fig. 30.9
843
Absorption profiles.
product (see also Schaller-Povolny et al., 2000). This means that a wafer with inulin can contain more moisture and stay crisp. 30.5.8 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% native inulin with addition of sugars, cocoa powder, milk proteins and hazelnut paste makes it possible to produce a delicious low-fat (water continuous) hazelnut spread. Compared with the conventional product a calorie reduction of 45% can be achieved.
30.6 Regulatory status Regulations differ from country to country. Because inulin is part of our daily diet (Table 30.1), it is considered as a food ingredient, so it has no E-number in the EU (it is not an additive) and it can be used ad libitum in all food categories. Inulin has the status of dietary fibre in almost every country. As described above inulin can be used as a fat replacer and this creates the possibility to make nutrition claims based on the reduced fat or energy content of the food product. Table 30.5 shows such claims and conditions applying to them from EU 1924/2006, the latest legislation in the 27 member states of the EU (European Commission, 2006). As inulins are also a dietary fibre for labelling purposes, the fibre claims as mentioned in the Table 30.5 are equally applicable. Also in the USA nutrition claims are possible, but in contrast to the EU where the percentage in the food is the basis, the concentration of inulin per serving size is the basis in the USA for making claims. As an example, a `low fat' claim is allowed when the fat content is less than 3 g, whereas a `fat free' claim requires less than 0.5 per serving (see FDA, 1999). Dietary fibre claims, such as `excellent source of fibre', are based on a certain percentage of the reference daily intake of 25 g/d (FDA, 1999).
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Table 30.5 Overview of nutrition claims from EU 1924/2006 Claim
Conditions
Low energy
Less than 40 cal (170 kJ)/100 g for solids, or 20 kcal (80 kJ)/ 100 ml for liquids. For table-top sweeteners a limit of 4 kcal (17 kJ)/portion applies
Energy-reduced
Energy value reduced by at least 30% (with an indication of the characteristic(s) which make(s) the food reduced in energy)
Energy-free
Less than 4 kcal or 17 kJ per 100 ml, for table-top sweeteners less than 0.4 kcal (1.7 kJ) per portion
Low fat
Less than 3 g of fat per 100 g for solids, or 1.5 g of fat per 100 ml for liquids (1.8 g per 100 ml for semi-skimmed milk)
Fat-free
Less than 0.5 g per 100 g or 100 ml (X% fat-free is prohibited)
Source of fibre
At least 3 g of fibre per 100 g (or 1.5 g per 100 kcal)
High fibre
At least 6 g of fibre per 100 g (or 3 g per 100 kcal)
Reduced [ingredient]
Reduction of at least 30% compared to a similar product
Increased [ingredient]
Increase of at least 30% compared to a similar product
Light/lite
Conditions as for `reduced' and an indication of the charcteristic(s) which make(s) the food light/lite
For further details, see European Commission (2006).
Health claims based on inulin, such as based on the prebiotic or other effects described above, are also regulated in EU 1924/2006. A list of approved health claims describing the effect of an ingredient on a physiological function will be published at the latest on 31 January 2010. These Article 13 claims will be based on generally accepted scientific evidence. It seems very likely that (some of) the health effects of inulin will be on this list; for instance, a bifidogenic gut health claim is already approved in France (AFSSA, 2005) and the Netherlands (Rombouts et al., 2002). For so-called Article 14 claims (risk of disease reduction claims or claims aimed at growth and development of children) pre-market approval is required. The procedure to obtain such approval is described in EU 1924/2006 (European Commission, 2006). In the USA, so-called structure-function claims, which describe the effect of an ingredient on the structure or physiological function, do not require premarket approval. Care should be taken to avoid mentioning a disease in this type of claim, and that the necessary evidence is available, for instance to claim the effect of inulin on calcium absorption.
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845
30.7 Future trends In view of the oncoming obesity pandemic, it is to be anticipated that development of low caloric food products with an optimal taste and texture will continue at an increasing speed. Inulin on its own or in combination may be an important ingredient for these developments as it is low caloric and provides numerous health benefits, some of which are clearly relevant for the trends described, in particular the ability to improve satiety. Also the interactions with hydrocolloids are important as these may contribute to proper food texture and flavour release in a variety of products. In connection with this, it might be interesting to look also for new tools to investigate these aspects; instead of focusing on the bulk rheological properties of the product, the interaction of the product in the mouth, with palate and tongue, might provide more clues to the assessment of texture and its sensory attributes, such as creaminess (e.g., Dresselhuis et al., 2007). This means that future developments are to be aimed at providing more insight into the mechanisms behind the interactions, which will contribute to building better food products. By including inulin the health properties of such products might be further enhanced. Further research to provide the scientific evidence for the health benefits of inulin will create the possibility of making health claims on inulin-containing foods to convey a credible message to the consumer.
30.8 Acknowledgements The work of following collaborators from Cosun Food Technology Centre is gratefully acknowledged: S. Bink and B. Walraven for isolating the inulin from Cynara scolymus, P. Kip and J. de Wolf (now with Givaudan) for their research on low-fat yoghurt.
30.9 References and (2005) A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. Am. J. Clin. Nutr. 82: 471±476. AFSSA (2005) Avis de L'Agence FrancËaise de securite sanitaire des aliments relatif aÁ l'eÂvaluation des alleÂgations nutritionnelles concernant l'effet bifidogeÁne de l'inuline sur la flore intestinal humaine. AFSSA, Maisons-Alfort cedex, France. ARCHER, B.J., JOHNSON, S.K., DEVEREUX, H.M. and BAXTER, A.L. (2004) Effect of fat replacement by inulin or lupin-kernel fibre on sausage patty acceptability, postmeal perceptions of satiety and food intake in men. Br. J. Nutr. 91: 591±599. BEYLOT, M. (2005) Effects of inulin-type fructans on lipid metabolism in man and in animal models. Br. J. Nutr. 93: S163±S168.
ABRAMS, S.A., GRIFFIN, I.J., HAWTHORNE, K.M., LIANG, L., GUNN, S.K., DARLINGTON, G. ELLIS, K.J.
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and AGTEROF, W.G.M. (2004) Influence of crystallisation conditions on the large deformation rheology of inulin gels. Food Hydrocolloids 18: 547±556. CANI, P.D., JOLY, E., HORSMANS, Y. and DELZENNE, N.M. (2006) Oligofructose promotes satiety in healthy human: a pilot study. Eur. J. Clin. Nutr. 60: 567±572. CAUSEY, J.L., FEIRTAG, J.M., GALLAHER, D.D., TUNGLAND, B.C. and SLAVIN, J.L. (2000) Effects of dietary inulin on serum lipids, blood glucose and the gastrointestinal environment in hypercholesterolemic men. Nutr. Res. 20: 191±201. CUMMINGS, J.H., CHRISTIE, S. and COLE, T.J. (2001) A study of fructo oligosaccharides in the prevention of travellers' diarrhoea. Aliment. Pharmacol. Therapeut. 15: 1139± 1145. DAVIDSON, M.H., MAKI, K.C., SYNECKI, C., TORRI, S.A. and DRENNAN, K.B. (1998) Effects of dietary inulin on serum lipids in men and women with hypercholesterolemia. Nutr. Res. 18: 503±517. DEN HOND, E., GEYPENS, B. and GHOOS, Y. (2000) Effect of high performance chicory inulin on constipation. Nutr. Res. 20: 731±736. DRESSELHUIS, D.M., HOOG, E.H.A. DE, COHEN STUART, M.A. and AKEN, G.A. VAN (2007). Tribology as a tool to study emulsion behaviour in the mouth. In: Food Colloids: Self-assembly and Material Science (Dickinson, E. and Leser, M.E., eds.). Springer Verlag, Berlin, pp. 451±461. EUROPEAN COMMISSION (2006) Regulation for nutrition and health claims made on Foods (EU 1924/2006), available at: http://eur-lex.europa.eu/LexUriServ/site/en/oj/2007/ l_012/l_01220070118en00030018.pdf. FDA (1999) Center for Food Safety and Applied Nutrition. A Food Labeling Guide, available at: http://www.cfsan.fda.gov/~dms/foodlab.html.
BOT, A., ERLE, U., VREEKER, R.
FEMIA, A P., LUCERI, C., DOLARA, P., GIANNINI, A., BIGGERI, A., SALVADORI, M. CLUNE, Y. COLLINS, K.J., PAGLIERANI, M. and CADERNI, G. (2002) Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in rats. Carcinogenesis 23: 1953±1960. FRANCK, A. (2000) Prebiotics and Probiotics Ingredients Handbook, Leatherhead Food Publishing, Reading. GIBSON, G.R. and ROBERFROID, M.R. (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125: 1401±1412. HARRIS, D.W. and DAY, G.A. (1993) Structure versus functional relationships of a new starch-based fat replacer. Starch/StaÈrke 45: 221±226. HEBETTE, C.L.M., DELCOUR, J.A., KOCH, M.H.J., BOOTEN, K., KLEPPINGER, R., MISCHENKO, N. and REYNAERS, H. (1998) Complex melting of semi-crystalline chicory (Cichorium intybus L.) root inulin. Carbohydr. Res. 310: 65±75. KIM, S.-H, LEE, D.H. and MEYER, D. (2007) Supplementation of baby formula with native inulin has a prebiotic effect in formula-fed babies. Asia Pac. J. Clin. Nutr. 16: 172±177. KIM, Y. and WANG, S.S. (2001) Kinetic study of thermally induced inulin gel. J. Food Sci. 66: 991±997. KIM, Y., FAQIH, M.N. and WANG, S.S. (2001) Factors affecting gel formation of inulin. Carbohydr. Polym. 46: 135±145. KIP, P., PETERS, B. and MEYER, D. (2003) Improving mouth feel and texture of stirred yoghurt by the addition of inulin. Abstract P37 3rd NIZO Dairy Conference. Dynamics of texture, process and perception (11±13 June 2003, Papendal, the Netherlands).
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and JELLEMA, R.H. (2006) Inulins improve sensoric and textural properties of low fat yoghurts. Int. Dairy J. 16 (9): 1098±1103. KLEESSEN, B., SYKURA, B., ZUNFT, H. J. and BLAUT, M. (1997) Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. Am. J. Clin. Nutr. 65: 1397±1402. KOLIDA, S., MEYER, D. and GIBSON, G.R. (2007) A double-blind placebo-controlled study to establish the bifidogenic dosage of inulin in healthy humans. Eur. J. Clin. Nutr. 61: 1189±1195. MCDEVITT-PUGH, M. and PETERS, B. (2005) Weight management supported by tasty inulinbased products. AgroFood Ind. Hi-tech 16: 36±37. MEYER, D. (2007) Inulins for product development of low GI products to support weight management. In: Dietary Fibre Components and Functions (H. Salovaara, F. Gates and M. Tenkanen, eds). Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 257±270. MEYER, D. and STASSE-WOLTHUIS, M. (2006) Inulin and bone health. Curr. Top. Nutraceut. Res. 4: 211±226. MEYER, P.D., WOLF, J. DE and OLIVIER, P. (2007) Inulin und Fructooligosaccharide. In: Handbuch SuÈûungsmittel (K. Rosenplenter and U. NoÈhle, eds). Behr's Verlag, Hamburg, pp. 155±193. È M, B. and AMELSVOORT, J.M.M. VAN (1997) The effect of ingestion PEDERSEN, A., SANDSTRO of inulin on blood lipids and gastrointestinal symptoms in healthy females. Br. J. Nutr. 78: 215±222. KIP, P., MEYER, D.
RAFTER, J., BENNETT, M., CADERNI, G., CLUNE, Y., HUGHES, R., KARLSSON, P.C., KLINDER, A., O'RIORDAN, M., O'SULLIVAN, G.C., POOL-ZOBEL, B., RECHKEMMER, G., ROLLER, M.,
and COLLINS, J.K. (2007) Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients. Am. J. Clin. Nutr. 85: 488±496. ROBERFROID, M.B. (1999) Caloric value of inulin and oligofructose. J. Nutr. S1436±S1437. ROMBOUTS, F.M., BRANDT, P.A. VAN DEN, NAGENGAST, F.M. and SCHAAFSMA, G.J. (2002) Assessment report according to the Code of Practice for assessing the scientific evidence for health benefits. Netherlands Nutrition Centre, The Hague, available at: (http://www.voedingscentrum.nl/NR/rdonlyres/85938396-FFD6-44B0-8143769E3B25A14C/0/beoordelingsrapport_vitaalbrood_florapdf.pdf). SCHALLER-POVOLNY, L.A. and SMITH, D.E. (1999) Sensory attributes and storage life of reduced fat ice cream as related to inulin content. J. Food Sci. 64: 555±559. SCHALLER-POVOLNY, L., SMITH, D.E. and LABUZA, T P. (2000) Effect of water content and molecular weight on the moisture isotherms and glass transition properties of inulin. Int. J. Food Prop. 3: 173±192. SCHOLZ-AHRENS, K.E., ACIL, Y. and SCHREZENMEIR, J. (2002) Effect of oligofructose or dietary calcium on repeated calcium and phosphorus balances, bone mineralization and trabecular structure in ovariectomized rats. Br. J. Nutr. 88: 365±378. Õ SENSUS (2002) Frutafit inulin interactions with hydrocolloids. Brochure. Õ Õ SENSUS (2003a) Stability of Frutafit and Frutalose inulin/FOS in beverage applications. Brochure. Õ SENSUS (2003b) Frutafit inulin gels. Preparation and opportunities. Brochure. Õ Õ SENSUS (2005) The Frutafit and Frutalose product ranges, Brochure. SILVA, R.F. (1996) Use of inulin as a natural texture modifier. Cereal Foods World 41: 792±794. SUZUKI, M. (1993) History of fructan research: Rose to Edelman. In: Science and ROWLAND, I., SALVADORI, M., THIJS, H., LOO, J. VAN, WATZL, B.
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Technology of Fructans (M. Suzuki and N.J. Chatterton, eds). CRC Press, Boca Raton, FL, pp. 22±39. VAN LOO, J., COUSsem*nT, P., LEENHEER. L. DE, HOEBREGS, H. and SMITS, G. (1995) On the presence of inulin and oligofructose as natural ingredients in the western diet. Crit. Rev. Food Sci. Nutr. 35: 535±552. VERDONK, J.M.A.J., SHIM, S,B., LEEUWEN, P. VAN and VERSTEGEN, M.W.A. (2005) Application of inulin-type fructans in animal feed and pet food. Br. J. Nutr. 93: S12±S138. WELTERS, C.F.M., HEINEMAN, E., THUNNISSEN, F.B.J.M., BOGAARD. A.E.J.M VAN DEN, SOETERS,
and BAETEN, C.G.M.I. (2002) Effect of dietary inulin supplementation on inflammation of pouch mucosa in patients with an ileal pouch-anal anastomosis. Dis. Colon Rectum 45: 621±627. P.B.
31 Chitin and chitosan hydrogels R. A. A. Muzzarelli, Muzzarelli Consulting, Italy and University of Ancona, Italy and C. Muzzarelli, University of Ancona, Italy
Abstract: A large number of hydrogels of modified chitins and chitosans have become available during the last few years for a variety of applications in research and technology. The simplest example of hydrogel formation is the treatment of a chitosan acetate solution with carbodiimide to restore acetamido groups. A large variety of substituted amides have been obtained by reacting chitosan with anhydrides and acyl chlorides. Hydrogels are formed by polyelectrolyte complexation as in the case of chitosan salts treated with pentasodium tripolyphosphate or other polymeric anionic substances including DNA, polysaccharides, proteins, inorganic or synthetic polymers, but also according to more sophisticated approaches such as the use of tyrosinase, laccase and other enzymes, or crosslinking agents like glycidoxypropyltrimethoxysilane. While the controlled and targeted delivery of drugs is obtained today with chitosan-based vectors capable of forming safe and bioresorbable hydrogels, on the other hand intelligent articles and sensors are currently manufactured from chitosan hydrogels as well. The biochemical significance of chitin and chitosan in the food area is based on the role that chitosan gels play when interacting with bile salts (cholesterol lowering, overweight control), and with intestinal bacteria (control over bacterial flora, and enhanced absorption). The preservation of fruit and vegetables from microbial spoilage with the aid of chitosan gels and films is becoming more and more important, because today chitosan is recognised as safe and it is approved for human consumption in many countries. Key words: chitin, chitosan, drug delivery, nutritional value, cholesterol lowering, antimicrobial activity, polyelectrolytes.
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Introduction
Hydrogels, composed of water-soluble or swelling macromolecules, exhibit high water content, biocompatibility and desirable mechanical properties; the xerogels resulting after dehydration exhibit large specific surface area, and a network suitable for the incorporation of biomolecules. Present and future microgel applications require control over properties that include novel functionality, controlled particle size and tunable biodegradability. Major advances in the area of microgel/nanogel particles deal with drug delivery carriers for biological and biomedical applications (Oh et al., 2008), but food applications have long testified to a great deal of interest in natural and semisynthetic hydrogels. This chapter provides information on some chitin- and chitosan-based hydrogels suitable for applications in the food and welfare areas. Among polysaccharides, chitin is known for a large variety of hydrogels that can be obtained through chemical or enzymatic modification. 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 chitons and limpets (Shaw et al., 2008) and the cuticles of arthropods; sponges possess chitin as a component of their skeletons (Ehrlich et al., 2007). -chitin is present 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. Basic information on chitin and chitosan is available in books, encyclopedias and review articles (Enescu and Olteanu, 2008; Goosen, 1996; Kumar et al., 2004; JolleÁs and Muzzarelli, 1999; Kurita, 2006; Mano et al., 2007; Morimoto et al., 2002; Mourya and Inamdar, 2008; Muzzarelli, 1977; 1985a,b; 1993; 1996a,b; 2000; 2001; 2005; 2009a,b; Muzzarelli et al., 1986; Rinaudo, 2006a,b; RuelGariepy and Leroux, 2006; Terbojevich and Muzzarelli, 2000; Varghese and Elisseeff, 2006; Vinsova and Vavrikova, 2008; Yalpani, 1988). Today, chitins and chitosans of various grades are commercially available in large quantities from producers all over the world. Chitin isolates differ from each other in many respects, including 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 one million Da, but chitin isolates have lower values due to partial random depolymerisation occurring during the chemical treatments and depigmentation steps. The presence of a substantial quantity of nitrogen is a point of difference from other abundant polysaccharides: the fact that chitin contains four elements instead of three is often ignored when chitin is compared to cellulose. Chitosans are a family of deacetylated chitins. In general, chitosans have nitrogen content higher than 7% and degree of acetylation lower than 0.40. The
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removal of the acetyl group from chitin is a harsh treatment usually performed with concentrated NaOH. 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. Chitin and chitosan are not present in the human tissues, but acetylglucosamine and chitobiose are found in glycoproteins and glycosaminoglycans. Since chitosan is biodegradable, non-toxic, non-immunogenic and biocompatible in animal tissues, much research has been directed toward its use in medical applications such as drug delivery, artificial skin and blood anticoagulants.
31.2 Chitosan chemistry 31.2.1 Chemical structure and molecular characterisation The degree of acetylation for commercial samples is about 0.20. Experimental chitosan samples can be deacetylated almost completely, giving polyglucosamine. The evaluation of the degree of acetylation can be carried out by a number of techniques, depending on the amount of acetylated units. A total of 22 analytical methods were proposed for the determination of the degree of acetylation (Hein et al., 2008), including one based on UV spectrophotometry (Muzzarelli and Rocchetti, 1985; Wu and Zivanovic, 2008). High-resolution 1H and 13C NMR spectroscopy can provide information not only on the degree of acetylation in solution and solid state, but also on the sequence structure: in particular, random and block arrangements of GlcNAc and GlcN units are detected. In order to evaluate the average molecular weight of chitosan, viscometric and gel permeation chromatographic techniques are used. In contrast to the static light scattering 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, light scattering measurements are difficult to perform and sometimes the data are difficult to interpret, in the presence of aggregation and association. In summary, the molecular weight characterisation of chitosan remain problematic: 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±Sakurada equation is correctly used with sets of values as reported in the literature (Kasaai, 2007), for various aqueous salt systems in which the viscometric measurements are performed. Average molecular weight for chitosans can reach values of 500 kDa or more. Gel permeation chromatography methods are available to evaluate the molecular weight distribution of chitosan samples: calibration is performed by
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means of commercial standards (pullulan, for instance) or chitosan samples (Terbojevich et al., 1993). A good improvement is obtained by using a lowangle laser light scattering instrument as an on-line detector (Beri et al., 1993). Samples of chitosan with polydispersity index ca. 1.2±1.5 were obtained by preparative gel permeation chromatography; no variation of 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 ion exchange 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. Depolymerisation of chitosan 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 (Terbojevich et al., 1992). In the presence of nitrous acid, nitrosating species attack the glucosamine, but not the N-acetylglucosamine moieties and a 2,5-anhydro-D-mannose 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, proteases and pectinases are also effective (Yalpani and Pantaleone, 1994). This unspecific activity is possibly due to the simplicity of the enzyme±substrate complex formation. Almost all the cellulases produced by different kinds of micro-organisms can degrade chitosan to chitooligomers, thus they advantageously replace chitinases that are very expensive. The existence of bifunctional enzymes with cellulase and chitosanase activity is one of the reasons for cellulase efficacy on chitosan hydrolysis (Xia et al., 2008). In order to prevent the browning of the chitooligomers prepared via enzymatic hydrolysis, the chitooligomers were reduced with potassium borohydride to improve their chemical stability. The reduced chitooligomers had no oral acute toxicity, and they had almost the same biological significance in mice when injected intraperitoneally as the fresh chitooligomers (Qin et al., 2008).
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31.2.2 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 deacetylation of chitin is studied as a function of NaOH concentration, reaction time and temperature as determined by alkalimetric titration. Deacetylation is performed by traditional heating or under microwave irradiation. The maximum degree of deacetylation (98%) can be obtained after 30 min of thermal heating at 135 ëC in 50 wt% NaOH solution (Leonhardt et al., 2008). The shells of the brine shrimp Artemia urmiana are a rich source of chitin (29.3±34.5% of the shell dry weight). Artemia chitosan exhibited a medium molecular weight (450±570 kDa), regular degree of deacetylation (67±74%) and relatively low viscosity (29±91 centiposes). The physico-chemical characteristics (e.g., ash, nitrogen and molecular weight) and functional properties (e.g., water-binding capacity and antibacterial activity) of the prepared Artemia chitosans were enhanced, compared to crab control and commercial samples (Tajik et al., 2008). Chitosan of fungal origin in the molecular range 5±10 kDa was firstly prepared directly from the Absidia coerulea mycelia. To improve the low molecular weight chitosan production, the solid-state fermentation media were optimised to investigate the influence of substrate and supplemental medium components on low molecular weight chitosan production. The low molecular weight chitosan was obtained after treatments with 2% NaOH and 10% acetic acid. Maximal low molecular weight chitosan production was 6.12 g/kg substrate. Gel permeation chromatography combined with laser light scattering gave a molecular weight of 6.4 kDa with Mw/Mn of 1.09. Fourier transform infrared (FTIR), X-ray diffraction and 13C NMR spectra of the product showed typical peak distributions the same as those of standard chitosan which confirmed the extracted product to be chitosan-like. The method provided a new, simple and green technology to produce low molecular weight chitosan directly (Wang et al., 2008). The existence of chitosan in nature was discovered when it was isolated from the yeast Phycomyces blakesleeanus (Kreger, 1954). Chitosan occurs as the major structural component of the cell walls of certain fungi, mainly of the Zygomycetes species (Davies and Bartnicki-Garcia, 1984). However, to date, chitosans have been produced commercially by alkaline deacetylation of crustacean chitins, but experimental samples are available from other animals, such as houseflies and honey bees (Draczynski, 2008).
31.3 Properties of chitosans and derivatives 31.3.1 Solubility The solubility of chitin is remarkably poorer than that of cellulose, because of the high crystallinity of chitin, supported by hydrogen bonds mainly involving
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the acetamido and alcoholic groups. The insolubility of chitin is also pointed out as a serious drawback, nevertheless all chitins become soluble with the aid of hypochlorite as 6-oxychitins (Muzzarelli et al., 1999), and beta-chitin becomes soluble in a number of chemicals such as calcium chloride saturated methanol, among others (Nagahama et al., 2008). 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; the solubility of chitosan ascorbate depends on the preparation conditions. Under particular conditions chitin and chitosan can give hydrophilic 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. 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 usually being neutral or anionic. Chitosan is also soluble in alkaline solutions (ammonium bicarbonate, pH 10) where it forms glycosylamine functions (Muzzarelli et al., 2003). The selective introduction of saccharide residues at the chitosan amino group facilitates the conversion of the water-insoluble chitosan into various branched derivatives soluble in both neutral and slightly acidic (pH 5±6) aqueous media, with solubility being attained for some products with degree of substitution as low as 0.14. Chitosan can be reacetylated with acetic anhydride to obtain also water-soluble partially reacetylated chitin (Hirano and Horiuchi, 1989). 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. As reported for polyelectrolyte chains, the intrinsic viscosity of chitosan samples in aqueous solutions depends upon the ionic strength of the medium: plots of intrinsic viscosity vs. the reciprocal of the square root of the intrinsic viscosity are linear, and the intrinsic viscosity decreases with increasing salt concentration in the system is due to the shielding effect of the counter-ions. The chitosan molecule is rather stiff, less than DNA but more than polyacrylate; increasing acetylation leads to a more extended conformation and an even stiffer chain. It was confirmed that this is due to intra-residue hydrogenbonding between the carbonyl oxygen of the N-acetyl group and H6 in the next unit. A persistence length of ca. 22 nm was found, which is lower than that of chitin (ca. 35 nm) and the existence of a cholesteric mesophase was demonstrated (Terbojevich et al., 1991). Hydrophobic derivatives of chitosan, containing a small number of hydrophobic side chains, can be obtained from long chain acyl chlorides or
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anhydrides. A series of N-alkylchitosans (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). In aqueous solution, above a certain polymer concentration, intermolecular hydrophobic interactions lead to the formation of associations. As a consequence, these copolymers exhibit thickening properties equivalent to those observed for higher molecular weight hom*opolymers and play important roles as viscosity modifiers in a variety of waterborne technologies. N-Alkylated chitosans were synthesised by Michael addition reaction of chitosan and hydroxyethylacryl. The 1H NMR results indicated that the degree of substitution was from 0.18 to 1.2. The amorphous chitosan derivatives exhibited an excellent solubility in water. The thermal stability of the derivatives was lower than that of chitosan and hydroxyethylacryl-chitosan with degree of substitution higher than 1 decomposed at 226 ëC. In the presence of lysozyme, the initial degradation rate of the chitosan derivatives was dependent on the molecular weight. The antimicrobial activity of the chitosan derivatives was lower than that of chitosan (Ma et al., 2008).
31.3.2 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 depend on polyelectrolyte complexation mechanism and molecular weight of the enzymatically hydrolysed chitosan (Mi et al., 1999). 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 media than the chitosan-tripolyphosphate gel beads. Acylation 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 pre-treatment methods. Mixed N- and O-acylated products are prepared under similar conditions, using 10-fold excess anhydride (Yalpani, 1988). One of the simplest ways to prepare a chitin gel is to treat chitosan acetate salt solution with carbodiimide to restore acetamido groups. Thermally irreversible gels are obtained by N-acylation of chitosans: N-acetyl-, N-propionyl- and
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N-butyryl chitosan gels are prepared using 10% aqueous acetic, propionic and butyric acid as solvents for treatment with appropriate acyl anhydride. Both Nand O-acylation are found, but the gelation also occurs by selective N-acylation in the presence of organic solvents, as methanol, formamide and 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 N-acylation. Probably the gelation is due to the aggregation of chitosan chains through hydrophobic bonding. 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 1-naphthol-4-sulphonic acid or 1-naphthylamine-4-sulphonic acid, the mixture 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 (Lapasin et al., 1996). Chitosan gel beads could be prepared in amino acid solutions of about pH 9, despite the requirement for a pH above 12 for gelation in water (Kofuji et al., 1999). 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 pH 9. Gelation of the chitosan beads required about 25±40 minutes depending on the species of amino acid. pH-Sensitive hydrogels were synthesised (Qu et al., 1999a,b) by grafting D,Llactic 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 (pH 2.2) or simulated intestinal fluid (pH 7.4) at 37 ëC 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. 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 crosslinking with residual free amino groups. Gels were also obtained with 3hydroxybenzaldehyde, 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde (Muzzarelli and Ilari, 1994; Muzzarelli et al., 1994; Chen et al., 2003). As an extension of these works, a mushroom tyrosinase was observed to catalyse the oxidation of phenolic moieties of the synthetic polymer poly(4hydroxystyrene) in water-methanol. Although oxidation was rapid, in the order
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of minutes, only a small number of phenolic moieties (1±2%) underwent oxidation. Enzymatically oxidised poly(4-hydroxystyrene) was observed to undergo a subsequent non-enzymatic reaction with chitosan, based on UV spectra (Shao et al., 1999; Kumar et al., 1999). Further progress (Edwards et al., 1999a,b) 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. Dilute solutions of chitosan with 3,4-dihydroxyphenylethylamine (dopamine) become hydrogels within a short time by virtue of the tyrosinase-catalysed oxidation of dopamine to quinone: the viscosity of the chitosan solutions greatly increases as a result of the immediate reaction of quinone with the chitosan amino groups that confers water-resistant adhesive properties to the gel (Yamada et al., 2008). NaCl particles (porogen) and gamma-glycidoxypropyltrimethoxysilane (crosslinking agent) were used to make chitosan-silica porous hybrid membranes with optimal chitosan/crosslinker weight ratio 1. Macropores (ca. 10±202 m) in the membrane matrix, and micropores (8±10 nm) within the macropores were observed. Histidine, glutamic acid, tyrosine, L-DOPA, and p-aminobenzoic acid were individually linked onto said material with the aid of genipin: the paminobenzoate chitosan membrane exhibited the best affinity adsorption of tyrosinase from a crude Agaricus bisporus solution (Chao, 2008). Hydrogels of chitosan crosslinked with genipin appear to be most biocompatible and safe as indicated in a recent review article (Muzzarelli, 2009c). A biosensor was fabricated by immobilising tyrosinase on the surface of multiwalled carbon nanotubes±chitosan composite modified glassy carbon electrode. The composite film provided a biocompatible platform for the tyrosinase to retain the bioactivity and the nanotubes possessed excellent inherent conductivity to enhance the electron transfer rate. The biosensor showed high sensitivity (412 mA/M), broad linear response, low detection limit (5.0 nM) and good stability for determination of phenol. The biosensor was further applied to rapid detection of the coliforms, represented by Escherichia coli that could be detected as low as 10 colony-forming units (CFU) per ml (Cheng et al., 2008). Reactions with aldehydes The Schiff reaction between chitosan and aldehydes and ketones gives the corresponding aldimines and ketimines, which can be converted to N-alkyl derivatives 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
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carbohydrates and dialdehydes, such as glutaraldehyde. In particular, Ncarboxymethyl chitosan was obtained in a water-soluble form by a proper selection of the reactant ratios, i.e., using equimolecular quantities of glyoxylic acid and amino groups (Muzzarelli, 1988). Fully substituted N,N 0 -dicarboxymethylchitosan was obtained by the alkylation of chitosan at pH 8±8.5 and 90 ëC using a monochloroacetic acid : chitosan weight ratio of 4:1. The water-soluble derivative is suitable for applications involving chelation (An et al., 2008). 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 degree of substitution 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 betacyclodextrin (Yalpani, 1988). Studies on intestinal permeation enhancers were also reviewed as they provide information on the mechanism of action and safety of polymers. The active polymers are classified into: polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin), polyanions [Ncarboxymethyl chitosan, poly(acrylic acid)], and thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates) (Di Colo et al., 2008). Glutaraldehyde is a very popular crosslinking agent for chitosan (Muzzarelli et al., 1976). Chitosan networks were obtained by reaction with glutaraldehyde in lactic acid solution (pH 4±5). The rheology of the chitosan-glutaraldehyde gel system was studied by Arguelles-Monal et al. (1998). By reaction of chitosan with aldehydes, N-alkylidene 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. In the search for biocompatible hydrogels based exclusively on polysaccharide chains, chitosan and the dialdehyde obtainable from scleroglucan by controlled periodate oxidation were linked together (Crescenzi et al., 1995); the reaction takes place at pH 10 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. Likewise, -cyclodextrinmodified chitosan was synthesised via the Schiff base reaction with 6-O-(4formylphenyl)- -cyclodextrin (Liu et al., 2008), as well as a variety of chitosans containing aromatic groups (Sajomsang et al., 2008); the use of glutaraldehyde in most diverse applications does not decline at all. A semi-interpenetrating network was synthesised with poly(ethylene oxide) and chitosan, and crosslinked with glyoxal (Khalid et al., 1999). Swelling studies were performed on the chitosan/poly(ethylene oxide) semi-
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interpenetrating network and on the reference hydrogel (crosslinked chitosan) at pH 1.2 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 semi-interpenetrating network had elastic properties. Poly(ethylene glycol) 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 water-soluble 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 (DalPozzo et al., 2000). A continuation of this work was provided by Deng et al. (2007) who reported on the reaction with the counter-ion tripolyphosphate, and formation of nanoparticles.
31.3.3 Polyelectrolyte complex formation At high degree of protonation of the amino groups, chitosan spontaneously forms macromolecular complexes by reaction with anionic polyelectrolytes. These complexes are generally water insoluble and make hydrogels. Several reviews on polyelectrolytes have been published (Tsuchida and Abe, 1982; Kubota and Kikuchi, 1999; VanTomme et al., 2008). 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 diverse physical and chemical properties of the complexes. 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 (Sakiyama et al., 1999): the maximum volume of the complex gel was observed in a dilute NaOH solution at pH 10.5, and was about 300 times as large as the volume at pH values below 9 (Jiang et al., 1999). Nanoparticle made of two bioadhesive polysaccharides, hyaluronan and chitosan, were obtained by a very mild ionotropic gelation technique. The nanoparticles had a size in the range of 100 to 235 nm and a zeta-potential of ÿ30 to 28 mV. Toxicological studies were conducted in human corneal epithelial and conjunctival cell lines. The confocal images indicated that the nanoparticles were internalised by fluid endocytosis and that this endocytic process was mediated by the hyaluronan receptor CD44 (DeLaFuente et al., 2008a,b). Hyaluronan-chitosan nanoparticles made of low molecular weight chitosan (10±12 kDa) led to high levels of expression of secreted alkaline phosphatase in
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the human corneal epithelium model. Following topical administration to rabbits, these nanoparticles entered the corneal and conjunctival epithelial cells and were assimilated by the cells. More importantly, the nanoparticles provided an efficient delivery of the associated plasmid DNA inside the cells, reaching significant transfection levels (DeLaFuente et al., 2008a,b). Hydrogels composed of poly(N-isopropylacrylamide) with chitosan (Fang et al., 2008) or , -glycerophosphate with chitosan (Zhou et al., 2008) can be mentioned as examples of temperature-sensitive hydrogels with transition at 37 ëC.
31.4
Chitin as a food component
Crustaceans, insects, rodents, frogs, snails, earthworms, spiders, scorpions, centipedes, and millipedes have been used as food sources for millennia. A close examination of the world insect consumption shows that more than 2,000 edible species have been utilised as a food source, although this is deemed to be underreported and research will increase this number (Paoletti, 2005; Backwell and D'Errico, 2001; DeFoliart, 1992, 1999). Currently preferred insects are listed in Table 31.1. On the other hand, of the 15 million plants, animals and microbes on Earth, more than 90% of the world's food supply comes from just 15 crop species and 8 livestock species. Chitin digestion requires chitinolytic enzymes: chitinases are present in plant foods, and human and bacterial chitinolytic enzymes occur in the human digestive apparatus. The bibliographic data confirm the presence of chitinolytic enzymes in plant organs, and thus vegetables and fruits that are consumed by humans could help in the digestion of chitin. Although the level of plant chitinases is often correlated to the presence of stress, such as infections, these enzymes also occur in healthy plant tissues and above all in fruits under maturation, which are most often used in human nutrition (Taira et al., 2004; Truong et al., 2003). For examaple, the phyla Leguminosae have lectins, chitinases and glycohydrolases as major defence proteins in their seeds. Electrophoresis of the proteins of tamarind (Tamarindus indica L.) indicated the presence of a 34 kDa class III endochitinase (Rao and Gowda, 2008). Table 31.1 Consumption of insects as food in the world Continent
Species
Africa Central and South America
524 679
Asia
349
Australia
152
Most popular insects cooked and eaten Gonimbrasia belina (grub) Rhynchophorus spp (snout beetle) Aegiale hesperiaris (butterfly) Atta spp (ant) Locusta spp Bombyx mori (silk worm) Rhychophorus ferrugineus papuanus
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Chitin digestion by humans has generally been questioned or denied. Only recently have chitinases been found in several human tissues and their role has been associated with defence against parasite infections and to some allergic conditions. The gastric juices of 25 Italian subjects were tested on the artificial substrates 4-methylumbelliferyl- -D-N,N 0 -diacetylchitobiose and fluorescein isothiocyanate chitin to demonstrate the presence of a chitinase activity currently called acidic mammalian chitinase. It was found to be present in gastric juices of 20 out of 25 Italian patients in a range of activity from 0.21± 36.27 nmol/ml.hÿ1. In the remaining five gastric juices, the chitinase activity was almost absent. The allosamidine inhibition test and the measurement at different pH values confirmed that this activity was characteristic of a chitinase. The absence of activity in 20% of the gastric juices may be a consequence of virtual absence of chitinous food in the western diet (Paoletti et al., 2007). In the excreta of healthy people there is at least a bacterial species able to hydrolyse chitin to N-acetyl glucosamine. Clostridium paraputrificum in the human colon is able to synthesise and secrete chitinases and -N-acetyl glucosaminidase, that could take part in the digestion of chitin. Similarly, chitotriosidase, a chitinase produced by activated macrophages is able to hydrolyse chitin (Malaguarnera, 2006; Piras et al., 2007; Gianfrancesco and Musumeci, 2004; Renkema et al., 1995, 1998). An inherited deficiency in chitotriosidase activity is frequently reported in the plasma of Caucasian subjects, whereas this deficiency is rare in the African population. The study by Musumeci et al. (2005) compares chitotriosidase activity in the colostrum of 53 African women and 50 Caucasian women. Elevated chitotriosidase was found in the colostrum of African women on the first day after delivery (1230 662 nmol/ml.hÿ1) which decreased with time. The chitotriosidase activity on the first day after delivery in the colostrum of Caucasian women, however, was significantly lower (293 74 nmol/ml.hÿ1) and decreased to 25 20 and 22 19 nmol/ml.hÿ1 on the second and third day, respectively. The chitotriosidase activity in the plasma of African women was also higher (101 80 nmol/ml.h ÿ1 ) than that of Caucasian women (46 16 nmol/ml.hÿ1), but no correlation was found between the plasma and colostrum activities. The elevated chitotriosidase activity in the colostrum of African women is indicative of the presence of activated macrophages in human milk, consistent with the genetic characteristics of the African population, and their chitin eating habits. In fact, a chitin-rich diet induces the secretion of human chitinases.
31.5 Nutritional and health effects A number of European countries as well as the United States and some oriental countries (Japan, South Korea) have approved the sale of chitosan-based nutraceuticals as over-the-counter products for the control of obesity, hypercholesterolemia and hypertension. Chitosan, in this context, is generally
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regarded as safe, being a partially deacetylated chitin, i.e. a polysaccharide widely present in nature, in particular in human food such as certain cheeses and marine animals including crustaceans and squids. The chitosans used to prepare dietary supplements come, in fact, from crustaceans, mainly shrimps. The freshly caught crustaceans are peeled in the canning and freezing factories, and while the meat is canned or packed, the shells are collected and treated by chemical or microbiological means, in order to extract chitin. It has been verified that seasonal fluctuations and varieties of shrimps and squids caught during fishing activities introduce negligible differences in the chitosans resulting from the chitin extraction (Lavall et al., 2007); novel technologies have been experimented with, but with limited success (Kjartansson et al., 2006). The exposure of chitin to acids, alkali, surfactants, solvents for the extraction of carotenoids, and the submission of the chitosan flakes to drying, milling, sieving and other operations, introduce more diversity among various lots of chitosan. Thermal drying, for instance, may introduce a limited degree of crosslinking while the degree of deacetylation of the final chitosan may depend on the grain size of the chitin powder exposed to hot alkali during the deacetylation process. To date, there is no chitosan standard for any application. In other words, even though various grades are available such as technical, food and medical grades, no clear recommendation has been made and accepted for adopting a chitosan standard in food applications. As a consequence the chitosan currently used to manufacture chitosan tablets for human consumption are chosen mainly on the basis of their constant supply and economical convenience. In the eyes of today's producers, their characteristics are a secondary aspect and in general only the degree of acetylation, the viscosity and the microbiological contamination are certified. The producers do not declare the degree of crystallinity, the polydispersity of the molecular weight, the presence of amino acids and metals. Therefore it is difficult to predict the performances of the dietary supplements. A further aspect of uncertainty is introduced by the preparation of the tablets: the tendency is to adopt the most usual tabletting process regardless of the consequences that certain excipients have on chitosan activity in vivo. For example, tabletting involves the use of a binder in order to hold together the poorly compressible chitosan powder. Magnesium stearate, a slightly soluble compound, once coated on the chitosan powder, deeply alters the capacity of chitosan to react or effectively contact other compounds. Site-specific controlled release systems have been extensively investigated during the last decade. Chitosan salts with succinic acid, adipic acid, and suberic acid were prepared by spray-drying and were coated with stearic acid by the same technique. In a study the carriers were characterised in terms of morphology, size, swelling, mucoadhesive properties and drug loading, and focused on the in vitro influence of chitosan salts on the release behaviour of vancomycin hydrochloride from the uncoated and coated systems at pH levels of 2.0, 5.5 and 7.6 (Bigucci et al.,
Chitin and chitosan hydrogels
863
2008). The results of the study supports the view that chitosan tablets containing stearate salts might lack full anti-hypercholesterolhaemic activity simply because the stearate excipient prevents hydrogel formation in the stomach and effective contact with bile salts.
31.5.1 Cholesterol lowering in humans Chitosan was first shown to reduce serum cholesterol in humans in 1993, when adult males fed chitosan-containing meals for two weeks (3 g/d for week 1, 6 g/d for week 2) experienced 6% decrease in total cholesterol (Maezaki et al., 1993). The subjects also exhibited a 10% increase in high density lipoprotein cholesterol. Two studies have reported serum cholesterol reductions with chitosan treatment. Obese women consuming 1.2 g of microcrystalline chitosan for 8 weeks demonstrated significant reductions of low density lipoprotein, although not total serum cholesterol. A total of 84 female subjects with mild to moderate hypercholesterolemia receiving 1.2 g of chitosan per day experienced a significant decrease in total serum cholesterol (Bokura and Kobayashi, 2003). Metso et al. (2003) observed 83 middle-aged men and women without severe disease and with a total cholesterol of 4.8±6.8 mmol/l and triglycerides below 3.0 mmol/l. Their conclusion was that treatment with microcrystalline chitosan had no effect on the concentrations of plasma lipids or glucose in healthy middle-aged men and women with moderately increased plasma cholesterol concentrations. For oral administration to humans, chitosan is generally recognised as safe (Harrison, 2002; Kumar et al., 2004). Some 21 overweight normocholesterolemic subjects were fed a supplement containing equal amounts of glucomannan and chitosan for 28 days: the observed serum cholesterol reduction was mediated by increased faecal steroid excretion and was not linked to fat excretion. Greater faecal excretion of neutral sterols and bile salts was observed. The topic has been reviewed by Muzzarelli and Muzzarelli (2006) and by Pittler and Ernst (2004). In Sprague±Dawley rats, chitosan did not affect food intake but decreased body weight gain and significantly increased faecal fat and cholesterol excretion, reduced the lipid level in plasma and liver, increased liver hepatic and lipoprotein lipase activities (Zhang et al., 2008). To understand the mechanism of cholesterol lowering by chitosan, one should consider first that bile salts are formed from cholesterol in the liver and are secreted into the duodenum by the enterohepatic circulation: the bile salt pool is maintained stable, the newly ingested cholesterol compensating the excreted quantities. If, however, bile salts are sequestered by any suitable compound, some cholesterol is oxidised to produce more bile salts. Bile is produced at the rate of 700±1200 ml/day, bile salts accounting for 1.24±1.72%, cholesterol for 0.86±1.76 g/l; the average pH is 7.3. For the various intestinal tracts, the pH values are as follows: duodenum 4.7±6.5, upper jejunum 6.2±6.7, lower jejunum 6.2±7.3, ileum 6.1±7.3, colon > 7.3. It is worth noting that these
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values tend to keep the bile salts in solution, while depressing the chitosan solubility (chitosan pK = 6.3). The uptake of bile salts into chitosan-alginate gel beads was observed by Murata et al. (1999): the presence of weak acids (orotic, citric, folic and ascorbic) does not hinder the uptake; rather, chitosan orotate salt was found to enhance it. Various chitosans and heavily modified ChitopearlÕ chitosans were studied by Murata et al. (2003) and found to have capacities in the range 0.53± 1.20 mmol taurocholate per gram of chitosan (various degrees of acetylation) and 0.2±0.9 mmol taurocholate for ChitopearlÕ products; the capacity of QuestranÕ was ca. 1.0 mmol taurocholate/g. Higher capacities were observed for taurodeoxycholate (1.8 mmol taurodeoxycholate/g chitosan). Of course, these capacities depend on the initial bile salt concentration, grain size and other parameters. One millimole of taurocholate corresponds to 515 mg, thus the weight ratio taurocholate/chitosan orotate is 0.515 : 1.000, which appears to be a high ratio, notwithstanding the uncertainties related to the description of the experimental conditions. By using chitosan cinnamate and analogue compounds such as those of vanillic acid, hydroxycinnamic acids, cumaric acids and rosmarinic acid, the bile acid adsorption and the release of cinnamate were investigated in vitro. When chitosan cinnamate was soaked in a taurocholate solution, it adsorbed the bile acid while simultaneously releasing cinnamate. The amount of cinnamate analogue released was 0:286 0:001 mmol/g, practically coincident with the amount of taurocholate adsorbed. The amount of released cinnamate analogue was altered extensively by the species of analogue used for gel-bead preparation. Results were indicative of high affinity of taurocholate for chitosan, and suggest that those compounds may be of interest for complementary medicine to prevent lifestyle-related diseases (Murata et al., 2006). Eleven chitosan samples were evaluated for their fat- and bile acid-binding capacities, physico-chemical properties, and the correlations between each binding capacity and individual physico-chemical properties. The bile acid- and fat-binding capacities were estimated using in vitro assays, whereas the measured physico-chemical properties were deacetylation degree, swelling capacity, and solution viscosity. Chitosan samples might differ in their binding capacities against fat and/or individual bile acids. The bile acid-binding capacities were 0.20±0.61, 0.43±1.63 and 0.61±1.61 micromol/g chitosan for cholic, deoxycholic, and chenodeoxycholic acids, respectively. Stronger binding capacity of chitosan against a selected bile acid did not imply greater binding capacity for other bile acids (Zhou et al., 2006). Thongngam and McClements (2005 a,b) provided thermodynamic data by isothermal titration calorimetry on the binding of Na taurocholate to chitosan. At 30 ëC, Na taurocholate binds strongly to chitosan to form an insoluble complex containing ca. 4 mmol Na taurocholate/g chitosan at saturation, that means taurocholate : chitosan molar ratio ca. 2 : 3 and weight ratio ca. 2 : 1. Ionic strength had scarce influence; the enthalpy changes went from endothermic (at 10 ëC) to exothermic (at 40 ëC) indicating the importance of changes of
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hydrophobic interactions leading to the formation of micelle-like clusters within the chitosan structure. The binding capacities of sodium glycocholate to chitosan, diethylaminoethyl chitosan, quaternised diethylaminoethyl chitosan, and cholestyramine were 1.42, 3.12, 4.06 and 2.78 mmol/g, respectively. The capacity of dialkylaminoalkyl chitosans increased with the number of carbons in the alkyl groups, indicating that hydrophobic interaction plays a major role in the sequestration of bile acids (Lee et al., 1999); similarly, the capacity of 6oxychitosan for cholic acid decreases with increasing degree of oxidation, i.e., loss of cationicity (Yoo et al., 2005). Among the chitosan salts that instantly form from chitosan and bile acids in vitro, chitosan taurocholate appears to possess infrared spectral characteristics that qualify it as a real ionic, water-insoluble, poorly crystalline, hydrophobic chitosan salt. In chitosan taurocholate and hom*ologues, chitosan accounts for no more than one half by weight, therefore the hydrophobic nature prevails when contacting lipids. Butter and oils are collected to high extents with no discrimination of their components, including cholesterol and tocopherols. For these chitosan salts the lipid uptake is much higher than for plain chitosan. The salts studied in the present work are scarcely hydrolysed by a variety of hydrolases, and it is presumed that their complexes with lipids would be even more resistant to enzymatic attack. If the above findings are extrapolated from the in vitro model to the physiological environment, it seems reasonable to speculate the following: · Chitosan glycocholate and chitosan taurocholate insoluble salts subtract bile salts from the circulation thus forcing the organism to replete the bile pool at the expenses of cholesterol. · The activity of lipases on triglycerides is depressed as a consequence of the poor emulsification of lipids due to the lowered availability of taurocholate, the emulsifier. It is known that the pancreatic lipases require a certain dimension of the oil droplets in the emulsion in order to hydrolyse triglycerides: now, when the bile salts become scarce, inadequate emulsions are formed and then limited hydrolysis of triglycerides takes place. Ample information on digestive lipases supports these views (Mukherjee, 2003) that have been experimentally confirmed (Helgason et al., 2008). Lipases work thanks to the presence of bile salts that in one case activate the bile saltsdependent lipases, and in the other case provide the emulsion necessary to the pancreatic lipases for enzymatic activity. Moreover, as soon as the bile salt availability decreases due to chitosan ingestion, the bile salts-dependent lipases are poorly activated, and assimilation of lipids by the organism decreases sharply. · While pectinase is one of the most representative bacterial enzymes in the intestine, the resistance of the chitosan taurocholate, glycocholate and taurodeoxycholate salts to hydrolysis by this enzyme, as well as by other hydrolases, would certainly be an indication of the capacity of these hydrophobic salts to be excreted, presumably with accompanying adsorbed lipids.
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Table 31.2 Time (min) necessary for total digestion of freeze-dried chitosan salts suspended in aqueous solutions of animal, fungal and plant enzymes at 20 ëC and optimum pH Enzyme
Cellulase, Trichoderma reesei Alpha-amylase, barley malt Alpha-amylase, porcine pancreas Pectinase, Aspergillus niger Lysozyme, hen egg white Lipase, wheat germ Lipase, porcine pancreas
CTC
CGC
CTDC
pH
min
pH
min
pH
min
6.8 7.1 6.8 7.1 7.3 6.7 7.3
60 1440 no no 30 no no
6.6 7.0 6.8 6.9 7.0 6.6 7.0
90 1440 no no 4.5 no no
6.5 4.5 7.0 6.4 7.0 6.7 6.9
no no no no no no no
CTC = chitosan taurocholate; CGC = chitosan glycocholate; CTDC = chitosan taurodeoxycholate. No = No appreciable digestion after 48 hours. Source: Muzzarelli et al. (2006).
Upon mixing a solution of Na taurocholate with a solution of chitosan acetate, both at pH values close to 4, immediate precipitation of chitosan taurocholate salt is observed. The salt obtained with nearly stoichiometric amounts of both reagents was characterised by infrared spectrometry, and found to exhibit new bands indicative of its ionic nature. Similar compounds were obtained from Na taurodeoxycholate and Na glycocholate. When exposed to a number of hydrolases at pH values close to neutrality and 20 and 37 ëC, they were found to be poorly susceptible to enzymatic degradation: only Trichoderma reesei cellulase, egg white lysozyme and barley malt -amylase were effective on chitosan taurocholate and glycocholate, as shown in Table 31.2. The observed capacities of the freeze-dried salts for olive oil were 22 g oil/ g of chitosan taurocholate, 60 g oil/g of chitosan glycocholate, and 27 g oil/g of chitosan taurodeoxycholate. The capacities were 22.1 g butter oil/g of chitosan taurocholate and 22.1 g of corn oil/g chitosan taurocholate. These data, substantially much higher than similar data published for plain chitosan and various oils, mean that the lipid uptake takes place mainly by hydrophobic interactions with the insoluble salts formed by chitosan upon contact with bile (Muzzarelli et al., 2006).
31.5.2 Overweight control Fourteen published trials including a total of 1,131 participants met the inclusion criteria in a study by NiMhurchu et al. (2005a,b) published in review format by Jull et al. (2008). Analyses including all trials indicated that chitosan preparations result in a significantly greater weight loss (weighted mean difference ÿ1.7 kg; 95% confidence interval (c.i.) ÿ2.1 to ÿ1.3 kg), decrease in total cholesterol (ÿ0.2 mmol/L; 95% c.i. ÿ0.3 to ÿ0.1), decrease in systolic (ÿ5.9 mmHg; 95% c.i. -7.3 to -4.6) and diastolic (ÿ3.4 mmHg; 95% c.i. ÿ4.4 to
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ÿ2.4) blood pressure compared with placebo. If recalculated in terms of mg/dl, the loss of cholesterol is 3-4%, i.e., clinically significant. It was remarked that the quality of certain studies was sub-optimal. The conclusions were that evidence exists that chitosan is more effective than placebo in the short-term treatment of overweight and obesity. NiMhurchu et al. (2004) also conducted a 24-week randomised, double-blind, placebo-controlled trial, with a total of 250 participants (82% women; mean (s.d.) body mass index, 35.5 (5.1) kg/m2; mean age, 48 (12) y). The chitosan group lost more body weight than the placebo group (mean (s.e.), ÿ0.4 (0.2) kg (0.4% loss) vs +0.2 (0.2) kg (0.2% gain), P = 0.03) during the 24-week intervention. Studies of the effect of chitosan on human adiposity suggest that results may differ depending on whether the subjects are eating ad libitum or are on a weight loss diet. Overweight subjects consuming 2.4 g/day of chitosan for 28 days, also consuming their normal diet, showed no change in body weight during the trial (Gallaher et al., 2002). A six-week study was conducted on obese adults who lost more body weight (-2.3 vs 0.0 kg), body fat (-1.1 vs 0.2%) and absolute fat mass (-2.0 vs 0.2 kg). The combination of glucomannan, chitosan, fenugreek Gymnema sylvestre and vitamin C were therefore effective (Woodgate and Conquer, 2003). Gades and Stern (2005) tested the fat-trapping capacity of a chitosan product in 12 men and 12 women. Faecal fat excretion increased with chitosan by 1:8 2:4 g/day in males, but did not increase with chitosan in females: therefore fat-trapping claims should not be generalised. Chitosan greatly increases faecal fat excretion when consumed in sufficient amounts and can accelerate weight loss when subjects follow a low calorie diet, but will be ineffective in those consuming their habitual diets.
31.6 Food industry applications Chitosan, whose applications in the food industry have been reviewed by Rudrapatnam and Farooqahmed (2003) exhibits antimicrobial activity against a range of food-borne micro-organisms and consequently has attracted attention as a potential natural food preservative (Chen et al., 1998; El Ghaouth et al., 1992; Shahidi et al., 1999). Binding of trace metals and effect on membrane permeability have been postulated to be the main mechanisms for its antibacterial action (Helander, 2001; Zheng and Zhu, 2003). Muscle foods have low oxidative stability and are very susceptible to rancidity during production and storage. Numerous studies have indicated that lipid oxidation in meat and meat products may be controlled or minimised through the use of antioxidants (Gray et al., 1996; Nissen et al., 2004; Rhee, 1987). However, chitosan has a bitter taste, which may limit the use of artificial antioxidants in meat and meat products, when flavour is an important consideration. The antimicrobial and antioxidant potential of spices and herbs, such as basil, thyme, rosemary, garlic, clove, coriander, ginger, mustard and pepper are well
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known (Georgantelis et al., 2007a,b; Sebranek et al., 2005; Tipsrisukond et al., 1998). Mint (Mentha spicata) is used extensively (Choudhury et al., 2006). The mint extract has very good antioxidant potential, comparable to that of the synthetic antioxidant butylated hydroxy toluene (Kanatt et al., 2007, 2008). Mint extract did not show any antibacterial activity, though essential oils of some Mentha species have been reported to have antibacterial activity (Marino et al., 2001; Moreira et al., 2005). Mixed mint and chitosan efficiently scavenged superoxide and hydroxyl radicals and was particularly effective against Grampositive bacteria. The shelf-life of pork co*cktail salami, as determined by total bacterial count and oxidative rancidity, was enhanced in mint + chitosan-treated samples stored at 0±3 ëC. Chitosan films prepared with oregano essential oil were applied on bologna slices. Release of the essential oil compounds during film preparation and application on the meat product and consumer acceptability of bologna enriched with oregano essential oil were found satisfactory (Chi et al., 2007). Antimicrobial and physico-chemical properties of chitosan films and chitosan films enriched with essential oils were determined in vitro and on processed meat. Antimicrobial effects of pure essential oils of anise, basil, coriander and oregano, and of chitosan-essential oil films against Listeria monocytogenes and Escherichia coli were investigated. The films have the potential to be used as active biodegradable films with strong antimicrobial effects. Edible films can provide supplementary and sometimes essential means of controlling physiological, morphological and physico-chemical changes in food products. High density polyethylene film, a common packaging material used to protect foods, has disadvantages like fermentation due to the depletion of oxygen and condensation of water, which promotes fungal growth. Due to their filmogenicity, chitin and chitosan are satisfactorily used as food wraps. Semipermeable chitosan films modify the internal atmosphere, decrease the transpiration and delay the ripening of fruits. For the preparation of chitosan/ pectin laminated films and chitosan/methylcellulose films several approaches have been used, including simple coacervation. Chitosan films are tough, flexible and tear-resistant; moreover, they have favourable permeation characteristics for gases and water vapour. Chitosan is also suitable as a texturising agent for perishable foods: for instance, high viscosity chitosan solutions were used to prepare tofu, a widely consumed oriental food, for which the organoleptic properties did not vary appreciably while shelf-life was extended (Kim and Han, 2002; No et al., 2002). Processing of clarified fruit juices commonly involves the use of clarifying agents, including gelatin, bentonite, tannins, potassium caseinate and polyvinyl pyrrolidone. Chitosan is a dehazing agent used to control acidity in fruit juices, as well as being a good clarifying agent for grapefruit juice with or without pectinase treatment, besides apple, lemon and orange juices, and a fining agent for apple juice, which can afford zero turbidity products with as little as 0.8 kg/m3 of chitosan. No impact on the biochemical parameters of the juices was found (Chatterjee et al., 2004). Apple juice can be protected from fungal spoilage with the aid of modest additions of chitosan glutamate (Roller and Covill, 1999).
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Chitosan has a good affinity for polyphenolic compounds such as catechins, proanthocyanidins, cinnamic acid and their derivatives that can change the colour of white wines due to their oxidative products. By adding chitosan to grapefruit juice (15 g/l), the total acid content (citric, tartaric, malic, oxalic and ascorbic acid) was sharply reduced. Jang and Lee (2008) demonstrated the stability of tripolyphosphate-chitosan nanoparticles for ascorbic acid during heat processing and suggested the use of ascorbate-loaded chitosan nanoparticles to enhance antioxidant effects because of the continuous release of ascorbate from the nanoparticles in food processing.
31.7 Applications in drug delivery The usefulness and versatility of chitosan hydrogels is particularly evident when the field of drug delivery is considered. Plain chitosan, a safe biopolymer, has been extensively applied to the human body by all possible means and for a variety of purposes: chitosan is a functional ingredient of cosmetics, a biocompatible coating for prosthetic materials, a drug delivery item, a gene vector, a dietary supplement, and a wound healing dressing capable of restoring the native histoarchitecture. In a large group of materials used to enhance absorption of drugs, chitosan ranked first in terms of maximum drug bioavailability (enhanced absorption) and minimum damage to the membranes (Illum and Davies, 2005). Of course, the limitations of plain chitosan, mainly its scarce solubility at neutral and alkaline pH values, can be overcome by selecting adequate chemical or enzymatic modifications free from adverse effects on the living tissues: for example, the derivatisation of chitosan with the aid of glyoxylic acid (the simplest aldehydoacid) leads to N-carboxymethyl chitosan that can be interpreted as a glycine derivative as well. Because glycine is one of the most abundant amino acids in the human body, in collagen for instance, N-carboxymethyl chitosan is safe in principle, as confirmed experimentally; indeed it is a functional ingredient of hydrating creams present on a niche market for more than a decade. Pyruvic acid exemplifies the ketoacids from which aminoacid glucans can be derived via ketimine formation with chitosan. Such modified chitosans were developed in the 1980s (Muzzarelli et al., 1985; Muzzarelli and Zattoni, 1986) and have found applications in various areas (Sun et al., 2007). The safety of carboxymethylated chitosans has been demonstrated by Janvikul et al. (2007). According to Sun and Wan (2007), to qualify as a drug delivery aid and absorption enhancer, a modified chitosan should exhibit hydrogel characteristics, particularly because it should remain in contact with the mucosae long enough to achieve maximum effect, and any consequence on the mucosae should be completely reversible. Other requirements are also satisfied by chitosan hydrogels, because they are generally compatible with drugs, have no odour, are non-irritating, non-toxic and non-allergenic. From the pharmaco-
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logical standpoint, chitosans are inert at the concentrations required in practical use: they exhibit mild anti-inflammatory action. In fact, when chitosans contact the mucosal tissue a reaction takes place between the chitosan's glucosamine units and the sialic acid units of the mucin and epithelial cell membrane (Deacon et al., 1999). Therefore a decreased clearance of administered formulations and an increased absorption are observed. In a sheep model, chitosan was far superior to diethylaminoethyl (DEAE) dextran in terms of absorption enhancing effect. A reason for the difference between the two polymers was that the DEAE dextran is randomly and scarcely substituted, is a branched polysaccharide as opposed to linear chitosan that carries one primary amino group on each anhydroglucose ring. Some of the nitrogen atoms are hidden in the DEAE dextran network and are therefore unavailable for interaction with the mucosae (Harding et al., 1999). Experimental studies have led to the development of gastroretentive bioadhesive microsphere formulations and to devices for nasal delivery as well as for various mucosal routes. For example, chitosan-tripolyphosphate and chitosan± tripolyphosphate-chondroitin sulfate core-shell type microspheres were prepared by a continuous method using a multi-loop reactor under sterile conditions. All the types of microspheres produced were spherical in shape and had a porous structure; their mechanical resistance increased in the presence of chondroitin sulfate, which toughened the microsphere shell structure. For a drug release application, the preparation included the dissolution of ofloxacin, an antibiotic, in the chitosan solution before complex formation (Vodna et al., 2007). The study of the bioadhesive characteristics of chitosan led to the important discovery that this polysaccharide is not only bioadhesive so that it can be used to alter the intestinal transit of formulations and nasociliary clearance, but also has exceptional absorption effects when given nasally to rat and sheep models together with highly polar molecules such as insulin, calcitonin and morphine. The impressive increase in the bioavailability of the drugs administered together with chitosan could not be explained simply by a prolonged period of residence: it is now generally accepted that the mechanism of action of chitosan in enhancing the transport of drugs across the mucosal membranes is due to the combination of bioadhesion and the transient opening of the tight junctions between the cells of the mucosal membrane (Illum, 2003). Chitosan gels releasing insulin in response to glucose concentration have also been developed (Kashyap et al., 2007). For these purposes, chitosans can be formulated either as viscous solutions, as spray-dried powders, or as microspheres or nanoparticles. In most cases the effects of chitosan powders and microspheres are superior in providing enhancement of the nasal absorption of polar drugs as compared to chitosan solutions (nearly fivefold bioavailability of insulin, for instance). This may be justified by the formation of more effective hydrogels by spray-dried chitosans (see below). As a polar compound, morphine is not readily absorbed via the nose when administered in simple formulations (bioavailability in humans ca. 10%). A nasal chitosan morphine solution formulation can give rapid absorption of the
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drug with the peak plasma concentration within 10 min and 60% bioavailability that can be optimised at 80%, offering patients rapid and efficient pain relief by a non-injectable route (Illum et al., 2002). Chitosan microparticles produced by emulsification, precipitation, complex coacervation or solvent evaporation are optionally stabilised by crosslinking or by polyelectrolyte complex formation. The drug is either encapsulated during production or absorbed into the particles after production. The freeze-drying techniques are generally preferred (Kas, 1997). For the production of chitosan nanoparticles, one may rely on the ability of chitosan to gel in contact with the negatively charged counterparts, for example tripolyphosphate ions. The definition of the experimental conditions for the formation of the chitosantripolyphosphate nanoparticles has been made by Pan et al. (2002).
31.7.1 Trans-dermal drug delivery The use of chitosan has great potential as an aid to trans-dermal drug delivery and has been shown to mediate trans-dermal drug availability. The reported widening of tight junctions by chitosan observed in Caco-2 cells in vitro may be partially responsible for the ability of chitosan to improve trans-dermal drug delivery. Tight junctions are dynamically regulated protein complexes that link adjacent epithelial cells in such a manner as to prevent the passage of molecules through the paracellular space. They are composed of known trans-membrane proteins (occludin and claudin) and several associated intracellular proteins (Tsukita et al., 2001). Tight junction integrity can be investigated in numerous ways including measuring the trans-epithelial electrical resistance across confluent Caco-2 monolayers. The quaternary derivatives of chitosan have been shown to have penetration enhancement properties because they open the tight junctions of the intestinal epithelia at neutral and alkaline pH values. The use of nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Trimethyl chitosan and diethylmethyl chitosan, both with quaternisation degree of ca. 50%, were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and polyelectrolyte complexation. The latter nanoparticles had higher insulin loading efficiency and zeta potential than those made by ionotropic gelation. Trimethyl chitosan was also loaded with antigens for intranasal vaccination (Amidi et al., 2007). In vivo studies performed with trimethyl chitosans in rabbits confirmed the transdermal permeation enhancement, which increased with increasing degree of quaternisation (He et al., 2008). The polymers in free form had higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form (Sadeghi et al., 2008). Previous studies have shown chitosan-mediated decreases in electrical resistance of up to 80% across Caco-2 monolayers. This is accompanied by an increase in the permeability of the monolayers to inert hydrophilic protein
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markers such as inulin or mannitol (Borchard et al., 1996; Dodane et al., 1999; Kotze et al., 1998; Ranaldi et al., 2002; DeBritto and Assis, 2007). A chitosanmediated increase in permeability to inert markers points to passive transport that is possible only through the paracellular space (Artursson et al., 1996). As the chitosan-mediated changes in resistance do not appear to be a result of cell toxicity, it is supposed to act through an effect on the barrier properties of the cellular tight junctions. Also quaternised chitosan has been studied extensively as an absorption enhancer and proved to be non-toxic, mucoadhesive and capable of opening the tight junctions between epithelial cells. Changing the chitosan concentration or increasing the number of crosslinks by using chitosan derivatives with branching side chains can modify the rate of release of drug from these formulations. Once applied, the chitosan present in this formulation mediates prolonged contact with the epithelium via electrostatic interaction of the positively charged chitosan and the negatively charged glycoprotein residues on the cell surface. Passive diffusion of the drug down its concentration gradient onto the underlying epithelium over a prolonged period results in absorption of drug (Park et al., 2000; Ramanathan and Block, 2001). There are several advantages of trans-dermal drug delivery as opposed to oral delivery. For example, trans-dermally delivered drugs avoid the hostile environment of the gastrointestinal tract (hydrochloric acid and enzymes), and avoid first pass metabolism (the skin empties into the venous circulation and the drug is not routed directly through the liver where much drug metabolism occurs as with orally applied drugs). In addition, drugs delivered by the trans-dermal route show sustained plasma profiles over long periods of time, thus minimising the risk of fluctuations of drug plasma levels at night or between oral doses. Other attractive features lead to better patient compliance. The most popular and commercially available trans-dermal drug delivery system is the nicotine patch for nicotine replacement during smoking cessation. Chitosan gel-mediated trans-dermal drug permeation may be enhanced by the simultaneous application of other technologies known to aid trans-dermal drug delivery. The application of an electric current to aid the passage of drugs across the epithelium, along with dermal application of chitosan-drug hydrogels, resulted in a greater drug flux across the skin barrier compared to either method individually. This represents an enormous increase in the amount of permeated drug across the usually impermeable skin.
31.7.2 Spray-drying As a drug carrier, chitosan offers certain advantages over other polymers, such as cationicity, bioadhesion via interaction with mucin, degradability by lysozyme and lipases, generation of safe oligomers and monomers (glucosamine and N-acetylglucosamine), absence of allergic reactions, and immunostimulating activity. Chitosan helps overcome insolubility and hydrophobicity of drugs, but the semi-crystalline powder does not lend itself to direct compression; however, amorphous chitosan microspheres obtained by spray-drying
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are free-flowing and compressible powders, most suitable for drug delivery (He et al., 1999; Rege et al., 1999, 2003; DeLaTorre et al., 2003; Huang et al., 2003; Muzzarelli et al., 2004). Spray-drying of chitosan salt solutions provides chitosan microspheres having diameters close to 2±5 micron and improved binding functionality. While chitosan was often spray-dried, the following are significant examples. Betamethasone disodium phosphate-loaded microspheres demonstrated good drug stability (less 1% hydrolysis product), high entrapped efficiency (95%) and positive surface charge (37.5 mV). Formulation factors were correlated to particulate characteristics for optimising the pulmonary delivery. The betamethasone release rates were influenced by the drug/polymer ratio in the manner that an increase in the release percentage and burst release percentage was observed when the drug loading was decreased. The in vitro release of betamethasone showed a dose-dependent burst followed by a slower release phase that was proportional to the drug concentration in the range 14±44% w/w (Huang et al., 2002). Chitosan microspheres containing chlorhexidine diacetate, an antiseptic, were prepared by spray-drying. Chlorhexidine from the chitosan microspheres dissolved more quickly in vitro than chlorhexidine powder. The minimum inhibitory concentration, minimum bacterial concentration and killing time showed that the loading of chlorhexidine into chitosan could maintain or improve the anti-microbial activity of the drug, the improvement being particularly high against Candida albicans. It should be noted that the drug did not decompose despite its thermal lability above 70 ëC. An inclusion complex composed of progesterone and hydroxypropyl- cyclodextrin was prepared by spray-drying and freeze-drying methods. Progesterone alone and its inclusion complex with hydroxypropyl- -cyclodextrin were incorporated into chitosan by spray-drying and freeze-drying. Release data showed significant improvement of the dissolution rate of progesterone and controlled release was obtained in the presence of chitosan (Cerchiara et al., 2003).
31.7.3 Pulmonary drug delivery Chitosan is able to enhance the surface activity of 0.5 mg/ml of bovine lipid extract surfactant and to resist albumin-induced inactivation at an extremely low concentration of 0.05 mg/ml, one thousand times smaller than the usual concentration of poly(etylene glycol) and 20 times smaller than for hyaluronan (Zuo et al., 2006). Learoyd et al. (2008) described the preparation of highly dispersible dry powders for pulmonary delivery that display sustained drug release characteristics; they were prepared by spray-drying 30% aqueous ethanol formulations containing terbutaline sulfate as a model drug, chitosan as a drug release modifier, and leucine as an aerosolisation enhancer. The aerosol properties of the spray-dried powders were investigated. By selecting the average molecular
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weight of chitosan it was possible to exert some control over the rate of drug release: upon increasing the molecular weight of chitosan a more sustained release profile was obtained. For example, the low molecular weight chitosan released the totality of terbutaline after 30 min, whereas the high molecular weight chitosan required 2 h. Of course, the in vitro dissolution tests of this nature do not take into consideration the environment that would be encountered following inhalation, such as the relatively low quantity of lung secretions, the large surface area, the lungs' clearing mechanism such as the mucociliary escalator and the mucus layer and the presence of lung surfactant. The powders are expected to deposit predominantly in the central and peripheral regions of the lung following inhalation, with minimal oropharyngeal concurrent deposition. By virtue of their hydrogel behaviour in the lung, these powders would display delayed rather than instantaneous drug release thus offering the opportunity to reduce dosing frequency. Ventura et al. (2008) incorporated moxifloxacin, a wide spectrum antimicrobial active against common respiratory pathogens, in spray-dried chitosan microspheres, and observed that its intrapulmonary concentration was superior to that generated in plasma. By this means, they could overcome the common adverse events associated with the oral administration of this drug such as nausea and diarrhoea due to the impact on the human intestinal microflora. Uncrosslinked microspheres rapidly swelled in the lungs and released some chitosan that altered the biomembrane permeability to the drug. Glutaraldehydecrosslinked microspheres, on the other hand, did not exhibit this property. Therefore the microspheres retarded the absorption of moxifloxacin, and within 6 h the cumulative amount of permeated drug was ca. 18, 11 and 7% w/w for free drug, loaded crosslinked microspheres, and loaded uncrosslinked microspheres, respectively. Microspheres of methylpyrrolidinone chitosan, a modified chitosan amply recognised as one of the least cytotoxic materials, was found suitable for the intranasal delivery of metoclopramide HCl (Giunchedi et al., 2002). In their further study (Gavini et al., 2008) attention was devoted to the hydrogel formation from methylpyrrolidinone chitosan. The microspheres obtained by spray-drying were contacted with buffer solutions: when HCl/KCl or acetate buffers are used, gel forms and then dissolves upon dilution with water. However, when phosphate buffers are used, a hydrogel forms which does not dissolve in water regardless of the amount used, but dissolves in diluted HCl solutions. Hydrogel formation is therefore dependent on the medium, but not on the presence of a drug. The phosphate anion at pH values 5.5±7.4 seems to bridge the chitosan chains leading to the formation of an ionically interconnected hydrogel. Because methylpyrrolidinone chitosan microspheres retain adhesion properties and lend themselves to incorporation of a variety of drugs, it appears that they are most suitable for biomedical applications involving drug delivery.
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31.7.4 Drug delivery to the intestine Neutron activation-based gamma scintigraphy was used to evaluate the gastroretentive properties of formulations containing chitosan (Mw 150 kDa) in humans. At the same time, the transit of the formulations (40 or 95% of chitosan) was monitored in the small intestine: although the chitosan studied had exhibited marked mucoadhesive capacities in vitro, retention of the chitosan formulations in the upper gastrointestinal tract was not sufficiently reproducible and the duration of retention was relatively short (Sakkinen et al., 2006). Therefore, chitosan tablets should be protected with an enteric coating or similar, if they are expected to reach the lower intestine without damage. In this light, there is interest in preparing chitosan-Ca-alginate microparticles that can effectively deliver 5-aminosalicylic acid (5ASA) to the colon after per os administration. A solution containing 5ASA and sodium alginate was spraydried to obtain microspheres smaller than 10 m, that were crosslinked and coated into a solution of CaCl2 and chitosan. Of importance, 1H NMR and UVvis spectra of 5ASA showed no degradation when working under light protection with freshly prepared solution, and using nitrogen to prevent the oxidative self-coupling of 5ASA moieties. The microspheres showed acceptable morphology, and prevailing localisation of chitosan in the particle wall, while alginate was hom*ogeneously distributed throughout the particle imparting anionic character. The IR spectra of 5ASA-loaded Ca alginate microparticles indicated absence of covalent bonds between the polymer and the drug that was molecularly dispersed within the chitosan alginate microspheres during the production process (Mladenovska et al., 2007). Chitosan alginate nanoparticles were investigated as mucoadhesive vehicle for the prolonged topical ophthalmic delivery of an antibiotic, gatifloxacin. A modified coacervation or ionotropic gelation method was used to produce gatifloxacin-loaded nanoparticles with average particle size 205±572 nm and zeta potential 17.6±47.8 mV (Motwani et al., 2008). A combination of spray-dried chitosan acetate and hydroxymethylpropyl cellulose was tested as a new compression coat for 5ASA tablets. In a simulated system, such tablets were able to pass through the stomach, pH 1.0, and the small intestine, pH 6.8. The delayed release was controlled owing to the swelling with gradual dissolution of chitosan and modified cellulose at low pH and depressed solubility at neutral pH. After reaching the colon, the dissolution of chitosan triggered the 90% drug release within 14 hours. Moreover, the chitosan was found to undergo degradation by unspecific activity of -glucosidase in the colonic fluid thus enhancing the drug release (Nunthanid et al., 2008). Because the chitosan powder can be easily compressed into a tablet, it was demonstrated that 5ASA can be admixed with the plain chitosan powder and the resulting tablet can be coated with convenient enteric coatings: the antiinflammatory action of chitosan enhances the curative properties of 5ASA while being degraded by enzymes provided by the colonic flora (Muzzarelli, 2005). Following a more traditional approach, chitosan hydrogel beads were prepared by the crosslinking method followed by enteric coating with
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EudragitÕS100. The size of the beads was ca. 1:04 0:82 mm. The amount of the drug released after 24 h from the formulation was ca. 97% in the presence of extracellular enzymes as compared with ca. 64% and 96% release of drug after 3 and 6 days of enzyme induction, respectively, in the presence of 4% cecal content (Jain et al., 2007). Uncoated and EudragitÕ-coated chitosan-Ca alginate microparticles efficiently loaded with budesonide, with bioadhesive and controlled release properties in the gastrointestinal tract, were prepared by spray-drying. Microspheres had mean size 4.05±5.36 m, narrow unimodal distribution and positive surface charge. CaCl2 limited the swelling ratio, while the swelling behaviour of coated beads was mainly determined by the EudragitÕ enteric coating. The controlled release properties were suitable for local treatment of inflammatory bowel diseases (Crcarevska et al., 2008). The water-soluble N-(2-carboxybenzyl)chitosan (Muzzarelli et al., 1982) was crosslinked with glutaraldehyde and its swelling characteristics were studied in different pH buffer solutions. The swelling ratio decreased with an increase in the amount of glutaraldehyde, and the swelling was more evident in alkaline solutions than in acidic media, showing the lowest swelling ratio at pH 5.0. The latter increased in alkaline solutions with the raising of the degree of substitution, but no significant change was observed in an acidic environment. The crosslinked carboxybenzyl chitosan showed swelling reversibility when alternately soaked in pH 1.0 and 7.4 buffer solutions. Results qualified the crosslinked carboxybenzyl chitosan as a potential pH-sensitive carrier for colonspecific drug delivery system (Lin et al., 2007).
31.8
Conclusion
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, support the expectation that chitosan gels will expand their current range of applications in the near future. This versatile biopolymer, remarkable for its cationicity, has attained a unique position among the hydrocolloids.
31.9
References
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and MCCLEMENTS D J (2005a), `Influence of pH, ionic strength and temperature on self-association and interactions of sodium dodecyl sulfate in the absence and presence of chitosan', Langmuir, 21, 79±86. THONGNGAM T and MCCLEMENTS D J (2005b), `Isothermal titration calorimetry study of the interaction between chitosan and bile salt (sodium taurocholate)', Food Hydrocolloids, 19, 813±819. TIPSRISUKOND N, FERNANDO L N and CLARKE A D (1998), `Antioxidant effects of essential oil and oleoresin of black pepper from supercritical carbon dioxide extractions in ground pork', Journal of Agricultural and Food Chemistry, 46, 4329±4333. TRUONG N H, PARK S M , NISHIZAWA Y, WATANABE T, SASAKI T and ITOH Y (2003), `Structure, heterologous expression, and properties of rice (Oryza sativa L.) family 19 chitinases', Bioscience, Biotechnology and Biochemistry, 67, 1063±1070. TSUCHIDA E and ABE K (1982), `Interactions between macromolecules in solution and inter-macromolecular complexes', Advances in Polymer Science, 45, 83±213. TSUKITA S, FURUSE M and ITOH M (2001), `Multifunctional strands in tight junctions', Nature Reviews, 2, 285±293. VANTOMME S R, STORM G and HENNINK W E (2008), `In situ gelling hydrogels for pharmaceutical and biomedical applications', International Journal of Pharmaceutics, 355, 1±18. VARGHESE S and ELISSEEFF J H (2006), Hydrogels for musculoskeletal tissue engineering, Polymers for Regenerative Medicine, 2006, 95±144. VENTURA C A, TOMMASINI S, CRUPI E, GIANNONE I, CARDILE V, MUSUMECI T and PUGLISI G (2008), `Chitosan microspheres for intrapulmonary administration of moxifloxacin: interaction with biomembrane models and in vitro permeation studies', European Journal of Pharmaceutics and Biopharmaceutics 68, 235±244. VINSOVA J and VAVRIKOVA E (2008), `Recent advances in drugs and pro-drugs design of chitosan', Current Pharmaceutical Design, 14, 1311±1326. VODNA L, BUBENIKOVA S and BAKOS D (2007), `Chitosan based hydrogel microspheres as drug carriers', Macromolecular Bioscience, 7, 629±634. WANG W P, DU Y M, QIU Y L, WANG X Y, HU Y, YANG J H, CAI J and KENNEDY J F (2008), `A new green technology for direct production of low molecular weight chitosan', Carbohydrate Polymers, 74, 127±132. WOODGATE D E and CONQUER J A (2003), `Effects of a stimulant-free dietary supplement on body weight and fat loss in obese adults: a six-week exploratory study', Current Therapeutic Research ± Clinical and Experimental, 64, 248±262. WU T and ZIVANOVIC S (2008), `Determination of the degree of acetylation of chitin and chitosan by an improved first derivative UV method', Carbohydrate Polymers, 73, 248±253. XIA W S, LIU P and LIU J (2008), `Advances in chitosan hydrolysis by non-specific cellulases', Bioresource Technology, 99, 6751±6762. YALPANI M (ed.) (1988), Polysaccharides: Synthesis, Modifications and Structure/ Property Relations, Amsterdam, Elsevier. YALPANI M and PANTALEONE D (1994), `An examination of the unusual susceptibility of aminoglycans to enzymatic hydrolysis', Carbohydrate Research, 256, 159±75. YAMADA K, AOKI T, IKEDA N, HIRATA M, HATA Y, HIGASHIDA K and NAKAMURA Y (2008), `Application of chitosan solutions gelled by melB tyrosinase to water-resistant adhesives', Journal of Applied Polymer Science, 107, 2723±2731. YAMADA T and KAWASAKI T (2005), `Microbial synthesis of hyaluronan and chitin: new approaches', Journal of Bioscience and Bioengineering, 99, 521±528. THONGNGAM T
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and LEE H G (2005), `Effects of selective oxidation of chitosan on physical and biological properties', International Journal of Biological Macromolecules, 35, 27±31. ZHANG J L, LIU J N, LI L and XIA W S (2008), `Dietary chitosan improves hypercholesterolemia in rats fed high-fat diets', Nutrition Research, 28, 383±390. ZHENG L Y and ZHU J F (2003), `Study on antimicrobial activity of chitosan with different molecular weights', Carbohydrate Polymers, 54, 527±530. ZHOU H Y, CHEN X G, KONG M, LIU C S, CHA D S and KENNEDY J F (2008), `Effect of molecular weight and degree of chitosan deacetylation on the preparation and characteristics of chitosan thermosensitive hydrogel as a delivery system', Carbohydrate Polymers, 73, 265±273. ZHOU K Q, XIA W S, ZHANG C and YU L (2006), `In vitro binding of bile acids and triglycerides by selected chitosan preparations and their physico-chemical properties. LWT-Food Science and Technology, 39, 1087±1092. ZUO Y Y, ALOLABI H, SHAFIEI A, KANG N X, POLICOVA Z, COX P N, ACOSTA E, HAIR M L and NEUMANN A W (2006), `Chitosan enhances the in vitro surface activity of dilute lung surfactant preparations and resists albumin-induced inactivation', Pediatric Research, 60, 125±130. YOO S H, LEE J S, PARK S Y, KIM Y S, CHANG P S
32 Konjac mannan S. Takigami, Gunma University, Japan
Abstract: Konjac mannan is a main component of tubers of konjac which is a perennial plant of Araceae. It is a heteropolysaccharide consisting of -Dglucose (G) and -D-mannose (M), with a G/M ratio of 1 to 1.6. Konjac mannan contains very small amounts of acetyl groups and the viscosity of its aqueous solution is quite high. Deacetylation occurs with alkali treatment and a chewy irreversible gel is prepared. The gel has been used as a traditional dietary food in Japan for a long time. Konjac mannan interacts synergistically with other polysaccharides and forms thermoreversible gels. In this chapter, the following subjects are explained: cultivation of the konjac tuber, the production process and purification of konjac flour, the chemical structure and molecular weight of konjac mannan, the component analysis of commercial konjac flour, the properties of mixture gel synergistically prepared by konjac mannan and other polysaccharides, the uses and applications of konjac flour, and regulatory status. Key words: konjac mannan, glucomannan, konjac tuber, cultivation, production process, purification, structure, gel, viscosity.
32.1 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 C. 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 C. Koch grows in Japan. They contain konjac mannan in their tubers. Konjac
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Fig. 32.1
Konjac plants.
mannan is a heteropolysaccharide consisting of -D-glucose (G) and -Dmannose (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 crossfertilisation. Figures 32.1 and 32.2 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 ~650 m 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. 150 m 5 m. 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
Konjac mannan 891
Fig. 32.2 Two-year-old konjac tubers.
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%. 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,
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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, texturing, and water binding. It may be used to provide fat-replacement properties in fat-free and low-fat meat products.
32.2
Manufacture
32.2.1 Cultivation Only Amorphophallus konjac C. 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.
32.2.2 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 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 lowerquality 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.
Konjac mannan 893 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.
32.2.3 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 32.3 and 32.4 show the SEM images of commercial konjac flour, with Fig. 32.4 being the one which has been highly purified. The surface of konjac flour shows scale-like patterns and seems to have been worn smooth (Fig. 32.3). After purification, the scale-like patterns are more clearly observed. Table 32.1 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 nitrogencontaining 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
Fig. 32.3 Scanning electron micrograph of konjac flour (Seiko).
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Fig. 32.4 Table 32.1 purification
Scanning electron micrograph of purified konjac flour.
Analytical results of components in konjac flour before and after 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
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.
32.3
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:
Konjac mannan 895 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 32.5 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 32.6 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 (1/2) of KM was carried out using partially methylated KM samples.20 The average values of Mw and 1/2 were determined to be 10105 and 110nm. It was also reported that both Mw and 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
Fig. 32.5 FT-IR spectrum of konjac mannan analysed by the attenuated total reflection (ATR) method.
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Fig. 32.6 Chemical structure of konjac mannan.
chromatographic (GPC) fractionation. The Mw, molecular dispersity and RMS were 13.2105, 2.1 and 130nm, respectively.
32.4
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 32.2 shows typical technical data of two types of commercial konjac flours and purified flour of them. The data is kindly given by 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. 32.7. The konjac flour, Rheolex RS, was characterised by a very fine mesh size (80 mesh sieve) and the measurements were carried out at 25 ëC 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. 32.7, 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
Konjac mannan 897 Table 32.2
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
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 490 ppm 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 85 ppm Less than 300/g negative
Chinese konjac tuber (bonded) Ordinary flour
Purified flour
13.5±13.6 18.0 69.9 68.9 8.4 5.3 3.0 1.5 0.9 0.3 82.2 90.0 0.6 0.8 4.9 2.1 2.1 g/kg 0.64 g/kg Not detected Not detected Not detected Not detected 760 ppm 170 ppm 420/g Less than 300/g negative negative
+ 1% konjac aqueous solution at 35 ëC after 4 h stirring at 90 rpm. * g/100 g of konjac flour. ** nitrogen-containing substances.
concentration, but is affected by pH of the solution. The effect of pH on viscosity change for 1% and 2% KM solutions is listed in Table 32.3. The viscosity of KM solution decreases with decreasing pH value. At a high pH, KM solution changes to gel.
Fig. 32.7
Relationships between viscosity of purified commercial konjac flour, Rheolex RS, and stirring time: () 1%, (·) 2%.
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Table 32.3 Effect of pH on viscosity change for 1% and 2% KM solutions Viscosity (cps)
KM* concentration Water (%) (no pH adjust.) 1 2
31,600 341,000
pH 4
pH 3
pH 2.5
31,800 340,000
29,900 301,000
18,600 251,000
* ± Rheolex RS.
Table 32.4 Viscosity of mixtures of KM and other gums with various composition. Total concentration of the mixtures is 1% KM* Other gums concentration 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 32.5 Gel strength of mixtures of KM and other gums with various compositions. Total concentration of the mixtures is 1% KM* Other gums concentration 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
Gel strength (g) Xan
LBG
Gel
Pec
Car
Aga
GG
CMC
± 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 ±
± ± ± ± ± ±
± ± ± ± ± Ð
* ± Rheolex RS; Xan ± xanthan gum; LBG ± Locust bean gum; Gel ± Gelatin; Pec ± Pectin; Car ± carrageenan; Aga ± Agar; GG ± Guar gum; CMC ± Carboxymethyl cellulose.
Konjac mannan interacts synergistically with other polysaccharides and forms thermoreversible gels. The viscosity of the mixtures and the gel strength are listed in Tables 32.4 and 32.5, respectively. The synergism is observed for the combination of KM and xanthan gum, KM and carrageenan, and KM and agar. Table 32.6 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
Konjac mannan 899 Table 32.6 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 32.7 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.
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 32.7 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.
32.5 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,
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Table 32.8 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
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 32.8.
32.6
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.
Konjac mannan 901
32.7 References 1. 2.
3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
(ed.), Science of Konjac, Keisuisha, Hiroshima (1984). 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). R. TAKAHASHI, I. KUSUKABE, S. KUSANO, Y. SAKURAI, K. MURAKAMI, A. MAEKAWA 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. S. OKIMASU
S. TAKIGAMI
INDEX
Index Terms
Links
A A. xylinum BPR2001
725
A. xylinum BPR3001E
725
Absidia coerulea
853
Acacia senegal
253
726
258
260
258
260
702 amino acid composition
261
carbohydrate component of gum
263
characteristics
261
collecting gum arabic
253
molecular mass distribution
262
Acacia seyal
253
amino acid composition
261
characteristics
261
molecular mass distribution
262
Acacia seyal var. fistula
255
Acacia seyal var. seyal
255
Acetobacter xylinum
725
acidic mammalian chitinase
861
728
actigum, see Scleroglucan Aerobacter levanicum
603
affinity chromatography
314
afzelia africana
539
agar
540
541
82 agarophyte seaweed for production use
84
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
477
Index Terms
Links
agar (Cont.) applications
96
cultural world regions
99
different uses and agarophytes used
97
food applications
96
industrial application
103
insect culture
102
microorganism culture media
103
vegetable tissue culture formulations
102
chemical structure
89
agarose
90
future trends
104
easily soluble agar comparative analysis gelation
105 91
agarose gelation
94
gelling and melting temperatures
93
typical gel temperatures
93
historical background
83
manufacture
86
freezing-thawing method
87
syneresis method
87
Agaricus bisporus
857
agarobioses
88
agaropectins
91
agarophyte seaweed
84
Gelidium sesquipedale microphotograph taxonomic classification α-L-arabinofuranosyl
86 85 654
664
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Albumen, see egg white albumin
391
Alcalase
658
Alcaligenes faecalis var. myxogenes
568
alcohol
194
aldobiouronic acid
480
alginates
807
composition and sequential parameters
811
foods, nutrition and health
823
applications in food products
394
823
nutritional aspects and health benefits
823
future trends
824
gels and gelling technologies
817
alginic acid gels
822
diffusion setting
819
elastic properties as function of average G-block length
820
internal setting
821
ionic crosslinking
818
parameters controlling kinetics and final properties principal methods of manufacturing
817 819
isolation from seaweeds
809
manufacture
808
regulatory status
824
structural characteristics
810
structure and physical properties
809
chemical composition and sequence
809
molecular weight
812
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
alginates (Cont.) selective binding ions
812
source dependence
811
technical data limitations for use
813 813
properties and applications in liquid phase
816
alginic acid gel
813
alkalimetric titration
853
alkanamines
524
allosamidine inhibition test
861
alternan
606
applications
608
manufacture
606
properties
607
regulatory status
609
structure
607
structure of a portion
608
alternansucrase
606
alternase
607
Amadori rearrangement
607
44
American Association of Cereal Cemists
52
Amorphophallus aldus
892
Amorphophallus konjac
892
Amorphophallus rivieri
892
amphotericin B
524
amyloglucosidase
636
amylopectin
594
Anogeissus dhofarica
479
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Anogeissus latifolia
477
Anogeissus leiocarpus
479
Anogeissus pendula
479
Anogelline
479
anthrone-sulfuric reaction
602
AOAC method
281
Aquasorb cmc type A-500
721
Arabidopsis AtCesA gene
621
arabifuranohydrolase
669
arabinofuranosidases
660
arabinoxylans
xxiv
analysis and detection
479
68
621
669
apparent viscosity effect of shear rate
673
effect of shear rate and polymer concentration applications
674 679
cryostabilisers
680
film formation
680
surface active agents
680
content in various fractions of barley
663
effect of concentration on volume of baked bread
678
elastic modulus development during gelation
676
and ethanol co-production from wheat
662
extraction, isolation and purification
658
aqueous extraction from endospermic tissue
658
physical grain fractionation
662
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
653
Index Terms
Links
arabinoxylans (Cont.) strategies of extraction from agricultural by-products
660
xylooligosaccharides production
664
molecular and physico-chemical characteristics molecular structure
675 664
arabino(glucurono)xylans general structure
666
general molecular features
664
phenoxy radicals of ferulic acid
667
structural elements present
665
structural heterogeneity
667
neutral arabinoxylans general structure
654
neutral monosaccharides and phenolic acids
657
occurrence and content
655
physico-chemical properties
671
gelation
675
molecular weight
671
molecular weight variation of arabinoxylan preparation solution properties physiological effects
671 672 681
procedure for large-scale isolation and purification
659
role in bread-making
677
total and water soluble, in whole grains and grain tissues arachin
656 391
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Arako
891
arrum
410
Artemia urmiana
853
Asakura-Oosawa depletion potential
892
38
Ascophyllum nodosum
814
asialoglycoprotein receptors
525
Aspergillus aculeatus
664
Aspergillus niger
660
ASTM D-897
792
ASTM D-1002
792
ASTM D-2094
792
ASTM D-3528
792
ASTM E96-93
778
Astragalus gossypinus
496
Astragalus gummifer
496
Astragalus membranaceus
521
Astragalus microcephalus
496
Astragalus mongholicus
521
astragalus root
521
Astralagus gossypinus
502
Astralagus gummifer
502
atomic force microscopy
368
584
Aureobasidium pullulans
593
699
autohydrolysis
172
avenin
392
Avicel AC-4125
743
Avicel BV-1518
743
Avicel BV-2815
743
Avicel BV 2815
747
Avicel CL-611
742
775
746
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Avicel CM 2159
746
Avicel LM-310
755
Avicel PH
754
Avicel RC-501
742
Avicel RC-581
742
Avicel RC-591
742
Avicel RT 1133
742
Avicel-plus SD 4422
749
Avicel-plus stabiliser
749
avidin
364
axlewood
479
Ayurveda
487
Azotobacter vinelandii
808
Aztec marigold
523
749
746
B bacterial cellulose biosynthesis
724 726
mechanism
727
pathway
726
functional properties
730
crystallinity and degree of polymerisation
730
macromolecular structure
730
mechanical strength
731
miscellaneous properties
732
water-holding capacity
732
generalised model of ribbon assembly in A. xylinum historical overview
727 725
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
bacterial cellulose (Cont.) manufacture
728
agitated culture
728
stationary culture
728
mechanical properties
732
regulatory status
736
structure
729
uses and applications
733
applications in paper and paper products
734
audio components development
734
biomedical applications
734
food applications
733
other applications
735
bacteriocin
764
Bakli
479
Bancroft’s rule
401
bassorin
502
benzoic acid
772
Bernoullian statistics
810
Beta Trim
628
betamethasone disodium
873
β-glucan synthase
621
β-glucanase
535
β-glucopyranose
711
1,3-β-glucosidic linkages
567
Bifidobacterium lactis
453
bioelectrodes
792
Biofills
735
biological response modifier
586
773
561
636
774
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
660
Index Terms
Links
Bioprocess
735
BioZate
338
bipolar membrane electroacidification
313
Blanose cmc type 7HOF
719
Blanose cmc type 7H3SXF
718
Blanose cmc type 7HXF
718
Blanose cmc type 7HXFMA
720
Blanose cmc type 9M31F
720
Blanose cmc type 9M31XF
718
bloom strength
147
β-mercaptoethanol
308
Botox
632
bovine serum albumin
366
Brewer’s grain
660
720
430
818
571
572
C Caco-2 cells
871
cadoxen
570
caffeine
522
calcitonin
870
Calpis
338
Calvin–Benson–Bassham cycle
394
capsaicin microcapsules
459
carboxymethyl cellulose
700
applications
717
bakery products
720
frozen products
718
general
717
instant products
718
low pH milk products
721
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
carboxymethyl cellulose (Cont.) soft drinks
720
table sauces and dressings
719
idealised unit structure
717
properties
713
general
713
interaction with proteins
714
viscosity effect CMC-casein complex carob bean gum
715 237
carrageenan food applications
177
applications in dairy products
183
applications in water
181
dairy applications
181
water gels
178
furcelleran and other seaweed-derived products manufacture
164 165
manufacturing process
165
raw materials
165
physical properties acid stability
170 171
cation concentration effect on gelling temperatures gel properties
174 173
gel properties of pure and blended carrageenans
175
kappa carrageenan-kappa casein milk protein interaction pH stability
176 172
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
carrageenan (Cont.) properties of solution
171
summary of properties
177
synergism with other gums
175
synergy with locust bean gum and konjac glucomannan thixotropic rheology regulatory status E407 structure
176 173 167 167 169
alkali modification
170
repeating units
171
caseinomacropeptide
328
caseinophosphopeptide
328
caseins
299
338
301
-based protein products industrial reparation and caseinates
304 303
and casein-derived milk protein products composition
306
fractionation
307
physico-chemical characteristics
299
302
Casocidin-I
329
339
casokinins
327
339
casomorphins
326
339
casopiastrin
328
casoplatelins
328
casoxins
326
Cellulase
636
Cellulon
735
339
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
‘cellulose gel’ network
748
cellulosics
710
applications
714
carboxymethyl cellulose
717
hydroxypropyl cellulose
716
methyl cellulose and hydroxypropyl methyl cellulose methyl ethyl cellulose cellulose structure
714 717 712
formulations bake-stable filling
716
buttermilk drink pH4.5–4.6
721
fruit cake mix
721
ice cream
719
instant chocolate drink
718
instant fruit drink powder
718
ketchup
720
milk orange juice beverage pH4.5–4.6
722
potato croquettes
715
soya burgers
716
topping for whipping
717
water ice or ripple
719
idealised unit structure of cellulose gum
712
manufacture manufacturing process
711
raw material
711
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
cellulosics (Cont.) properties
712
carboxymethyl cellulose
713
general
712
hydroxypropyl cellulose
713
methyl cellulose and hydroxypropyl methyl cellulose
713
methylethyl cellulose
713
regulatory status
722
ingredient declarations
723
names and serial numbers
722
permitted use levels
723
structure
711
cereal arabinoxylans
654
cereal β-glucans
615
biosynthesis
621
botanical distribution
617
chromatogram from lichenase hydrolysis
618
commercial products
628
extraction and purification
622
food applications
632
future trends
638
general structure
617
health benefits
624
alleviate blood pressure
627
alleviate diabetes
626
antimicrobial action
627
cancer prevention
627
626
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
cereal β-glucans (Cont.) changes in serum total and LDLcholesterol
625
cholesterol-lowering effect
624
delay gastric emptying
627
626
interactions that may form junction zones between molecules
637
molecular weight and apparent viscosity
620
Nutrim-OB micrograph
629
processing
635
regulatory status
637
structure and analysis
617
variation in molecular weight
619
variation in sequence of triosyl units
618
wheat vs barley baked breads
633
cereals
386
chemical composition
388
composition of various grains
388
nutritional and health effects
414
protein composition and structure
394
Cerogen
626
CesA gene
621
chaconine
414
chaotropic agents
631
637
96
chaotropic salts
673
chemical peeling process
238
Chinese Kombuchar
733
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
chitin and chitosan hydrogels
849
applications in drug delivery
869
drug delivery to intestine
875
pulmonary drug delivery
873
spray-drying
872
trans-dermal drug delivery
871
chitin as food component
860
chitosan chemistry
851
chemical structure and molecular characterisation
851
chitosan production
853
depolymerisation of chitosan
852
consumption of insects as food
860
food industry applications
867
nutritional and health effects
861
cholesterol lowering in humans
863
overweight control
866
properties of chitosans and derivatives
853
gelation
855
polyelectrolyte complex formation
859
solubility
853
total digestion time of freeze-dried chitosan salts
866
chitinases
852
chitobiose
851
Chitopearl
864
chitosanase
852
chitotriosidase
861
chlorhexidine diacetate
873
chloropropanols
417
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
chlorosulphonic acid
637
Choice dm
336
chondroitin sulfate
870
Chondrus crispus
165
chúcata
499
chymosin
320
Clarifoil
770
Clarisol
770
claudin
871
clearogel, see Scleroglucan clinical nutrition
69
enteral feeds
70
supplements
69
CMC, see carboxymethyl cellulose Coacervates, see protein-polysaccharide complexes coalescence
34
Codex Alimentarius Commission
5
Cohn process
395
colonic health
69
256
415
459
461
Committee on Food Additives and Contaminants
5
complex coacervation
422
confocal scanning laser microscopy
444
continuous centrifugal separators
399
conversions
119
acid hydrolysis
119
dextrinisation
119
enzyme hydrolysis
120
oxidation
119
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
convicilin
393
Corynebacterium laevaniformans
603
cosmetics
161
co-solubility
421
creaming
31
Cross equation
37
cross-linking
115
cryo-TEM
443
Cryptococcus albidus
664
C-trim
628
629
C-trim20
629
632
C-trim30
629
632
C-trim50
629
Cucumis L. cv. ‘Laura,’
562
cupin
415
curdlan
567
applications
637
585
food applications
585
other applications
586
13
580
chemical structure
568
degree of thermo-irreversible gelation
583
C NMR spectra of curdlan gels
118
569
DSC heating curves 5% aqueous dispersions after heating
583
aqueous dispersions at various concentrations electron micrograph of curdlan granule
582 570
frequency dependence of storage and loss modulus
573
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
curdlan (Cont.) functional properties aqueous suspension properties
570 573
solution properties and conformations thermal and morphological analysis gel network
570 582 579
gel strength concentration dependence
575
effect of urea
576
heating temperature effect
576
and syneresis at 30°C
577
and syneresis at 32°C
577
gelation
574
gel formation
574
gel properties
575
molecular conformations
577
580
high temperature structure at high humidity
581
at low humidity
581
hydrogen bond types in curdlan triple helix
579
light scattering and viscosity data
571
manufacture process
569
molecular weight dependence of meansquare radius of gyration
572
NaOH concentration dependence of intrinsic viscosity
572
native curdlan
568
neutralised curdlan gels morphologies
584
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
curdlan (Cont.) production
568
regulatory status
588
room temperature structure
581
X-ray fibre diffraction pattern of anhydrous form
578
cyanoethylated pullulan
596
cystein-sulfuric acid reaction
602
D Dahlquist criterion
787
Dahurian larch, see Larix dadurica dairy products
181
dalarelin
458
D-arabitol
728
Davedi
479
Dawn multi-angle laser light scattering method
895
deacetylation
853
de-arabinosylation
669
debranning
661
dehydrodiferulic acid
668
denaturation temperature
153
dental moulds
103
desolventizer-toaster
399
detarium
539
540
dextran
609
671
applications
611
chemical structure
610
manufacture
609
663
541
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
542
Index Terms
Links
dextran (Cont.) properties
611
regulatory status
612
structure
610
dextransucrase
610
D-glucono-δ-lactone
822
Dhanta
479
Dhao
479
Dhawra
479
Dho
479
diclofenac-sodium
522
dietary fibre
52
effects on metabolism and health colonic health
55
57
68
69
gastrointestinal tract and impact on other nutrients
57
metabolic syndrome : obesity appetite and anti-inflammatory effects
61
diethylaminoethyl dextran
870
diethylmethyl chitosan
871
differential scanning calorimetry
679
diffusion theory of adhesion
784
diguanylate cyclase
727
dimethylformamide
594
dimethylsulfoxide
570
Dindal
479
Dindiga
479
dithiothreitol
308
68
594
598
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
611
Index Terms
Links
E E407
167
E407A
167
Eastern larch, see Larix laricina edible fibre
99
insoluble fibres
99
soluble dietetic fibres
99
egg proteins
359
egg white foams
369
egg yolk emulsions
365
gels
373 basic principles
373
egg white
375
egg yolk
374
II/A isotherms of different lipid constituents
368
LDL adsorption mechanism at oilwater interface
369
mean droplet diameter and creaming index
367
parameters of kinetic of diffusion towards air-solution interface physico-chemistry and structure
371 360
egg white
363
egg yolk
360
technofunctional uses egg white foams egg white foams
359 363 369 370
372
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
egg white (Cont.) formation and stabilisation mechanisms
369
interfacial properties of proteins
370
key parameters
372
gels
375
glucidic and mineral functions
365
hen egg white composition proteins
364 364
avidin
364
composition and physico-chemical and functional properties
365
interfacial characteristics
372
lysozyme
365
ovalbumin
364
ovomucin
365
ovomucoïde
364
ovotransferrin
364
egg yolk
360
chemical composition
360
emulsions
365
basic principles
365
importance of assemblies
366
role of constituents
366
gels
368
374
hen egg yolk composition
361
fractionation of plasma and granules
361
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
egg yolk (Cont.) physical state of granules as function of pH and ionic strength repartition of constituents macrostructure and main constituents
363 362 360
‘egg-box’ model
813
electrical model theory
784
electron beam irradiation
680
electron microscopy
392
electrostatic interactions elsinan
27 600
applications
602
manufacture
600
properties
601
regulatory status
603
segment structure
601
structure
601
Elsinoe leucospila
600
Emivirine
521
emulsifier E473
774
emulsifiers
xxiv
emulsifying agent
774
24
emulsion stability and hydrocolloids
23
adsorbing hydrocolloids effect
42
non-adsorbing hydrocolloids effect
35
oil-in-water emulsion instability mechanism principles
25 26
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
emulsion stability (Cont.) theoretical reaction potential of spherical emulsion droplets
27
endoglucanases
607
endoxylanases
660
Ensure
336
enteral feeds
70
enzymes
194
epichlorohydrin
605
epigallocatechin gallate
548
551
Escherichia coli
857
868
EU food legislation
415
Eucheuma denticulatum
165
Eudragit S100
876
European larch, see Larix deciduas Evolus
338
exoglucanases
607
exudate gums
xxiv
F fats
124 and oils
127
ferulic acid esterase
660
feruloylation
668
flash desolventizer system
399
flocculation depletion and serum separation
31 38
Flory plot
540
Flory universal viscosity constant
571
fluorescein isothiocyanate chitin
861
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
FMC BioPolymer
809
food hydrocolloids
1
commercially important hydrocolloids
2
food products containing hydrocolloids
3
hydrocolloid fibres
19
fermentation product effect
20
future trends
21
health benefits
20
physical effect
19
regulatory aspects
5
European system
6
international
5
International Numbering System for Food Additives other trade blocks synergistic combinations
8 7 18
interactions in hydrocolloid mixtures
18
thickening characteristics
8
disaccharide repeat units
13
main hydrocolloid thickeners
14
viscosity–shear rate profile
11
10
viscosity–shear rate profile for 1% guar gum
12
viscosity–shear rate profile for 1% xanthan gum 1% CMC 1% guar gum 20% dextran and 30% gum arabic
12
zero shear viscosity log as a function of polymer concentration
10
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
food hydrocolloids (Cont.) viscoelasticity and gelation
14
18
frequency dependence for 1% xanthan gum solution and 1.5% amylose gel
15
frequency dependence for guar gum solutions at concentrations of 0.5% and 2.0%
14
gel texture comparison
16
main hydrocolloid gelling agents
17
world market for hydrocolloids formamide
5 611
637
formulations 30% oil dressing with roasted sesame seeds and Dijon mustard
200
bake-stable fruit preparation
225
chilled liquid cake mix
199
cooked ham with 30% added brine
179
dairy ice cream mix
182
dessert jelly
215
flan dessert
181
fluid gel for beverages
217
fruit juice jelly
214
fruit-flavored water dessert jelly
178
jelly sweets
222
jelly sweets using gellan gum and thin boiling starch
222
peach yogfruit
224
pulp suspension beverages
218
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
formulations (Cont.) reduced sugar jam
224
vinaigrette-style salad dressing
180
Fourier transform infrared spectroscopy
440
Fox-Flory theory
544
freeze-fracture microscopy
442
freezing-thawing method
87
French wheat flours
668
fructan
604
fructo oligosaccharides
838
fructofuranosyl rings
604
Fry Shield
767
FT-Raman spectroscopy
670
full press method
398
Furcellaria
165
670
853
374
furcelleran carrageenan and other seaweed-derived products
164
G galactomannans
195
228
annual consumption of certain hydrocolloids
230
future trends
250
manufacture
237
carob bean gum
237
fenugreek gum
242
guar gum
239
guar splits and gum powder
240
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
895
Index Terms
Links
galactomannans (Cont.) raw materials and structure
229
aleurone cell layers
232
Dreiding model of galactomannan
234
233
235
GPC chromatogram of xanthan, guar and carob bean gum
236
guar seed cross section
233
model in carob bean gum
233
theoretical repeating units of galactomannan
234
regulatory status
249
technical data
243
carob bean gum analysis
249
245
cold swelling carob bean gum power law data
248
fenugreek seek analysis
246
guar solutions acid stability
248
gum content in fenugreek
246
other guar products power law data
248
polynomial equation coefficients
247
uses and applications
249
carob bean gum functional properties
250
gastric emptying
57
gastrointestinal tract
57
colonic effects
58
prebiotic effects and interactions
60
production and effects of short chain fatty acids
59
second meal effects
58
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
236
Index Terms
Links
gastrointestinal tract (Cont.) small intestine effects
57
upper gut effects – gastric emptying
57
Gas-X Thin Strips
770
gatifloxacin
875
GATIFOLIA
480
GATIFOLIA SD
488
Gaviscon
822
gel permeation chromatography
260
314
485
895 gelatin
142
applications
158
bloom values, concentration and function in some food
159
confectionery
159
cosmetics
161
foods
159
nutritional and health proprieties
161
pharmaceutical and medical
160
photography
162
chemical composition and physical properties
149
10% bovine, warm and cold water fish gelatin gelling kinetics amino acids
155 150
bloom maturing process for 6.67% mammalian gelatin
156
collagen and gelatin chemical composition
149
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
851
Index Terms
Links
gelatin (Cont.) collagen and gelatin physical properties
152
helix amount in 2% cold water fish gelatin solution
154
molecular weight distribution
151
thermoreversible gelling process
154
types A and B gelatins with similar Bloom values gelatin derivatives
152 157
chemically modified gelatin
158
cold water soluble
157
hydrolysates
157
manufacturing
143
acid pre-treatment
145
alkaline pre-treatment
145
from extraction to final product
145
gelatin manufacturers
146
polypeptide chains with varying molecular weight
146
raw material consumption for the gelatin production raw material sources
144 143
regulations, technical data and standard quality test methods
147
bloom strength
147
quality control
149
viscosity
148
gelatinisation Gelidiella
112 84
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Gelidium amansii
84
gellan gum
4
future trends
225
manufacture
205
regulatory status
225
structure
205
primary structure technical data
204
205 206
calcium, sodium and potassium effects
210
common sequestrants
208
dispersion
206
effect of dissolved salts on hydration temperature
207
gel texture comparison
212
gelation
208
211
gum blend ratio effect on modulus and brittleness
213
gum gel properties for gel formation in 60% sucrose
210
gum gel properties for gel formation in water
210
hydration
206
sodium citrate effect
207
texture
211
texture profile high and low acyl gum gels uses and applications
212 213
dairy
218
dessert jellies
214
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
gellan gum (Cont.) effect of sugar on textural properties
221
formulation guidelines
217
fruit preparations
221
223
high and low acyl gum gel properties
214
other applications
224
potential processes for fluid gels
216
pre-gelation prevention
223
raw fruits ionic composition
223
sucrose concentration effect on modulus, hardness and brittleness
220
sugar confectionery
219
suspending agent
215
gelling memory
94
Gengiflexs
735
genipin
857
Gigartina
165
gliadin
392
394
globulins
391
394
Glucagel
631
632
glucansucrases
606
Glucerna
332
glucomannan
894
glucomannan fibre
637
336
68
glucomannans
195
Gluconoacetobacter xylinum
725
glucopyranosyl monomers
617
glucose oxidase-peroxidase reagent
620
glucurono(arabino)xylans
665
728
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
glutamate glucan
858
glutaraldehyde
858
glutelins
392
gluten
394
glycerolmonostearate
768
Glycine max
694
glycinin
389
glycomacropeptide
314
Glyloid
537
glyoxal
858
glyoxylic acid
869
Gouda cheese
455
Gracilaria green leaves and fruits
393
84 387
chemical composition
389
food application
412
nutritional and health effects
414
processing
399
protein composition and structure
394
Grinsted Xanthan 200
189
Grinsted Xanthan CLEAR 200
189
Grinsted Xanthan EASY
189
Grinsted Xanthan SUPRA
189
Grinsted Xanthan ULTRA
189
guanosine triphosphate
726
guar gum
239
derivatives
242
guar pods and seeds
240
seed components
241
Guinier plots
391
594
542
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms gum arabic
Links 43
252
488
703 applications
270
beverages
271
confectionery
270
dietary fibre
271
flavour encapsulation
271
arabinogalactan component
263
arabinogalactan protein complex structure caramel-type products formulation
264 270
definition Codex Alimentarius Advisory Specification for Gum arabic
258
EU Gum Arabic Specification
258
European Pharmacopoeia
258
FAO Food and Nitrition Paper No. 49 1990
256
51st meeting of Joint Expert Committee on Food Additives
257
49th meeting of Joint Expert Committee on Food Additives United States Food Chemical Codex
256 259
United States Pharmacopeia and the national Formulary
259
effect of shearing time on viscosity of gum arabic solutions
267
EU Gum Arabic Specification
258
European Pharmacopoeia
258
new comprehensive regulatory status
259
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
702
Index Terms
Links
gum arabic (Cont.) United States Food Chemical Codex
258
United States Pharmacopoeia and the national Formulary flavour encapsulation formulation
259 271
gel permeation chromatography Elution profiles
268
UV absorbance elution profiles
269
grades of Sudanese gum
254
manufacture
255
marshmallow formulation
270
properties
265
regulatory aspects
256
stabilisation of oil droplets
268
storage and loss moduli
267
structure
260
supply and market trends
254
twisted hairy rope proposal AGP
505
viscosity as function of concentration
265
vs xanthan gum vs sodium CMC viscosity shear rate gum ghatti
266 477
formulations
488
butter cream
490
dressings
490
mayonnaise type dressing
489
MCT
488
orange oil, ester gum, β-carotene MCT orange oil–ester gum
489 489
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
gum ghatti (Cont.) gum ghatti nodules
478
manufacture
480
regulatory status
491
Asia
491
Australia
493
Japan
492
Russia
493
South Africa
493
South America
491
United States
492
structure
480
technical data
483
emulsification
486
molecular weight
484
physico-chemical parameters
483
rheology
486
solubility
483
uses and applications
487
beverage emulsion
488
butter cream
490
dressings
490
mayonnaise type dressing
489
viscosity as function of concentration
487
vs gum arabic amino acid composition
484
elution profile after fractionation
485
emulsification performance and stability
488
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
gum ghatti (Cont.) physico-chemical and molecular weight parameters gum karaya
483 488
manufacture
498
product specification
510
regulatory status
526
structure
503
technical data
510
adhesive properties
513
compatibility
513
film-forming properties
513
heat stability
513
pH stability
512
preservative
513
rheological properties
512
solubility
511
viscosity
511
water-binding properties
513
uses and applications
521
food applications
521
industry
522
pharmaceutical uses
522
gum tragacanth grades in Turkey Bianca
497
Fior
497
Fior Extra
497
Pianto
497
major components
502
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
gum tragacanth (Cont.) manufacture
496
pectic component structure
502
product specification
508
regulatory status
525
structure
501
technical data
508
acid stability
509
compatibility
510
emulsification ability
510
heat stability
510
preservatives
510
rheological properties
509
solubility
508
surface activity
509
viscosity
508
uses and applications
519
food applications
519
non-food applications
520
pharmaceutical applications
520
Gummi indici
479
Gummi indicum
479
gummosis
496
Gymnema sylvestre
867
H hashab
253
heat-induced gelation
374
helianthinin
391
helices
111
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
hemicellulose
725
heparin
458
Hercules
767
high acyl gellan gum gelation
211
hydration
208
high methoxyl pectin
700
Hobart
752
hom*ogalacturonan
695
hom*opolysaccharide
593
hordein
392
Huang Ch’i
521
Humicola insolens
661
hydrocolloid emulsifying agents hydrocolloids
664
42 1
90° peel test curves
791
coated garlic
766
for coatings and adhesives
760
adhesion mechanisms
783
adhesion tests
788
adhesive hydrocolloid preparations
782
film-application techniques and stages
777
food uses and applications of adhesives future trends
784 792
inclusion of food additives in edible films
771
market estimates for edible films
779
methods for testing coatings
778
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
hydrocolloids (Cont.) next generation of edible films and possible research direction non-food coatings
779 776
non-food uses and application of adhesives
781
novel products
769
parameters to be considered in food coating
775
structure-function and hydrogeladherend relationships
786
uses and applications of bioadhesives
785
edible protective films
761
coatings for fried products as oil resisters edible packaging materials
766 761
gum coatings for fruits and vegetables
764
meat, seafood and fish coatings
762
miscellaneous coatings
767
emulsifying/stabilising agents and emulsion stability
834 23
gellan-sitosterol-coated garlic
766
gelling agents
834
health aspects
50
clinical nutrition
69
effects on metabolism and health
55
future trends
70
mechanism of action
56
57
68
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
hydrocolloids (Cont.) non-digestible carbohydrates in food
51
modified probe-back test
789
Oblate
767
thickening agents
834
toffee coated by edible film
767
as wet glues
790
hydrogels
783
hydrolysed oat flour
622
792
hydrolysed starch-polyacrylonitrile copolymers hydrophobic hydration
776 787
hydrophobically modified cellulosic thickeners
776
hydroxypropyl cellulose applications
716
properties
713
hydroxypropyl methyl cellulose applications
714
properties
713
hydroxypropyl-β-cyclodextrin
873
hyperentanglements
545
hysteresis
173
I immunoglobulins
314
immunoglobulins Y
452
incipient gel temperature
713
Indian gum
479
insulin
870
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
intelligent gels
855
International Numbering System for Food Additives
8
hydrocolloids
9
modified starches
8
Inula helenium
829
inulin
829
absorption profiles
843
applications
839
fat spreads and dairy spreads
840
fillings
842
low-fat and low-sugar ice cream
841
low-fat cake
842
low-fat hazelnut spread
843
low-fat mayonnaise/dressing
841
low-fat yoghurt
841
wafers
842
basic chemical structure
830
effect of shear treatment and seeding on firmness
837
effect on creaminess of skimmed yoghurt
841
effect on rheology of carrageenan gel
834
effect on starch viscosity
835
future trends
844
gel strength in relation to concentration
837
hydrolysis of different types at pH 3.5
833
inulin content of different crops
830
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
inulin (Cont.) long-term solubility at different temperatures
832
maximum solubility as function of temperature
832
nutritional and health benefits
838
overview of applications
840
overview of nutrition claims from EU 1924/2006
844
procedure for making inulin gel
836
production process
831
regulatory status
843
seeding process
837
technical properties
831
acid stability
833
heat stability
833
inulin and hydrocolloids
833
inulin as gelling agent
835
parameters affecting gel characteristics
836
solubility
831
viscosity
832
ion exchange chromatography
314
ionotropic gelation technique
859
Iranian grading system
497
Isogel
51
isomaltodextranases
607
isothermal titration calorimetry
864
isracidin
329
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
J Japanese larch, see Larix leptolepsis jelly mini-cups
824
Joint Expert Committee on Food Additives
5
256
296
594
595
723
744
824
K kallikrein-kinin system
327
kami
781
Kanten
86
Kappacin
329
Kappaphycus alverezii
165
Kardhai
479
KELCOGEL
205
KELCOGEL F
205
KELCOGEL LT
205
KELCOGEL LT100
205
Kenwood
752
Kerry Ingredients
767
ketoprofen
459
kibbling
255
Kitchenaid
752
Klebsiella pneumonaie
327
339
konjac flour analytical results of components
894
manufacture production process
892
purification
893
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
konjac flour (Cont.) purified analytical results of components
897
scanning electron micrograph
894
Rheolex RS, relationship between viscosity and stirring time scanning electron micrograph konjac mannan
897 893 889
applications and functional uses
900
chemical structure
896
effect of pH on viscosity change
898
FT-IR spectrum analysed by attenuated total reflection method
895
and κ-carrageenan with various compositions effect of salt concentration
899
effect of sugar concentration
899
konjac plants
890
manufacture
892
cultivation
892
production process
892
purification
893
and other gums with various composition gel strength
898
viscosity
898
purified, scanning electron micrograph
894
regulatory status
900
structure
894
technical data
896
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
konjac mannan (Cont.) two-year-old tubers
891
uses and applications
899
Kon-nyaku
891
Kuhn length
541
kuzu mochi
555
899
L lactalbumin
312
337
Lactobacillus acidophilus
453
Lactobacillus brevis
681
Lactococcus lactis
325
lactoferricin
329
336
339
lactoferrin
314
325
337
lactoferroxins
326
lactokinins
327
lactoperoxidase
314
lactorphins
326
lactotransferrin
337
Laminaria hyperborea
808
Langevin dynamics
424
Langmuir Blodgett transfer
368
Langmuir film balance
368
Langmuir monolayers
516
lap-shear test
788
325
larchwood arabinogalactan manufacture
500
product specification
518
regulatory status
527
structural features
507
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
larchwood arabinogalactan (Cont.) structure
505
technical data
517
moisture retention and shelf-life
519
mouthfeel
519
osmolality
518
solubility
518
stability to pH
518
viscosity
518
uses and applications
524
biomedicine
524
food
525
industry
524
508
Larix dadurica
501
Larix deciduas
501
524
Larix laricina
501
524
Larix leptolepsis
501
Larix occidentalis
500
501
Larix siberica
501
524
leaf protein concentrate
387
legumes
384
chemical composition
388
chemical composition of soybean defatted meal, concentrate and isolate
396
forage legumes
384
green legumes
384
farmed
food applications of main soybean protein preparations
409
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
legumes (Cont.) nutritional and health effects
413
popular grain legumes
385
protein composition and structure pea
393
soybean
393
protein structure or soybean 11S globulin
390
soymilk, soywhey, tofu and okara production
411
legumin
393
legumin-like globulins
391
Leuconostoc mesenteroides
606
levan
603
applications
605
chemical structure
604
manufacture
603
properties
604
structure
604
Lifsh*tz-Slezov-Wagner theory
609
35
lignin
725
lipophilic substitution
120
Listeria innocua
772
Listeria monocytogenes
764
Listerine PocketPaks
769
locust bean gum
175
locust bean gum-xanthan system
786
773
868
low acyl gellan gum gelation
209
hydration
206
211
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
low-methoxyl pectin
767
low-viscosity tamarind seed gum
556
LVSTX, see low-viscosity tamarind seed gum lysozyme
365
372
375
439
540
672
851
M Maltese Cross
112
maltodextrin
594
maltooligosaccharides
622
maltotetraose
593
maltotriose
593
Manchurian Tea
733
Manners’ method
568
Mark–Houwink equation
505
MCT, see edium chain triglyceride medium chain triglyceride
487
Mentha spicata
868
mercaptodextran
611
mesquite gum analytical parameters for Prosopis velutina and specifications for Prosopis laevigata
514
carbohydrate component structure
504
definition
499
manufacture
499
regulatory status
526
structure
503
505
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
mesquite gum (Cont.) technical data
513
compatibility
517
effect of pH
515
emulsification ability
516
encapsulation ability
516
film forming
517
preservatives
517
solubility
514
surface activity
515
viscosity
514
uses and applications flavour and colour emulsification
522 523
flavour and colour microencapsulation non-food applications metabolic syndrome
523 523 61
appetite regulation
62
blood glucose and insulin sensitivity
64
blood lipids
66
RCT on hydrocolloid effects
68
68
67
body weight reduction and weight management modulation of satiety-related hormones
61 64
subjective feelings of appetite and satiety systemic anti-inflammatory effects Metamucil
63 69 51
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
methyl cellulose
717
applications
714
properties
713
methylation
670
methylation analysis
617
methylethyl cellulose properties
713
methylpyrrolidinone chitosan
874 ′
4-methylumbelliferyl-β-D-N,N diacetylchitobiose
861
metoclopramide HCl
874
Michael addition reaction
855
microbial polysaccharides
xxiv
microcrystalline cellulose
740
Avicel characteristics
752
food applications and functionality
745
592
beverages (high temperature stability)
746
beverages (low pH stability)
747
beverages (suspension of solids)
745
bulking agent, fat replacer, flow aid tablet exipient
754
dressings, sauces and cooking cream
748
ice cream
752
vegetable fat whipping cream
749
whipping process
752
formulations acidified milk-juice beverage
749
an indulgent healthy chocolate everage
746
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
microcrystalline cellulose (Cont.) calcium fortified, recombined soy beverage
747
low fat ice cream
755
nougat style confections
756
retorted adult nutritional beverage
748
UHT dairy cooking cream
750
vegetable fat whipping cream
751
future developments MCC-based bulking agent for fat and sugar calorie reduction
757
MCC/food approved hydrocolloids
757
MCC/lipids
757
low fat ice cream air cell integrity
753
air cell structure
754
molecular structure of cellulose
741
nutritional and regulatory information
743
physical properties
744
raw materials and manufacturing process
741
strain sweep curve
745
thixotropy
745
viscosities obtained at different fat levels
750
microencapsulation
271
Microquick WC-595
743
milk proteins
298
application baked products
330
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
milk proteins (Cont.) beverages
333
confectionery products
335
convenience foods
340
dairy-type products
331
desert-type products
334
meat products
336
nutritional/medical/pharmaceutical applications
337
pasta products
334
textured products
341
biological activity
324
intact caseins
324
intact whey proteins
325
biologically active peptides
326
ace inhibitory peptides
327
antibacterial peptides
329
antithrombotic peptides
328
caseinophosphopeptide
328
immunomodulatory peptides
327
opioid peptides
326
composition of bovine milk
299
distribution of proteins in bovine milk
300
food uses
329
bakery products
329
beverages
332
confectionery
335
convenience foods
339
dairy products
331
dessert-type products
333
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
milk proteins (Cont.) films and coatings
341
meat products
336
nutritional/medical/phamaceutical applications
336
pasta products
334
textured products
340
functional properties
317
emulsifying and foaming properties
323
gelation and coagulation
319
hydration properties
320
solubility
317
surface active properties
322
viscosity
321
future trends
341
milk protein system
299
the milk protein system caseins
299
whey proteins
301
production of milk protein products
301
303
caseins and caseinates
303
casein-whey protein co-precipitates
315
fractionation of caseins
307
fractionation of whey proteins
312
milk protein concentrates/isolates
315
milk protein hydrolysates and biologically active peptide fractions
316
modified milk protein products
316
whey protein-enriched products
308
311
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms Miscellaneous Additives Directive
Links 6
modified probe-tack test
789
molecularly imprinted polymers
855
3-monochloropropane-1,2-diol
418
MonoQ anion exchanger
314
Monte Carlo simulation
434
Morgan-Elson reaction
602
morphine
870
moxifloxacin
874
multi-angle laser scattering
485
439
N N, O-carboxymethyl chitosan
765
N-acetylcysteine
771
N-acetylglucosamine
852
nanolaminates
780
nanotechnology
779
‘nano-whiskers’
779
1-naphthol-4-sulphonic acid
856
1-naphthylamine-4-sulphonic acid
856
naproxen
458
Nata de Coco
733
Natureal
630
N-carboxymethyl chitosan
856
Nigerian No. 2
255
Nikan Sui method
772
94
nimodipine
522
nisin
764
772
NMR spectroscopy
670
851
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms non-digestible carbohydrates in food
Links 51
definition and properties
51
other non-digestible carbohydrates
54
physico-chemical properties and physiological effects non-starch polysaccharides
54 53
nori
769
771
Novagel RCN
755
NovaMatrix
809
nuclear magnetic resonance
443
nuclear Overhauser effect
550
nutraceuticals
xxiv
Nutrim-5
633
Nutrim-OB
628
637
Nuture 1500
630
637
Oatrim
622
628
OatVantage
632
637
OatWell
637
OatWell 14% Oat Bran
630
OatWell 16% Oat Bran
630
OatWell 22% Oat Bran
630
Oblate
767
occludin
871
ofloxacin
870
oilseeds
386
O
chemical composition
389
nutritional and health effects
414
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
oilseeds (Cont.) sunflower oil and defatted meal manufacture
398
okara
693
orcinol-HCl
669
organic natural oat fibre
630
Origami Foods
770
oscillatory small-deformation rheology
512
Ostwald disproportionation
370
695
637
638
34
402
516
ovalbumin
364
370
375
ovomucin
365
375
ovomucin-lysozyme complex
364
ovomucoïde
364
375
ovotransferrin
364
375
oxidative- reductive polymerisation
815
6-oxychitins
854
oxygen transfer coefficient
729
oxygen transfer rate
729
Ostwald ripening
P pea, see legumes pectinase pectins
865 43
chemical nature
54
274
277
formulations bakers jam
287
baking stable fruit preparation
287
concentrated glaze
288
extra jam
284
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
439
Index Terms
Links
pectins (Cont.) fruit flavoured confectionery jelly with buffered pectin
295
fruit flavoured confectionery jelly with unbuffered pectin
295
fruit flavoured dessert jelly
294
fruit preparation for yoghurt
289
milk/fruit juice drink
292
orange marmalade
284
ready-to-use spray glaze
288
reduced sugar raspberry jam
285
reduced sugar strawberry jam
285
requirement for traditional jam at 65% soluble solids
283
set and stirred yoghurt
292
syrup for milk dessert
293
Turkish delight jelly
296
yoghurt drink
291
galacturonic acid, ester and amide units
275
gel strength variation of low methyl ester pectin gels
280
hypothetical structure of apple pectin
278
legal status
296
manufacture
275
production processes
276
raw materials
275
nutritional and health aspects
281
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
pectins (Cont.) properties and function
277
availability of pectin types
280
gelation properties
279
range of commercial non-amidated pectins uses and applications
281 282
dairy products
290
dissolving pectin
282
fruit preparations and fruit bases
289
293
fruit preparations for bakery products
286
high sugar jams, jellies, marmalades and preserves
283
industrial fruit preparations
286
lower sugar jams and jellies
283
optimising pectin formulations
282
other dessert products
293
other food applications
296
sugar confectionery
294
286
variation of gel strength and setting temperature of high methyl ester pectins
279
pediocin
772
peel test
778
pentosans
654
perturbation theory
439
788
PES, see processed Euchema seaweed phlorizin
560
phloroglucinol-HCl
669
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
phosphocaseinate
304
phthalic anhydride
776
phycocolloids
100
Phycomyces blakesleeanus
853
phytoalexin
561
Phytophthora megasperma
562
plant exudate gums
495
Plantago sp
665
Poisson–Boltzmann equation
433
polyacrylic acid
776
polydispersity index
812
polyethylene oxide
776
poly(4-hydroxystyrene)
857
polysaccharides porogen
772
44 857
potato, see root vegetables pregelatinisation
120
pre-press solvent extraction
398
pressure-sensitive adhesive
786
Prima Cel
735
processed Euchema seaweed
4
E407A
167
manufacturing process
166
Prodiet F200
335
Progesterone
873
prolamin
392
Pro-long
766
propylene glycol alginate
700
Prosopis juliflora
499
Prosopis laevigata
499
399
167
415
809
814
514
526
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Prosopis velutina
499
protein dispersibility index
410
protein hydrocolloids protein-polysaccharide complexes
xxiii
503
514
xxiv
420
external parameters and analytical techniques used for investigation
426
external parameters influencing formation
425
binding enthalpy evolution
436
ionic strength
432
pH
430
pH-induced evolution
431
protein to polysaccharide weight ratio
434
temperature, shearing and pressure
435
total biopolymer concentration
435
food applications
452
food products texturisation
455
interface stabilisation
456
microencapsulation
453
other food application
457
protein purification
452
whey protein isolate/acacia gum complexes in ice-cream mix formation energetics
457 422 423
exothermic signals upon titration of β-lactoglobulin with acacia gum
424
theoretical models
423
thermodynamic background
422
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
protein-polysaccharide complexes (Cont.) functional properties
445
adsorption at air/water interface and foaming properties
449
adsorption at oil/water interface and emulsifying properties
447
β-lactoglobulin/acacia gum complexes formation of thick interfacial layer solubility and rheological properties
451 445
internal parameters influencing formation
438
biopolymer charge density
438
biopolymer molecular weight
439
non-food applications
458
biomaterials
459
microencapsulation
458
structure, morphology and coarsening
440
aggregate-free β-lactoglobulin/acacia gum mixture
444
β-lactoglobulin and acacia gum aggregated complexes
442
coarsening mechanism and macroscopic level
443
mesoscopic level
441
microscopic level
443
molecular level
440
systems used for microencapsulation purposes in food and non-food applications
454
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms protein-polysaccharide emulsifiers
Links 44
effect of interaction on proteinstabilised emulsion
45
protopectins
277
Pseudomonas sp.
603
pseudoplasticity
171
Pterocladia pullulan
84 450
applications
595
cyanoethylated
596
manufacture
593
properties
594
regulatory status
596
segment structure
594
structure
593
pullulan PI-20
593
451
593
594
Q qemai
781
quantum satis principle
824
Quarg
332
Questran
864
R radix astragali
521
Rapid Viscosity Analyser
112
rasping machine
399
Regulan
51
renneting
445
629
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
699
Index Terms
Links
resistant starch
123
Resource
332
retrogradation
113
revitalised wheat gluten
416
rhamnogalacturonan
695
rhamnogalacturonoglycan
503
rheology
36
constant viscosity at low pH
194
flow behaviour comparison
192
189
flow curve of 0.5% xanthan gum solution in standardised tap water pH sensitivity
191 193
solution stability to acids at ambient temperature
194
structure/property relationship for xanthan gum
190
Rhynchelytrum repens
628
Ricotta cheese
332
rituximab
627
root vegetables
386
chemical composition
389
nutritional and health effects
414
protein composition and structure
394
rubisco
394
S Salmonella typhimurium
764
Schiff base
855
858
Schiff reaction
857
859
schizophyllan
598
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
Schizophyllum commune
598
Scientific Committee for Food
744
scleroglucan
596
applications
599
manufacture
597
properties
598
regulatory status
600
repeating unit
598
structure
597
858
sclerogum, see Scleroglucan Sclerotium glucanicum
596
Sclerotium rolfsii
597
screw press method
398
secalin
392
597
Seiko, see konjac flour Semperfresh
766
Sephadex
612
Shimla
483
shiruko
553
Siberian larch, see Larix siberica single cell protein
399
size exclusion chromatography
671
slow release protein
338
small angle neutron scattering
263
442
small angle X-ray scattering
392
443
Smith degradation
503
Soafil
764
Soageena
768
573
sodium carboxymethyl cellulose, see carboxymethyl cellulose This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
sodium cyanoborohydride
858
sodium-alginate coating
765
solanine
414
solubilised wheat gluten
416
soluble soybean polysaccharide
693
basic material properties and characteristics
696
adhesive strength and material property of film
699
antioxidative effect on soybean oil
699
antioxidative property
699
excellent adhesive and film-forming property high dietary fibre content
699 696
high solubility and stable viscosity against heat, acid and salts change in taste value of boiled rice
696 705
coating phase on surface of cooked noodles
706
foam stability test results
705
functional properties and applications
700
anti-sticking effect
704
emulsification test results
704
emulsifying stability
702
foam stabilising function
704
formulation of flavour emulsions
703
other applications
706
preparation of flavour emulsions
703
protein particles stabilisation
700
galacturonan region distribution
696
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
soluble soybean polysaccharide (Cont.) and HM-pectin effects on dispersion of acidic milk protein
702
main fraction structure
697
manufacture
694
molecular mass distribution
695
regulatory status
706
stabilising mechanism emulsion oil droplet
704
protein particles under acidic conditions structure
702 694
general composition
694
SSPS structure
695
viscosity change by heating at various pH ranges
698
comparison of various polysaccharide solution effect of various salts on
698 698
soy protein isolate
693
SOYAFIBE-S
693
functions and applications
700
manufacturing process
694
varieties
701
SOYAFIBE-S-DA 100 chemical composition
694
694 695
SOYAFIBE-S-LA200
700
soybean fibre
706
soybean hemicellulose
706
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
soybean polysaccharide
706
soymilk
693
Spiro
488
Spiro exudation
478
St. John’s bread
229
stabilisation
118
‘Stain Hall’ procedure
781
starch
108
manufacture extraction
109
sources
109
starch granule characteristics
110
modifications
115
118
chemical and biochemical modifications
118
conversions
119
cross-linking
115
lipophilic substitution
120
pregelatinisation
120
stabilisation
118
thermal treatment
121
type of starch modification
116
regulatory status modified starches as food additives
118
137 138
permitted food starches under European law
138
starches and modified starch product labelling
139
starches and modified starches as food ingredients
139
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
starch (Cont.) structure
110
advantages/disadvantages vs other hydrocolloids
114
gelatinisation, gelation and retrogradation helix formation
112 111
native starches gelatinisation properties
114
starch granule
111
starch polysaccharides
110
technical data
121
effect of crosslinking and stabilisation
122
nutrition
122
structure-function relationship
121
uses and applications
125
baked goods
128
batters and breadings
129
beverage emulsions and flavour encapsulation
130
confectionery
130
dairy products
131
effects of food processing
127
fruit preparations
132
gravies, soups and sauces
133
mayonnaise and salad dressings
135
meat products
136
savoury snacks
136
sensory attributes
126
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
starch (Cont.) starch selection
125
viscosity troubleshooting
137
starch polysaccharides
110
amylopectin
111
amylose
110
static light scattering microscopy
444
sterculia gum, see gum karaya Sterculia setigera
498
Sterculia striata
512
Sterculia urens
498
511
786
787
Sterculia villosa
498
steric interactions
29
Stokes’ Law
31
Streptococcus pyogenes
325
Streptococcus sp.
603
sucrose acetate isobutyrate
271
sucrose esters
774
sugar icings
101
sugars
126
supplements
501
sweet potato sweet
101
agar fabrication diagram synergies
512
521
279
319
69
Svedberg’s method
syneresis
511
87
167
88 94
acid and alkaline hydrolysis
96
agar-locust bean gum
95
gelling blockade by chaotropic agents
96
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
synergies synergies gelling blockade by tannic acid
96
sugar reactivity
95
synergism
175
T Takeda Chemical Industries Co.
568
Tal Pro-long
766
Talaromyces emersonii
660
talha
253
tamarind kernel powder
537
tamarind seed
539
tamarind seed xyloglucan
535
gelation
541
551
and LVTSX concentration dependence of steady shear viscosity preparation
556 537
see also yloglucan Tamarindus indica tannic acid
535
860
96
tapping mode atomic force microscopy
581
tensile-bond test
788
tetrasaccharide cellulosic unit
618
texture profile analysis
211
The National Starch and Chemical Co.
766
‘the vinegar plant’
725
theory of mechanical interlocking
783
Theraflu
770
thermal mechanical peeling process
238
Thermamyl
658
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
thermodynamic adsorption theory
784
thermodynamic incompatibility
421
Thin Strips
770
thixotropic
512
tilted plane method
784
tinzaparin
458
Tirman
479
Tobiko
892
tofu
693
tragacanthin
502
transmission electron microscopy
263
Triaminic
770
Trichoderma reesei
617
Trigonella foenum graecum
231
trimethyl chitosan
871
trimethylamine
893
triple helix formation
581
trisaccharide cellulosic unit
618
trypsin
623
584
664
866
TSX, see tamarind seed xyloglucan tyrosinase
856
tyrosine glucan
856
U Unani
488
uridine diphosphoglucose
726
US Institute of Medicine
53
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
V van der Waals electrostatic interactions
28
steric interactions
30
vanillin-containing coatings
772
vegetable protein isolates
383
antinutritional factors
413
application in food products
406
potato and green leaves
412
seed protein preparations
408
410
association-dissociation phenomena of legumin- and vicilin-like globulins cereals
392 386
chemical and enzymatic modification of protein products
404
chemical modification
405
enzymatic modification
404
chemical composition
387
cereals
388
legumes
388
oilseeds
389
potatoes, green leaves and fruits
389
choosing best functionality for application classification of storage proteins
406 390
albumins
391
globulins
391
prolamin and glutelins
392
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
vegetable protein isolates (Cont.) functional properties
399
functional properties for industrial application
401
dough formation
404
foams and emulsions
401
gels and related aspects
402
texturisation of proteins
403
water- and fat-holding capacity
404
green leaves and fruits
387
heat-induced gel formation
403
instability mechanisms of foams and emulsions
402
legumes
384
main processing operations
397
main sources
384
manufacture
394
potato and green leaves processing
399
seed protein extraction
396
nutritional and health effects
412
cereals
414
legumes
413
oilseeds
414
potato and green leaves
414
protein allergenicity
415
oilseeds
386
protein composition and structure
389
pea proteins
393
potato and green leaf proteins
394
soybean proteins
393
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
vegetable protein isolates (Cont.) wheat proteins regulatory status
394 415
required functional properties for food applications
407
root vegetables
386
Veis-Aranyi model
423
vicilin
393
vicilin-like proteins
391
Viscofiber
630
viscosity
148
effect of pH
192
effect of salts
192
effect of temperature
193
viscosity troubleshooting
137
vital wheat gluten
416
Vivapure Q Mini-H-column
313
432
637
638
W water gels
178
water vapour permeability
771
Wattle Blossom Model
263
703
Western larch tree, see Larix occidentalis wetting theory
784
wheat, see cereals Wheatpro
411
whey
768
whey protein isolate
769
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
whey proteins -derived milk protein products composition -enriched products lactalbumin
310 308 312
whey powders and modified whey powders
308
whey protein concentrates
311
whey protein isolates
311
fractionation
312
industrial isolation of protein products
309
physico-chemical characteristics
303
white talha
255
Wilhemly plate
515
311
X xanthan gum
439
Xanthom*onas campestris
186
xanthum gum
186
food product applications
195
baked goods, bakery and pie fillings
198
culinary products
200
dairy products
198
dry mixes
201
frozen foods
201
198
functionality and associated applications
197
stabiliser level for salad dressing formulation future trends
200 201
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
xanthum gum (Cont.) manufacture
187
regulatory status
201
structure primary structure technical data
187 188
compatibility
194
constant viscosity at low pH
194
flow behaviour comparison
192
flow curve of 0.5% xanthan gum solution in standardised tap water
191
gum and media ratio effect on gel strength
196
interactions with galactomannans/ glucomannans
195
pH sensitivity
193
rheology of xanthan gum solution
189
solution stability to acids at ambient temperature
194
structure/property relationship
190
theoretical and observed viscosities
196
viscosity development profile
190
xanthan gum solution preparation
188
XCells Antimicrobial Wound Dressing
735
XCells Cellulose Wound Dressing
735
x-ray diffraction
679
xylanase
677
678
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
xyloglucan
535
applications in food industry
553
application of low-viscosity tamarind seed gum
556
emulsion stabiliser
554
fat replacement
556
gelling agent
555
ice crystal stabilisation
555
starch modification
555
thickener or stabiliser
553
concentration dependence of viscosity
543
effects on plasma lipids hydrolysed xyloglucan
559
xyloglucan and hydrolysed xyloglucan
559
emulsifying properties of soy proteinpolysaccharide conjugates
555
and epigallocatechin gallate NOESY spectrum
551
estimated molecular characteristic parameters
541
food application of low viscosity TSX
557
food applications
554
frequency and temperature dependence of storage shear modulus
545
gel strength
547
interactions
546
gelation by addition of polyphenols
548
gelation by change of solvent
546
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
xyloglucan (Cont.) gelation of tamarind seed xyloglucan
551
iodine colour reaction
553
Mark–Houwink–Sakurada plots
540
and native xanthan temperature dependence
552
origin, distribution and preparation
535
other aspects and applications
561
drug delivery systems
562
plant growth and plant defense systems physiological effects
561 558
biological functions
560
carbohydrate metabolism
559
improved lipid metabolism
558
potential linkages with cellulose
536
regulatory status in food industry
557
repeating unit chemical structure
538
rheology and DSC results cooling and subsequent heating
550
heating
549
safety data
557
sol-gel transition temperature diagram
548
structure and fundamental properties
538
chemical structure
538
dilute solution properties
539
molecular weight
539
rheological properties at higher concentrations
542
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
Index Terms
Links
xyloglucan (Cont.) TSX and LVTSX concentration dependence of steady shear viscosity
556
and xanthan solutions steady shear viscosity
544
xyloglucan endotransglycosylase
561
xylooligosaccharides
664
Xylos
735
681
Y Yariv’s reagent
487
yokan
100
youkan
555
Young’s modulus
731
734
818
Z zein
392
Zisman plots
775
This p a g e ha s b e e n re fo rma tte d b y Kno ve l to p ro vid e e a sie r na vig a tio n.
