Maximising the Value of Marine By-products

Maximising the value of marine by-products

Related titles: Safety and quality issues in fish processing (ISBN-13: 978-1-85573-552-1; ISBN-10: 1-85573-552-0) `an interesting mix of thought-provoking philosophy, excellent up-to-date wellreferenced topic reviews, useful practical, technical advice, as well as very recent research results, plus ideas about future trends.' Food Science and Technology Environmentally-friendly food processing (ISBN-13: 978-1-85573-677-1; ISBN-10: 1-85573-677-2) With increasing regulation and consumer pressure, the food industry needs to ensure that its production methods are sustainable and sensitive to environmental needs. This important collection reviews ways of analysing the impact of food processing operations on the environment, particularly life cycle assessment (LCA), and techniques for minimising that impact. The first part of the book looks at the application of LCA to the key product areas in food processing. Part II then discusses best practice in such areas such as controlling emissions, waste treatment, energy efficiency and biobased food packaging. Details of these books and a complete list of Woodhead titles can be obtained by: · visiting our web site at www.woodheadpublishing.com · contacting Customer Services (email: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext. 30; address: Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB21 6AH, England)

Maximising the value of marine by-products Edited by Fereidoon Shahidi

Published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB21 6AH, England www.woodheadpublishing.com Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2007, Woodhead Publishing Limited and CRC Press LLC ß 2007, 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-13: 978-1-84569-013-7 (book) Woodhead Publishing Limited ISBN-10: 1-84569-013-3 (book) Woodhead Publishing Limited ISBN-13: 978-1-84569-208-7 (e-book) Woodhead Publishing Limited ISBN-10: 1-84569-208-X (e-book) CRC Press ISBN-13: 978-0-8493-9152-1 CRC Press ISBN-10: 0-8493-9152-0 CRC Press order number: WP9152 The publishers' policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elementary chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards.

Project managed by Macfarlane Production Services, Dunstable, Bedfordshire, England (e-mail: [email protected]) Typeset by Godiva Publishing Services Ltd, Coventry, West Midlands, England Printed by TJ International Limited, Padstow, Cornwall, England

Contents

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

xiii

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

Maximizing the value of marine by-products: an overview . . . . . . . . . . . . .

xxi

Part I 1

Marine by-products characterisation, recovery and processing

2

Physical and chemical properties of protein seafood by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Rustad, Norwegian University of Science and Technology, Norway 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Physical and chemical properties of protein-rich by-products ± seasonal, habitat, species and individual variations . . . . . . . . 1.4 Implication for by-products valorisation . . . . . . . . . . . . . . . . . . . . . 1.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical and chemical properties of lipid by-products from seafood waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. P. Kerry and S. C. Murphy, National University of Ireland, Cork, Ireland 2.1 Introduction to fish lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 3 4 11 16 17 17 18 22 22

vi

Contents 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Health benefits associated with fish lipids . . . . . . . . . . . . . . . . . . . Fatty acids found in fish muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fatty acids found in fish by-products . . . . . . . . . . . . . . . . . . . . . . . . Factors affecting the fatty acid composition of fish and their associated by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deterioration of fish lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implications for fish fat by-product valorization . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

On-board handling of marine by-products to prevent microbial spoilage, enzymatic reactions and lipid oxidation . . . . . . . . . . . . . . . E. Falch, M. Sandbakk and M. Aursand, SINTEF Fisheries and Aquaculture, Norway 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Deterioration of marine biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Handling and sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Conservation and stabilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 On-board processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Utilisation of by-products from gadiform species . . . . . . . . . . . . 3.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 Recovery of by-products from seafood processing streams . . . . . J. A. Torres, Oregon State University, USA, Y. C. Chen, Chung Shan Medical University, Taiwan, J. Rodrigo-GarcõÂa, Universidad AutoÂnoma de Ciudad JuaÂrez, Mexico and J. Jaczynski, West Virginia University, USA 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 State of global fisheries and by-products . . . . . . . . . . . . . . . . . . . . . 4.3 Basic properties of water, proteins and lipids in aquatic foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Recovery of functional proteins and lipids from by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Protein recovery from surimi processing water . . . . . . . . . . . . . . 4.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Increasing processed flesh yield by recovery from marine by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. D. A. Taylor and A. Himonides, University of Lincoln, UK and C. Alasalvar, TUBITAK, Turkey 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Recovery of flesh from filleting waste . . . . . . . . . . . . . . . . . . . . . . .

24 25 26 28 30 32 34 35 47 47 47 49 52 56 59 61 61 62 65

65 66 68 75 84 88 88 91 91 92

Contents 5.3 5.4 5.5 5.6 5.7

vii

Recovery of flesh from demersal species . . . . . . . . . . . . . . . . . . . . Quality and improvement of fish mince . . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of further information and advice . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

98 99 103 104 104

6 Enzymatic methods for marine by-products recovery . . . . . . . . . . F. Guerard, University of Western Brittany, France 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Overview of by-products extracted by enzymatic methods . . . 6.3 Enzymatic extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Traceability of by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Conclusions and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

107

7

Chemical processing methods for protein recovery from marine by-products and underutilized fish species . . . . . . . . . . . . . . . . . . . . . . H. G. Kristinsson, A. E. Theodore and B. Ingadottir, University of Florida, USA 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Chemical extraction: fish protein concentrate . . . . . . . . . . . . . . . . 7.3 Chemical hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Surimi processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Fish protein isolates: pH-shift processing . . . . . . . . . . . . . . . . . . . . 7.6 Other processes using low or high pH . . . . . . . . . . . . . . . . . . . . . . . 7.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8 References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Part II 8

107 108 109 134 135 136 136 144 144 146 148 149 152 161 162 163

Food uses of marine by-products

By-catch, underutilized species and underutilized fish parts as food ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Batista, Fish and Sea Research Institute, (IPIMAR), Portugal 8.1 Introduction: by-catch, discards and by-products . . . . . . . . . . . . . 8.2 Key drivers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Using the by-catch and underutilized species . . . . . . . . . . . . . . . . 8.4 Using underutilized fish parts as food and food ingredients . . 8.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 8.7 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171 171 173 174 179 189 190 191 191

viii

Contents

9

Mince from seafood processing by-product and surimi as food ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J.-S. Kim, Gyeongsang National University, South Korea and J. W. Park, Oregon State University, USA 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Manufacturing fish mince/surimi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Machinery for preparation of fish mince/surimi . . . . . . . . . . . . . . 9.4 Mince/surimi processing by-products . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Functional properties of fish mince/surimi . . . . . . . . . . . . . . . . . . . 9.6 Nutritional characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7 Storage stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.8 Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 Aquatic food protein hydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. G. Kristinsson, University of Florida, USA 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 The enzymatic hydrolysis process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Properties of fish protein hydrolysates . . . . . . . . . . . . . . . . . . . . . . . 10.4 Role in food systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Physiological role in humans and animals . . . . . . . . . . . . . . . . . . . 10.6 Role in plant growth and propagation . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Role as growth media for microorganisms . . . . . . . . . . . . . . . . . . . 10.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Engineering and functional properties of powders from underutilized marine fish and seafood products . . . . . . . . . . . . . . . . S. Sathivel and P. J. Bechtel, University of Alaska Fairbanks, USA 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Fish protein powder as bio-active ingredients . . . . . . . . . . . . . . . . 11.3 Functional properties of fish protein powders . . . . . . . . . . . . . . . . 11.4 Flow properties analysis of emulsion containing fish protein powders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Viscoelastic properties of emulsions containing fish protein powders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Thermal properties of fish protein powders . . . . . . . . . . . . . . . . . . 11.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12 Marine oils from seafood waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Shahidi, Memorial University of Newfoundland, Canada 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Oil from fish processing by-products . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Marine mammal oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196 196 197 200 204 210 216 217 219 223 229 229 230 234 234 239 241 242 242 243 249 249 250 250 251 253 254 256 256 258 258 261 265

Contents 12.4 12.5 12.6 12.7

ix

Algal oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marine oil manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health effects of PUFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268 269 271 272

13 Collagen and gelatin from marine by-products . . . . . . . . . . . . . . . . . J. M. Regenstein and P. Zhou, Cornell University, USA 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Key drivers of marine collagen and gelatin . . . . . . . . . . . . . . . . . . 13.3 Sources of marine collagen and gelatin . . . . . . . . . . . . . . . . . . . . . . 13.4 Manufacture of marine collagen and gelatin . . . . . . . . . . . . . . . . . 13.5 Properties of marine collagen and gelatin . . . . . . . . . . . . . . . . . . . . 13.6 Food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.7 Non-food applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.8 Improving the quality of collagen and gelatin . . . . . . . . . . . . . . . 13.9 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 13.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279

14 Seafood flavor from processing by-products . . . . . . . . . . . . . . . . . . . . C. M. Lee, University of Rhode Island, USA 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Aqueous extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.4 Enzymatic hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 Enzyme-assisted seafood flavors from processing by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.6 Flavor-imparting compounds and chemistry . . . . . . . . . . . . . . . . . 14.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Fish and bone as a calcium source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S.-K. Kim and W.-K. Jung, Pukyong National University, Republic of Korea 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Biochemical properties of fish bone . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 Utilization of fish bone calcium and organic compound . . . . . 15.4 In vivo availability of soluble calcium complex from fish bone 15.5 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Chitin and chitosan from marine by-products . . . . . . . . . . . . . . . . . . F. Shahidi, Memorial University of Newfoundland, Canada 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 Chemical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Applications of chitin, chitosan and their oligomers . . . . . . . . .

279 279 281 281 288 295 297 298 299 299 304 304 305 305 306 308 319 324 325 328 328 330 331 335 336 336 340 340 341 353

x

Contents 16.4 16.5

Safety and regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References and further reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

364 365

17 Marine enzymes from seafood by-products . . . . . . . . . . . . . . . . . . . . . M. T. Morrissey and T. Okada, Oregon State University Seafood Laboratory, USA 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Marine enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Producing enzymes from seafood processing by-products . . . . 17.4 Marine by-product enzyme utilization . . . . . . . . . . . . . . . . . . . . . . . 17.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

374

18 Antioxidants from marine by-products . . . . . . . . . . . . . . . . . . . . . . . . . . F. Shahidi and Y. Zhong, Memorial University of Newfoundland, Canada 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 Antioxidants from marine algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3 Antioxidants from marine animals and their by-products . . . . 18.4 Antioxidants from other marine sources . . . . . . . . . . . . . . . . . . . . . 18.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Pigments from by-products of seafood processing . . . . . . . . . . . . . . B. K. Simpson, Department of Food Science and Agricultural Chemistry, Canada 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 Pigment types and sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 Carotenoid pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4 Other pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5 Economic, environmental, and safety considerations . . . . . . . . . 19.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 19.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Part III 20

374 376 381 384 388 389 397 397 398 404 407 408 413 413 414 414 422 426 427 428 429

Non-food uses of marine by-products

By-products from seafood processing for aquaculture and animal feeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. J. Bechtel, University of Alaska, USA 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 Driving forces for utilization of by-products . . . . . . . . . . . . . . . . . 20.3 By-product components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4 Overview of different products produced from fish by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

435 435 436 437 438

Contents 20.5 20.6 20.7 20.8

Methods of producing hydrolysates and silage . . . . . . . . . . . . . . . Nutritional benefits and other properties of fish and animal feeds made from seafood processing wastes . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Using marine by-products in pharmaceutical, medical, and cosmetic products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Losso, Louisiana State University, USA 21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Squalamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5 Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.6 Protamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22

Bio-diesel and bio-gas production from seafood processing by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. Zhang and H. M. El-Mashad, University of California Davis, USA 22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Quantity and quality of various seafood processing by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Theories and technologies for production of bio-diesel and bio-gas fuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.4 Potential yields and quality of bio-diesel and bio-gas fuels . . 22.5 Problems encountered and possible approaches for overcoming them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.6 Future research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 22.9 List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 Composting of seafood wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M. Martin, Memorial University of Newfoundland, Canada 23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 Biodegradation of seafood wastes by composting . . . . . . . . . . . . 23.3 Composting operational parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 23.4 Characteristics of the composting of seafood wastes . . . . . . . . . 23.5 Technological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6 Biological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7 Vermicomposting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.8 Quality considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi 441 443 444 445 450 450 451 452 454 454 455 456 456 460 460 461 463 471 476 479 480 480 481 482 486 486 487 490 494 495 501 503 507

xii

Contents 23.9 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.10 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 23.11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

509 510 511

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

516

Contributor contact details

(* = main contact)

Editor Professor Fereidoon Shahidi Department of Biochemistry Memorial University of Newfoundland St. John's, Newfoundland Canada A1B 3X9 E-mail: [email protected]

Chapter 1 Dr Turid Rustad Department of Biotechnology NTNU 7491 Trondheim Norway E-mail: [email protected]

Chapter 2 Dr Joe P. Kerry and Sheila C. Murphy Department of Food and Nutritional Sciences University College Cork National University of Ireland Cork Co. Cork Ireland

Chapter 3 Dr Eva Falch,* Marit Sandbakk and Dr Marit Aursand SINTEF Fisheries and Aquaculture N-7465 Trondheim Norway E-mail: [email protected] [email protected]

xiv

Contributors

Chapter 4 Dr J. Antonio Torres* Food Process Engineering Group Dept. of Food Science & Technology Oregon State University Corvallis, OR 97331-6602 USA E-mail: [email protected] Dr Yi-Chen Chen School of Nutrition Chung Shan Medical University Taichung City 402 Taiwan

Food Research Centre University of Lincoln Brayford Pool Lincoln LN6 7TS UK E-mail: [email protected] Dr Cesarettin Alasalvar TUBITAK Marmara Research Centre Food Institute PO Box 21, 41470 Gebze-Kocaeli Turkey E-mail: [email protected]

E-mail: [email protected] Professor JoaquõÂn Rodrigo-GarcõÂa Departamento de Ciencias BaÂsicas Instituto de Ciencias BiomeÂdicas Universidad AutoÂnoma de Ciudad JuaÂrez Ciudad JuaÂrez Chih MeÂxico E-mail: [email protected] Dr Jacek Jaczynski Dept. of Animal and Veterinary Sciences West Virginia University Morgantown, WV 26506 USA E-mail: [email protected]

Chapter 5 Professor K. D. Anthony Taylor* and Dr Aristotelis Himonides Faculty of Technology

Chapter 6 Dr Fabienne Guerard ANTiOX Laboratory University of Western Brittany PoÃle universitaire P.J. Helias F-29000 Quimper France E-mail: [email protected]

Chapter 7 Dr Hordur G. Kristinsson, Ann E. Theodore and Bergros Ingadottir Laboratory of Aquatic Food Biomolecular Research Aquatic Foods Product Program Department of Food Science and Human Nutrition University of Florida Gainesville, FL 32611 USA

Contributors

Chapter 8 Dr Irineu Batista IPIMAR Irineu Batista Av. BrasõÂlia 1449-006 Lisbon Portugal E-mail: [email protected]

Chapter 9 Professor J.-S. Kim* Gyeongsang National University South Korea E-mail: [email protected] Professor Jae Park Oregon State University 2001 Marine Drive #253 Astoria, OR 97103 USA E-mail: [email protected]

Chapter 10 Dr Hordur G. Kristinsson Laboratory of Aquatic Food Biomolecular Research Aquatic Foods Product Program Department of Food Science and Human Nutrition University of Florida Gainesville, FL 32611 USA

Chapter 11 Dr Subramaniam Sathivel* Fishery Industrial Technology Center University of Alaska Fairbanks 118 Trident Way

xv

Kodiak, AK 99615 USA Dr Peter J. Bechtel USDA/ARS Subarctic Research Unit 245 O'Neill Building University of Alaska Fairbanks Fairbanks, AK 99775 USA

Chapters 12 and 16 Professor Fereidoon Shahidi Department of Biochemistry Memorial University of Newfoundland St. John's, Newfoundland Canada A1B 3X9 E-mail: [email protected]

Chapter 13 Professor Joe M. Regenstein* and Peng Zhou Department of Food Science Stocking Hall Cornell University Ithaca, NY 14853-7201 USA E-mail: [email protected]

Chapter 14 Professor Chong Lee Department of Nutrition and Food Sciences University of Rhode Island 530 Liberty Lane West Kingston, RI 02892 USA E-mail: [email protected]

xvi

Contributors

Chapter 15 Dr Se-Kwon Kim* and Won-Kyo Jung Marine Bioprocess Research Center Pukyong National University Busan 608-737 Republic of Korea

Macdonald-Stewart Building 21111 Lakeshore Road Ste. Anne de Bellevue Quebec, H9X 3V9 Canada E-mail: [email protected]

Chapter 17

Chapter 20

Dr Michael T. Morrissey* and Tomoko Okada Professor and Director Oregon State University Seafood Laboratory 2001 Marine Drive Rm. 253 Astoria, OR 97103 USA

Dr Peter J. Bechtel USDA-ARS Subarctic Research Unit 245 O'Neill Building University of Alaska Fairbanks Fairbanks, AK 99775 USA

E-mail: [email protected]

Chapter 18 Professor Fereidoon Shahidi* and Ying Zhong Department of Biochemistry Memorial University of Newfoundland St. John's, Newfoundland Canada A1B 3X9 E-mail: [email protected]

Chapter 19 Dr Benjamin K. Simpson Department of Food Science and Agricultural Chemistry 514.398.7737 (Office) Room MS1-034

Chapter 21 Professor Jack Losso Department: Food Science Department LSU AgCenter 111 Food Science Bldg. Louisiana State University Campus LA 70803 USA E-mail: [email protected] [email protected]

Chapter 22 Dr Ruihong Zhang* and Hamed M. El-Mashad Biological and Agricultural Engineering Department University of California Davis One Shields Avenue Davis, CA 95616 USA E-mail: rhzhang@ucdavis

Contributors

Chapter 23 Dr A. M. Martin Dept Biochemistry Memorial University of Newfoundland St John's NL, A1A 4A3 Canada E-mail: [email protected]

xvii

Preface

Aquatic species provide an important source of nutrients for human consumption. In addition, beneficial effects of seafoods and marine oils have contributed greatly to better appreciation of resources and encouraging of full utilization of the catch. Seafoods originating from the wild or cultivated species produce a large amount of by-products upon processing or as by-catch of targetted fishery that may not be put to full use. These by-products could serve as a rich source of a variety of biomolecules with potential health benefits. Thus, many niche products can provide a better commercial value and full utilization of raw materials. In this regard, marine oils, enzymes, hydrolysates, carotenoids and squalene, chitinous material, N-acetylglucosamine and glucosamine, among others, are of much importance. Many studies, for example, have shown the effects of marine oils in cardiovascular diseases, diabetes and rheumatoid arthritis as well as cancer prevention by different mechanisms at the cellular and subcellular levels. Therefore, it is imperative to maximize the value of processing by-products from aquatic species. This book is the first to provide a comprehensive account of value-added by-products from fisheries processing. It would serve as an important reference for researchers in academia, industry and government labs, both in terms of fundamental science and its application. I am grateful to the experts who have provided state-of-the-art contributions for inclusion in this book. I am also grateful to Woodhead Publishing personnel who provided me with much support that was essential for successful completion of this task. Fereidoon Shahidi

Maximizing the value of marine by-products: an overview F. Shahidi, Memorial University of Newfoundland, Canada

The world's annual catch of fish and marine invertebrates has been around 100 million metric tonnes in recent years. However, aquaculture developments have led to production of high quality products that have also assisted conservation strategies to be implemented. Of the total amount of harvest, a major portion remains unused or used for production of fish meal and fish oil. This is due to the fact that certain species might suffer from small size, high bone, skin and fat contents as well as unappealing shape. In addition, several species of fish may be used for their roe and production of caviar. The leftover carcass following roe extraction as well as those of their male counterparts may be discarded. Furthermore, processing discards from many species of fish and shellfish could be successfully processed for production of specialty enzymes, xanthophylls, chitin/chitosan, glucosamine and other value-added products. Thus, devising of strategies for full utilization of the catch and processing of discards for production of novel products is warranted (Shahidi, 2000).

Seafood processing by-products and their use The seafood processing industry is still producing a large quantity of byproducts and discards. These include heads, tails, viscera and backbone as well as shells. Utilization of these processing by-products may be exercised in different ways leading to 1.

production of animal and aquaculture feed, similar to that used for whole fish when producing fish meal and fish oil,

xxii

Maximizing the value of marine by-products: an overview

Table I.1

Physiological components from marine by-products

Ingredient

Application area

Proteins/biopeptides Minerals/calcium Chitosan, glucosamine Omega-3 oils Carotenoids/xanthophylls Chordprotein sulphate Squalene Specialty chemicals

Nutraceuticals, immune-enhancers Food, nutraceuticals Nutraceuticals, agriculture, food, water purification Nutraceuticals, dietary supplements, food Nutraceuticals, fish feed Supplements, arthritic pain relief Skin care Miscellaneous

2. 3.

production of food ingredients such as extraction of cheeks and tongue from cod and production of surimi from frames and production of novel and value-added products for nutraceutical, pharmaceutical and fine chemical industries (Table I.1).

Novel and specialty products with potential biological activity and/or functionality provide a means for value-added utilization of by-products. These may be used as food ingredients to take advantage of a specific flavour, such as those from cook water of crab and lobster (Jayarajah and Lee, 1999; Yang and Lee, 2000), or for rendering a specific functional property such as water-holding, foaming, emulsifying and gelling properties. The use of by-products as health food ingredients may be for nutritional purposes; these include proteins, lipids, mineral and vitamins. Finally, by-products may be employed for nutraceutical and specialty applications. In this category, protein hydrolyzates, fish oils, hormones, glucosamine, chitin/chitosan, flavourants and enzymes as well as other physiologically active ingredients may be included. The following sections provide a cursory account of current and potential uses of by-products in different applications and for rendering health benefits.

Proteins from seafoods and their by-products Seafood by-products are an excellent source of high quality proteins that may supply a major part of the essential amino acids that are required for a balanced nutrition. Recovery of proteins from by-products may be carried out by different processes using mechanical separation from frames, base extraction or hydrolysis. While hydrolysis of fish proteins by endogenous enzymes prior to or during primary processing may lead to fish quality deterioration, such processes may be intentionally carried out to produce specialty products. Thus, production of fish sauce and silage from fish and processing discards is commonplace. In addition, enzymes that are commercially available may be used to produce protein hydrolyzate that could be used in a variety of applications. Protein hydrolyzates are nearly colourless and appear like milk powder; they may be used in

Maximizing the value of marine by-products: an overview

xxiii

applications where water solubility and water-holding capacity are important. Protein hydrolyzates may possess biological activity in enhancing immune response and may also render antioxidant as well as angiotensin converting enzyme (ACE) inhibitory activity (Je et al., 2004) among others. Carotenoids (C40H56) and their oxygenated derivatives (xanthophylls) are another group of bioactives that are present in salmonid fish, crustaceans and their processing by-products, among others (Shahidi et al., 1998). These are often present in combination with proteins, known as carotenoproteins. Extraction and isolation of carotenoproteins as ingredients for potential use in salmonid fish aquaculture has been reported (Cremades et al., 2003). Digestive proteases from fish and shellfish processing discards may be used as industrial processing aids (Shahidi and Kamil, 2001). Suggested uses of digestive proteases from fish include acids for cheese making, herring fermentation, fish skinning, roe processing and production of specialty kits, as well as medical applications.

Lipids from processing by-products Seafood lipids provide unique health benefits to consumers, but also present a challenge to scientists and technologists for delivering their highly unsaturated fatty acids (HUFA) in an odour-free and appealing form. The oils originate from the body of fatty fish as a by-product of fish meal industry, liver of white lean fish such as those of cod and halibut, and finally blubber of marine mammals such as seals and whales. Viscera from fish also provide for a rich source of lipids. These lipids include a high proportion of long-chain polyunsaturated fatty acids (LC PUFA) belonging to the omega-3 family, namely eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). There is a rapidly growing body of literature illustrating the health benefits of HUFA. These effects include protection against cardiovascular disease, autoimmune and mental disorders, diabetes, arthritis and arrhythmia, among others (Shahidi and Finley, 2001; Shahidi and Miraliakbari, 2004, 2005). Marine lipids are highly prone to oxidation, hence their processing under relatively mild conditions and stabilization following refining, bleaching and deodorization is recommended. This is partly due to the fact that the refining process leads to the removal of endogenous antioxidants from the oil and hence replenishment with antioxidants, particularly those from natural sources is important. In addition, microencapsulation of the oils may prove useful when such oils are to be used in fortification of food and beverages.

Minerals and chitinous materials Seafood processing discards contain a large proportion of frames as well as shells that are primarily composed of calcium salts. Thus, the resultant calcium

xxiv

Maximizing the value of marine by-products: an overview

may be solubilized and potentially used for addressing concerns about bone health due to insufficient intake of calcium. Jung and co-workers (2006) have clearly demonstrated the solubilization of calcium from fish frames and their benefits. Most shellfish, especially those from shrimp, crab, lobster and crayfish contain a large amount of chitin that may be recovered following deproteinization and demineralization. The recovered chitin may be used for chitosan production using concentrated base or render pressure, glucosamine preparation or chitosan oligmers, short-chain copolymers of glucosamine and N-acetylglucosamine and derivatives thereof. Glucosamine, the monomer of chitosan, has been reported to possess benefits for joint health and build up as well in wound-healing, among others. The product is generally sold as glucosamine sulphate, but this is often a mixture of glucosamine hydrochloride and sodium or potassium sulphate. Furthermore, glucosamine may be sold in combination with chondroitin 4- and 6-sulphates. Chondroitins are mucopolysaccharides (MPs) with molecular weights of up to 50,000 Da and could be prepared from connective tissues of slaughtered animals and seafoods (Jo et al., 2005). In combination, while glucosamine helps to form proteoglycans that sit within the space in the cartilage, chondroitin sulphate acts as a liquid magnet (Shahidi and Kim, 2002).

Future trends and prospects Dwindling supply of seafoods from the wild dictates full utilization of the harvest. In addition, the advent of aquaculture allows a better control over the harvest time and hence better quality of products, including processing byproducts. A stricter environmental restriction on dumping of discards also serves as a further incentive to explore novel uses of products that might otherwise be considered uneconomical. Low temperature activity of enzymes as well as unique characteristics of products from processing discards might also catalyze new developments in value-added utilization of specialty products from processing lines.

References CREMADES, O., PARRADO, J., ALVAREZ-OSSORIO, M.C., JOVER, M., COLLANTES-DE-TERAN, L., GUTIERREZ, J.F. and BAUTISTA, J. 2003. Isolation and characterization of carotenoproteins from crayfish (Procambarus clarkii). Food Chem. 82, 559±566. JAYARAJAH, C.N. and LEE, C.M. 1999. Ultrafiltration/reverse osmosis concentration of lobster extract. J. Food Sci. 64, 93±98. JE, J.Y., PARK, P.J., KWEN, J.Y. and KIM, S.K. 2004. A novel angiotensin converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. J. Agric. Food Chem. 52, 7842±7845.

Maximizing the value of marine by-products: an overview

xxv

and PARK, D.C. 2005. Optimization of shark (squatina oculata) cartilage hydrolysis for the preparation of chondroitin sulfate. Food Sci. Biotechnol. 14, 651±655. JUNG, W.K., LEE, B.J. and KIM, S.K. 2006. Fish-bone peptide increases calcium solubility and bioavailability in ovariectomised rats. British J. Nutr. 95, 124±128. SHAHIDI, F. 2000. Seafood in Health and Nutrition ± Transformation in Fisheries and Aquaculture: Global Perspectives. ScienceTech Publishing Co., St. John's, NL, Canada. SHAHIDI, F. and FINLEY, J.W. 2001. Omega-3 Fatty Acids: Chemistry, Nutrition, and Health Effects. ACS Symposium Series 788, p. 330. American Chemical Society, Washington, D.C. SHAHIDI, F. and KAMIL, Y.V.A.J. 2001. Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci. Technol. 12, 435±464. SHAHIDI, F. and KIM, S-K. 2002. Quality management of marine nutraceuticals. In C-T. Ho and Q.T. Zheng (eds), Quality Management of Nutraceuticals. ACS Symposium Series 803, pp. 76±87. American Chemical Society. Washington, DC. SHAHIDI, F. and MIRALIAKBARI, H. 2004. Omega-3 (n-3) fatty acids in health and disease: Part 1 ± Cardiovascular diseases and cancer. J. Med. Food 7, 387±401. SHAHIDI, F. and MIRALIAKBARI, H. 2005. Omega-3 fatty acids in health and disease: Part 2 ± Health effects of omega-3 fatty acids in autoimmune diseases, mental health and gene expression. J. Med. Food 8, 133±150. SHAHIDI, F., METUSALACH and BROWN, J.A. 1998. Carotenoid pigments in seafoods and aquaculture. Crit. Rev. Food Sci. Nutr. 38, 1±67. YANG, Y. and LEE, C.M. 2000. Enzyme-assisted bioproduction of lobster flavor from the process by-product and its chemical and sensory properties. In Shahidi, F. (ed.), Seafood in Health and Nutrition ± Transformation in Fisheries and Aquaculture: Global Perspectives. ScienceTech Publishing Co., St. John's, NL, Canada, pp. 169±194. JO, J.H., DO, J.R., KIM, Y.M., KIM, D.S., LEE, T.K., KIM, S.B., CHO, S.M., KANG, S.V.

Part I Marine by-products characterisation, recovery and processing

1 Physical and chemical properties of protein seafood by-products T. Rustad, Norwegian University of Science and Technology, Norway

1.1

Introduction

Overexploitation of fish resources is a major problem as only 50±60% of the catch is used for human consumption. Globally, more than 91 million tonnes of fish and shellfish are caught each year. Some of the by-products are utilised but huge amounts are wasted. Annual discard from the world fisheries has been estimated to be 25% of the catch. Only a small amount of the by-products is used for human consumption, the rest is used for production of fishmeal, silage and animal feed. A list of valuable components in fish by-products is given in Table 1.1. Fish provides about 14% of the world's need for animal proteins and 4±5% of the total protein requirement (Venugopal, 1995). The amino acid composition and digestibility of fish proteins is excellent. It is a challenge both to increase the utilisation of the protein fractions from marine by-products and to use more of these valuable proteins as food ingredients. Use of by-products is not new. In the Nordic countries a lot of the byproducts have been and are still being used for various purposes. For instance fish skin has earlier been used to cover window openings, to make clothes, shoes, carrier packs and sacks. Some fish by-products that are used for human consumption include roe (canned, salted/marinated or as cod roe emulsion), liver (Eastern Europe), cleaned stomach, fried fish milt (a snack) and head products from Iceland (cheeks, tongues, dried heads). In 2000 a total of 251 000 metric tonnes of by-products were created by the Norwegian cod fisheries alone, of this 114 000 tonnes were dumped while 137 000 tonnes were utilised. Only 33 000 tonnes of the by-products were used for human consumption which amounts to about 13% of the total (RUBIN,

4

Maximising the value of marine by-products

Table 1.1

Valuable components of fish by-products

Lipids

Proteins

Other components

Oils Omega-3: EPA, DHA Phospholipids Squalen Vitamins Cholesterols

Hydrolysates Surimi Thermostable dispersions Peptides, amino acids Gelatine, collagen Protamines

Nucleic acids Calcium Bioactive compounds Colours

2001). The rest is used for production of fishmeal, silage and animal feed. A large part of the by-products that are dumped at sea are made up by heads (Stoknes and Hellevik, 2000). In Norway only about 20% of the cod heads are exploited. Of this 9000 tonnes goes to human consumption, mostly as dried heads to Nigeria. In some fisheries the tongues and cheeks are cut out. The rest is minced and used as feed for fur animals. Fisheries and fish industries are the single most important industry in Iceland. In 2001 the total catch was around 2 million tonnes, accounting for 62% of the value of exported products and around 48% of the foreign currency earnings that year. Fish meal and oil constitute the bulk of the volume of products from fisheries in Iceland or 63% of total, but their value is far less or only about 14% of the total value of exported seafood products. In 2001, Iceland exported about 45 500 tonnes of by-products with a value of US$73.5 million. Of a total available UK fish and shellfish resource of approximately 850 000 tonnes, only 43% end up as products for human consumption. The rest is categorised as `waste'. About 300 000 tonnes of this is produced on shore, whereas the rest is produced at sea (145 000 tonnes discarded fish and 43 000 tonnes processing waste). For cod-fish these numbers can be broken down to 154 000 tonnes on shore and 37 000 tonnes at sea. In the UK the major outlet for this raw material is fishmeal and oil production, only small quantities are used for other purposes (pet food, animal feed, fishing bait, etc.). In some areas of the UK the by-products primarily end up in landfill sites. Much of the waste production is concentrated in regional processing centres. Only very few fishing vessels utilise the by-products. The non-utilised by-products generated onboard are dumped at sea. The annual harvest of seafood in Alaska is over 2 million tonnes and yields more than 1 million tonnes of by-products, some of this is produced into fishmeal and oil but the majority is discarded (Crapo and Bechtel, 2003; Sathivel et al., 2004).

1.2

Overview

The protein-rich by-product fractions include cut-offs, backbones, heads, skin, roe, milt, stomachs, viscera and blood. The proportion of different by-products

Physical and chemical properties of protein seafood by-products

5

from different species is given in Table 1.2. For the industry to be able to utilise more of the by-products, data on the available amounts as well as on the chemical composition and the properties of both the protein and the lipid fractions as a function of species, season and fishing ground are needed. On average, production of cod fillets will generate two-thirds of the whole body weight as by-products. The data of Falch et al. (2006a) show that the viscera (all inner fractions) makes up 12±15% of the whole body weight of four Gadidae species (cod, saithe, haddock and tusk), caught in the Barents sea, the head 15±20% and the backbone and trimmings (cut-offs) make up 18±30%. In a study including 750 specimens from five different Gadidae species caught at three different fishing grounds in Europe, the viscera was found to constitute 3± 7% of the round weight of the fish. The highest proportion was found in the Norwegian Gadidaes and the lowest in the Irish (Falch et al., 2006b). For Gadidae species caught in Icelandic water, the proportion of intestines in tusk and ling was lower than in the other cod species (Thorarinsdottir et al., 2004). The proportion of liver, roe and milt were positively correlated, but negatively correlated to viscera and CF (condition factor). The proportion of head was similar for all cod groups (different sizes of cod), ranging from 27 to 33%. The proportion of head of tusk, ling, saithe and haddock was lower than that of cod. The proportion of backbone varied from 14.9 to 16.3% for cod and from 14.9 to 18.7% for haddock. The size of the cod did not affect the proportion of the backbone. The proportion of skin from cod fillets was in the range 5.1 to 7.7%. It was highest for ling (8.7 to 10.8%) and tusk (10.6 to 12.4%) at all seasons. Cut-offs in cod groups were in the range 9.5 to 12.0%, in haddock in the range of 7.7 to 11.7%, in saithe 10.5 to 15.6%, ling 12 to 15.4% and in tusk 11.5 to 13%. Seasonal effects were not significant. The global production of farmed Atlantic salmon was estimated to be 1025 000 tonnes in 2002 (Globefish, 2002) and more than 50% of this weight is regarded as by-products or waste. The largest fractions constitute the cut-offs (including backbone) (14%), viscera (13%) and head (10%), and these fractions might serve as a source of valuable marine lipids and proteins. Blood makes up 2% of the total weight of salmon. The composition of different by-product fractions from different species is given in Table 1.3. In different sources reporting protein content in various byproduct fractions, the protein content is usually given as crude protein, Nx6.25, this value also includes the NPN (non-protein-nitrogen). In some fractions this may constitute up to 25% of total N (Sikorski, 1994). In the meat of white fish, the NPN content is usually from 9 to 15% of total N. About 95% of NPN is made up of free amino acids, dipeptides, trimethylamine oxide (TMAO) and degradation products, urea, guanidine, nucleotides and degradation products of nucleotides. Liaset et al. (2000, 2002) reported that the protein content of cod frames was 16.9% while the content in salmon frames was 17.4%. In another study, the protein content in salmon frames was found to be 18.2% (Michelsen et al., 2004) and the protein content in salmon viscera was found to be 10.6%.

Table 1.2

Amount of different by-product fractions

Species

Cod Saithe Haddock Tusk Ling Atlantic salmon Carp, wild Carp, cultured 1

Includes bellyflap

By-product fraction amount of total fish weight (%) Head

Backbone/ frames

Cut-offs

Skin

Roe

Milt

Viscera

20.2 15.3 18.9 17.9 18.6 10.0 21±25 20±21

9.7 9.9 10.6 8.4

8.2 8.8 9.3 21.2

4.2 4.8 4.5 6.4

1.3 0.2 0.1 0.0

5±9 6±8

5.01 6±8 8±11

0.7 0.3 0.7 2.0 1.7

5.6 7.2 6.2 9.9 3.3 14.0 3±4 4±5

10.0

Reference Falch et al. (2006a) Falch et al. (2006a) Falch et al. (2006a) Falch et al. (2006a) Falch et al. (2006b) Sandnes et al. (2003) Bukovskaya and Blokhin (2004) Bukovskaya and Blokhin (2004)

Table 1.3

Protein content in different by-product fractions Protein content % of wet weight

Species Cod Saithe Haddock Tusk Ling Carp, wild Carp cultured Atlantic salmon Herring 1

Includes bellyflap

Head

11±13 13.1

Backbone/ frames

10±15

Cut-offs 13±23 15±19 15±18 17±23 15±20 14±22 12±17

Skin

8±121

Roe

Milt

14±27 19±25 18.0

Viscera

Reference

9±13 12±19 7±11 3±12 8±12 15±23 26 5±7

Sùvik (2005) Sùvik (2005) Sùvik (2005) Sùvik (2005) Sùvik (2005) Bukovskaya and Blokhin (2004) Bukovskaya and Blokhin (2004) Sandnes et al. (2003) Sathivel et al. (2004)

8

Maximising the value of marine by-products

Sikorski et al. (1984) have reported protein content in different fish skins to be between 18 and 35%, with a collagen content between 10.6 and 28.8%. Sikorski (1994) reported the crude protein content in different fractions of fish from New Zealand waters. The protein content in viscera varied from 7.5 to 23.9% while the content in skin varied between 11.9 and 29.6% and in frames the protein content was between 13.1 and 25.3%. Fish roe has high concentrations of proteins and lipids (Bledsoe et al., 2003). In general fish roe products are high in protein (16±30%). Crude lipid content can vary from less than 5% to 20% with an average of 10% in salmon roe. The protein quality of fish roe is high, either methionine/cystine or tryptophan/ tyrosine are the limiting amino acids. The backbone is one of the major by-product fractions, yielding about 15% of the fish weight (Gildberg et al., 2002). About 15% of the wet weight is pure bone, the rest is white muscle (Jeon et al., 2000). The bone makes up about onethird of the dry weight and consists of minerals (60±70%) and proteins (30%). The protein is mainly collagen (Lall, 1995; Nagai and Suzuki, 2000). Based on these values, it can be calculated that 80±85% of the protein in cod backbone fraction is muscle protein and the rest is collagen. Gildberg et al. (2002) found that the muscle proteins from the backbone fraction could be extracted using hydrolysis with commercial enzymes and the resulting bone could be used to recover gelatine using heat extraction. The gelatine extracted had a low molecular weight, but could be suitable for technical applications and nutraceuticals. Flesh from backbones and cut-offs may be a suitable raw material for production of fish mince, surimi and surimi-based products. Surimi is mechanically deboned fish flesh that has been washed with water or dilute salt solutions and to which cryoprotectants have been added. Surimi is used as the raw material for preparation of seafood analogues, such as shrimp, crab legs, scallop and lobster tail. In addition the surimi industry has the potential to develop new products. éines et al. (1995) found that the yield of fish mince that could be separated from salmon and white fish cut-offs and backbones was between 48 and 56% of the weight of the by-product fractions with a protein content ranging from 13 to 17%. The demand for collagen and gelatine from the industry throughout the world is considerable and still rising. By-products from fish processing are a potential source of collagen. In fish the largest collagen concentrations are found in the skeleton, fins and the skin (Norland, 1989; Sikorski and Borderias, 1994). Fish gelatine and collagen have been much less studied than mammalian gelatine and collagen (Norland, 1989; Gudmunsson and Hafsteinson, 1997). Mammalian collagen in its purified form has found a number of pharmaceutical and cosmetic applications. Similarly, gelatin, the hydrolysed form of collagen, is an ingredient extensively used in the food industry. Gelatin is used as a food additive to improve the texture, the water-holding capacity and stability of several food products (Borderias et al., 1994). Some documents also suggest that fish collagens were used in ancient times. Plinius (AD 23±79) speaks of `ichtyocolla' (fish glue) from Pondus (The Black Sea), and Plinius the Elder reports use of fish glue as a medicine against headaches and cramps (Solstad and Muniz, 2001).

Physical and chemical properties of protein seafood by-products

9

The quality and specific application of the extracted collagen and gelatine is highly related to their functional properties and to purity. Known problems with the extraction of collagen from fish skins are the abundance of pigments and the presence of fish odours, which would restrict its potential use. The uniqueness of fish collagen from cold water fish lies in the lower content of amino acids, proline and hydroxyproline (Haard et al., 1994). Although fish gelatine does not form particularly strong gels, it is well suited for certain industrial applications, as, for example, micro-encapsulations, light-sensitive coatings, and low-set-time glues. The extraction of native collagen, as described by van de Vis et al. (1996), instead of gelatine, which is the hydrolysed form of collagen, is strongly preferred, because native collagen provides more and better opportunities to modify the functional properties as well as the possible applications of collagen in the food ingredient industry. The skeleton, fins and skin constitute the main part of whitefish `offal'. The Norwegian fish industry produces approximately 600 000 metric tonnes of `waste' per year. This includes 10 000±12 000 tonnes of skins from white fish (cod, pollack, haddock, etc.) which could be used to produce at least 1500±2000 tonnes of fish gelatine (RUBIN, 2001). The search for new gelling agents to replace mammalian gelatine has led to patents for fish gelatine production (Grossman and Bergman, 1992; Holzer, 1996) as well as several published methods (Gudmunsson and Hafsteinsson, 1997; Nagai and Suzuki, 2000; Gomez-Guillen and Montero, 2001). Collagen from fish has just recently been identified as a potential allergen and this may be a potential problem for the use of fish gelatine in commercial products (Sakaguchi et al., 1999; Hamada et al., 2001). Fish milt is a product that is often wasted, even though canned milt from cod and herring is a traditional food product in England (Gildberg, 1999). The milt contains a high amount of protamine and nucleic acids and is used for industrial production of nucleic acids. The products are used in health food and cosmetics and the remainder used as feed supplement in aquaculture. Studies have shown that supplementation of cod milt cationic proteins to the feed of juvenile fish may improve their resistance to V. anguillarum infection (Pedersen et al., 2004). Real caviar is made from sturgeon, but a wide variety of other fish roes are consumed in their own right, as well as products sold as substitutes for sturgeon roe. Important commercial fish roes include salmon roe (Ikura), lumpfish roe, flying fish roe, whitefish roe, cod roe, mullet roe, herring roe (Kazunoko), pollack roe (Bledsoe et al., 2003). A wide variety of by-products have been used for making fish silage (Gildberg, 2002). Fish silage can be produced from all kinds of low-value fish and fish by-products and is almost entirely used for feed. Fish silage is normally made by mixing 2±3% formic acid into the minced raw material and storing at ambient temperatures till endogenous enzymes have dissolved the fish tissue. A well-preserved fish silage will normally have a pH of 3±4 which is the optimum pH for fish pepsins. The process usually takes a few days, provided that the raw material has a sufficiently high content of pepsins and other acid proteases

10

Maximising the value of marine by-products

(cathepsins). The silage may be used directly in feed or processed further by separation of the oil and evaporation to give a protein concentrate. The advantage of producing fish silage is the low capital investment and simple processing equipment. The disadvantage is the high transport costs due to the high water content. Norway is the major fish silage producer ± producing about 140 000 tonnes per year, mainly from aquaculture by-products (salmon). Fish silage is a low price product, but is a good alternative for utilising by-products that might otherwise have been wasted. A large proportion of the catch (~ 30%) is used for fish meal and animal feed because of its poor functional properties. One of the approaches for effective protein recovery from by-products is enzymatic hydrolysis, which can be applied to improve and upgrade the functional and nutritional properties of proteins (Gildberg, 1993; Liaset et al., 2000). Proteolytic modification of food proteins to improve palatability and storage stability of available protein resources is an ancient technology (Adler-Nissen, 1986). Hydrolysates can be defined as proteins that are chemically or enzymatically broken down to peptides of varying sizes. Today much of the fish flavour, fish soup and fish paste products available on the market are prepared by enzymatic hydrolysis (Shoji, 1990). Protein hydrolysates can be used as emulsifying agents in a number of applications such as salads dressing, spreads, and emulsified meat and fish products like sausages or luncheon meat (Badal and Kiyoshi, 2001). They can also be used in bacterial growth media (Poeronomo and Buckle, 2002). A large variety of different fish protein hydrolysates are being produced. The oldest is fish sauce which has long traditions in South-East Asia. Fish sauce, which is the major fermented fish product, was known in Ancient Greece and Rome (Corcoran, 1963). Fish sauce is produced in a quantity of about 250 000 tonnes per year. It is made by mixing three parts of fish raw material with one part of salt and storing at ambient tropical temperatures for 6±12 months. Both endogenous and microbial enzymes contribute to the degradation of the proteins in the fish and the resulting fish sauce is an amber liquid with 8±14% digested proteins and about 25% salt. The production is simple and requires little sophisticated equipment, but there is a need for a large storage space (Gildberg, 2002). Fish protein hydrolysates can be made with enzymes, acids or alkali. Enzymatic hydrolysis is strongly preferred over strictly chemical methods for producing hydrolysates for use in nutritional applications. Enzymatic hydrolysis can produce hydrolysates with well defined peptide profiles (Lahl and Braun, 1994). This approach gives an effective recovery of proteins, in addition to upgrading the functional and nutritional properties of the by-product proteins (Shahidi et al., 1995; Liaset et al., 2000; Slizyte et al., 2005). Enzymatic hydrolysis of fish by-products may be accomplished by an autolytic process, using the digestive enzymes of the fish itself or using added commercial enzymes, or by a combination of these (semiautolytic). The autolytic process lasts from a few days to several months. There are no enzyme costs involved and it is a simple operation. However, prolonged digestion may adversely affect the functional properties of the resulting hydrolysate, and such products are gener-

Physical and chemical properties of protein seafood by-products

11

ally used in feed formulations. Semiautolytic processes including the endogenous enzymes as well as commercial enzymes may be the most interesting, but require knowledge about the raw material composition and the activity, stability and specificity of the endogenous proteolytic enzymes regarding variations according to species, season and fishing ground (Slizyte et al., 2005; Dauksas et al., 2004). The development of fish protein concentrates (FPC) was one of the earliest attempts to recover fish protein from processing wastes. FPC has been produced using solvent extraction, resulting in a concentration of the proteins by removal of the water and oil from the raw material (Kristinsson and Rasco, 2000). By-products from cod species are a potential source for bioactive compounds. This may be both secondary metabolites as well as enzymes, lipids and heteropolysaccharides (Gudbjarnason, 1999). Protamine is a basic peptide containing more than 80% arginine. Protamine has been found in the testicles of more than 50 fish species. Protamine has the ability to prevent growth of Bacillus spores and may be used as an antibacterial agent in food processing and preservation. It has been reported that many proteins possess antioxidative activities, and fish protein hydrolysates have been found to be antioxidative (Amarowicz and Shahidi, 1997; Kim et al., 2001). Proteolytic enzymes from cold-adapted fish have received a lot of interest in recent decades. These enzymes have been found to be more catalytically active at relatively low temperatures compared to corresponding enzymes from mammals, thermophilic organisms and plant sources (De Vecchi and Coppes, 1996; Gudbjarnason, 1999). Enzymes with unique properties for industrial utilisation can be recovered from fish by-products (Haard et al., 1994). The Icelandic Fisheries laboratory has developed a process to recover trypsin-like enzymes from cod viscera (Stefansson and Steingrimsdottir, 1990). In Norway industrial processes for recovery of pepsin, trypsin, chymotrypsin, alkaline phosphatase and hyaluronidase from fish viscera have also been developed (AlmaÊs, 1990). Alkaline phosphatase is recovered from the thaw water from frozen shrimp (Olsen et al., 1990). This is used in diagnostic kits.

1.3 Physical and chemical properties of protein-rich by-products ± seasonal, habitat, species and individual variations It is a challenge to utilise more of the protein fractions from fish by-products as food ingredients. Many protein-rich marine by-products have a range of dynamic properties and can potentially be used in foods as binders, emulsifiers and gelling agents (Sathivel et al., 2004). Fish proteins have unique functional properties such as capacity to bind water, lipids, rheological properties, etc. but, due to lack of a suitable purification process to preserve protein functionality, fish protein has been lacking in the rapidly growing protein ingredient and health markets. Retaining the functional properties through preservation and processing

12

Maximising the value of marine by-products

operations is therefore of great importance. The functional properties of proteins are defined as `those physical and chemical properties which affect the behaviour of proteins in food systems during processing, storage, preparation and consumption' (Kinsella, 1976). The sensory properties of foods result from interactions between several functional ingredients. The physical and chemical properties that determine protein functionality include the size and the shape of the proteins, the charge and the distribution of charge and the flexibility as well as the ratio between the hydrophobicity and the hydrophilicity. Handling, processing and storage of the raw materials will all affect the functional properties and it is therefore important to both characterise the functional properties of the raw material and find out how the different processing steps will affect these properties. Fish is regarded as an excellent source of high-quality protein, particularly the essential amino acids lysine and methionine. Comparison of PER (protein efficiency ratio) from cod muscle and cod by-products shows that fish byproducts have a high content of essential amino acids and can be used to produce products with high nutritional value (Shahidi, 1994). Hydrolysates also have a high chemical score and the amino acid composition is generally similar to that of the raw material, except for content of sulphur amino acids, histidine and tryptophan which are affected during hydrolysis (Quaglia and Orban, 1987; Shahidi et al., 1995; Kim et al., 1997). The content and properties of the proteins in marine by-products vary with regard to species, fishing ground and season. In order to increase the utilisation of the protein fractions, knowledge about these variations is necessary. Seasonal differences in the amount of different by-product fractions were found, with a higher proportion of viscera during autumn and spring (Falch et al., 2006b). The Gadidae species caught in these waters spawn in the first six months of the year (January to June) and the proportion of gonads will therefore be highest during these months. The proportions of roe were, as expected, significantly higher in the autumn and spring. Higher proportions of roe were generally found in Gadidaes from Iceland and Ireland compared to Gadidaes from Norway. During the spawning season, the roe made up 2.2% of the round weight of fish on average. The lowest ratio of skin was found in spring and the highest in autumn for cod species from Icelandic waters (Thorarinsdottir et al., 2004). Also the chemical composition of the by-products varies according to species, season, fishing grounds and growth conditions. The protein content in by-product fractions from five cod species collected at three different fishing grounds and three different seasons was analysed. Total protein content was highest in the cut-off fraction and lowest in the viscera. All fractions showed variation in protein content with fishing ground, species and season. Amount of water-soluble protein was highest in liver and cut-offs and lowest in the viscera; the low content in viscera is due to the high degree of degradation in this fraction. The amount of free amino acids was generally lowest in the liver and highest in the viscera. The degree of hydrolysis was lowest in the cut-offs and

Physical and chemical properties of protein seafood by-products

13

highest in the viscera. In some liver samples a high degree of hydrolysis was also found (Sùvik, 2005). For cut-offs an inverse relationship was found between water and protein content (Thorarinsdottir et al., 2004); a similar relationship existed for roe. In carp by-products seasonal differences in both the amount and properties of the proteins were found for both wild and farmed carp. The amount and properties of the proteins also varied between wild and farmed carp by-products (Bukovskaya, unpublished results). The protein content in cut-offs and gonads were higher in the wild than in the cultured carp. During summer, the content of water-soluble proteins did not differ significantly between cultured and wild carp. In winter a substantial increase of water-soluble proteins content in intestine of wild carp was observed. Liver had the highest content of free amino acids followed by viscera and cut-offs, a substantially higher content of free amino acids was found in the liver and viscera of cultured carp compared to wild carp. This reflects the influence of the feeding on the composition not only in the fish fillets but also in the by-products. For products such as fish mince and surimi, the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters (Venugopal and Shahidi, 1995). Knowledge of how to retain these properties during storage and processing is therefore important. Seasonal variations in water-holding capacity in cut-offs from cod species was noted, with a lower water-holding capacity found in autumn, except for small cod, tusk and haddock. Minced meat is less stable than intact muscle. By-products from filleting have the same good quality as the main products (fillets), but unfortunately are not always treated in the same way, resulting in a rapid loss in quality. If the by-products are intended for use in food, the frames and cut-offs should be stored at 0ëC (éines et al., 1995). Freezing of the raw material will generally lead to loss of both water-holding capacity and gel forming ability, but freezing of the cut-offs/frames may result in smaller reduction in these parameters than freezing of mince. Both the freezing temperature and time as well as thawing conditions may influence the loss of functional properties. Gildberg (1993) found that the sauce fraction develops much faster if fresh intestines are used than if the material has been freeze-stored. The reason for this is not clear, but it is well known that freeze-storage of fish material implies a tougher tissue structure that may be less susceptible to digestion by enzymes. Another possible explanation is that bursting of lipid cells during freezing and thawing involves formation of a stable lipid-protein, emulsion which hinders a fast phase separation. The quality of by-products limits the possibilities for utilisation of the raw material, and enzymatic activities along with microbial degradation are the most important factors determining raw material quality. Variations in enzymatic activities and therefore quality are important when finding possible uses for the different by-product fractions. In processes utilising by-products, such as production of fish protein hydrolysates (FPH), minced and surimi-based products,

14

Maximising the value of marine by-products

extraction of lipids, enzymes and/or other bioactive compounds, the activity of the endogenous enzymes in the raw material needs to be controlled and knowledge about how these activities change according to temperature is important. Significant variations in quality parameters and enzymatic activities in byproducts from cod according to species, seasons and fishing grounds have been demonstrated (Sùvik and Rustad, 2004, 2005a, 2005b). The highest overall proteolytic activity in the by-product fractions was found in viscera at pH 3 (35ëC). Cut off and liver fractions also showed maximum activity at pH 3, 35ëC and 50ëC, respectively. Maximum median proteolytic activity in viscera is approximately 20 times higher than that in liver and 250 times higher than that in cut-off. Species affected the general proteolytic activity (pH 5 and 7), and activity of trypsin and chymotrypsin in viscera from cod species. Trypsin and chymotrypsin are the major proteolytic enzymes active at neutral pH in the viscera of cod species. Viscera from Atlantic cod, saithe and haddock had a higher proteolytic activity compared to tusk and ling. In cut-off and liver, general proteolytic activity (pH 5 and 7), activity of cathepsin B and collagenase varied according to species. Variations according to season were found in the activity of trypsin, chymotrypsin, elastase, cathepsin B, collagenase and lipase (pH 7) in the viscera from Atlantic cod. The results clearly indicated that viscera samples from the Icelandic sea had lower enzymatic activities in April±June compared to the other seasons. Cut-off samples from Icelandic Sea also had lower cathepsin B and collagenase activity in April±June compared to February± March; fishing ground influenced general proteolytic activity as well as activity of trypsin, chymotrypsin, elastase, cathepsin B, collagenase and lipase. Heat stability of the enzymes is also important and it has been shown that trypsin, chymotrypsin and cathepsin B in crude extracts from viscera from cod species lost 50% of initial activity after incubation for 10 min at 60ëC, while elastase, collagenase and lipase lost 50% of their initial activity after incubation for 10 min at 50ëC. The ratio between proteolytic activities in different by-products are similar in carp and cod and can be described as: INTESTINE > LIVER > CUTOFFS (Sùvik et al., 2004). In carp the difference in the levels of proteolytic activity in intestine, liver and cut-offs are less significant than in cod. Both in carp and cod, the total proteolytic activity in the intestine was highest at pH3 followed by pH 7 and lowest at pH 5. For cut-offs this correlation was similar between species: pH3 > pH5 > pH7 (Sùvik et al., 2004). The presence of proteolytic enzymes in the viscera of fish had a significant influence on the production of hydrolysates (Shahidi et al., 1995; Slizyte et al., 2005, 2006). When producing hydrolysates from capelin, endogenous enzymes alone gave a protein recovery of approximately 23% after 4 h at pH 3.0 (Shahidi et al., 1995). The hydrolysis of ground capelin by endogenous enzymes enhanced the overall extraction of fish protein at both acid and alkaline pH, since both acid and alkaline proteases are present in fish muscles and viscera. Pre-digestion of fish mince prior to the addition of exogenous enzymes might enhance the yield of protein extraction, however, autolytic enzymes may also

Physical and chemical properties of protein seafood by-products

15

bring about undesirable changes in the products, as it may be difficult to control the degree of hydrolysis during storage and processing. Furthermore, autolytic protease activity varies from species to species and depends on the season of harvest. Therefore, properties of functional protein hydrolysates may vary greatly under the same processing conditions. The chain length of the peptides formed during the hydrolysis process is important: this is one of the parameters determining both the functional and the organoleptic properties of the hydrolysate (Slizyte et al., 2005, 2006; Dauksas et al., 2004). The high collagen content in skeleton, fins and skin which constitute the main part of whitefish `offal' can become a problem when collagen turns into sticky fish glue in the production of fishmeal (Sikorski et al., 1984). In the production of fishmeal and oil from fish `offal' problems can arise due to gelatinisation of collagen into fish glue causing problems in concentration of stick water and drying of fishmeal. The content of collagen is thought to contribute significantly to the viscosity of the stickwater. Common problems connected with fish gelatin from cold water species are low gelling and melting temperatures and low gel modulus. The differences in physical and rheological properties between mammalian gelatine and gelatine from cold water species are due to a lower content of the amino acids proline and hydroxyproline (Sikorski et al., 1984). The quality and specific application of the extracted collagen and or gelatine is highly related to their functional properties and purity. There is also a market for non-gelling gelatine, which has a potential in the cosmetic industry as an active ingredient (i.e., shampoo with protein). Using fish collagen and gelatine generates new applications as a food ingredient, because it has properties different from mammalian collagen and because it can be used in food where mammalian gelatine from a cultural or safety point of view is not wanted. Generally, the enzymatic activities in by-products are high and it is therefore important that they are rapidly and continuously stored at cold temperatures. It is also important that the by-products are treated as valuable raw material in the same way as the main product, starting from the time the fish is caught. Separating the different by-product fractions, and fractions from the Lotidae family from the Gadidae family, will ensure that fractions with low enzymatic activity are not contaminated by fractions with higher enzymatic activities. Viscera from Atlantic cod caught in the Icelandic sea and from fish from the Gadidae family may be better raw materials for protein hydrolysates, especially if autolytic or semi autolytic processes are used or for extraction of proteolytic enzymes. Viscera samples from Atlantic cod caught in the Barents Sea have lower lipase activity and may therefore be a better raw material for extraction of marine oils compared to samples from the Icelandic Sea. Cut-off from ling and saithe, Atlantic cod caught in the Barents Sea (April±June and October± December) and Icelandic Sea (April±June) may be used for minced products or surimi-based products. Cut-off from haddock, Atlantic cod caught at the south coast of Ireland and in the Icelandic Sea (February±March) is a poor raw material for these kinds of products due to the high activity of cathepsin B

16

Maximising the value of marine by-products

(Sùvik, 2005). Results from this latter study also suggest that viscera should be kept separately. Contamination of liver and cut-off fractions with viscera fractions should be avoided.

1.4

Implications for by-products valorisation

To be able to produce value-added products from fish by-products for human consumption, it is necessary that the by-products are treated as a valuable raw material and the name should perhaps be `upgraded' to rest of raw material. Efficient on-board handling and sorting of specific by-products are important to reduce the rate of enzymatic degradation and microbial spoilage. The enzymatic activities in by-product fractions are high and it is important that they are rapidly and continuously stored at low temperatures. Rapid sorting and separation of different by-product fractions so that fractions with high enzymatic activity do not contaminate the fractions of low activity is also important. It is also possible to take the fish onshore ungutted and gut the fish in efficient processing lines onshore. In a Norwegian study, it was found that cod could be landed ungutted and gutted within 12 hours after catch without any negative effects on the quality of the fish or the by-products. Spawning cod with a low degree of filling in the stomach/intestines could be kept ungutted for up to 48 hours. Heavily feeding cod should not be stored ungutted for more than 12 hours. The quality of by-products (liver, gonads and stomach/intestine) from fish landed ungutted was found to be better compared to controls gutted immediately after catch and stored in bags on ice for up to 48 hours after catch (Akse et al., 2002). There is a need to develop methods to preserve different by-product fractions. Methods of preservation and utilisation depend upon the application of the byproduct. It is important to specify the shelf life at different levels of processing and product quality changes during storage. Potential preservation methods may include chilling, freezing, drying, fermentation and use of preservatives such as natural antioxidants. These methods should be tested for bulk- and final products and the different fractions (fat, protein/gelatine) from the most valuable byproducts. The final products should be evaluated for their usage in food applications. More research is also needed to develop processing methods to extract the fractions/biomolecules of interest. The processes developed should be able to handle variations on the basis of season, habitat and species. The processing steps should be optimised both regarding yield and product quality, but also about processing costs. Products should have as high quality as possible at the lowest possible price. In order to evaluate possible applications for products and conservation techniques of the rest of raw material, it is important that the raw material is characterised based on its chemical composition and enzymatic activity. Characterisation of variation in chemical compositions has mainly been carried

Physical and chemical properties of protein seafood by-products

17

out on fish muscle. Limited work has also been done on the chemical characterisation of some fishery by-products. To obtain a total picture of the applications and potential for new use, detailed characterisation including seasonal, species and habitat variations is still needed. For achieving profitable utilisation of by-products from the fish industry the final products require market interest. Knowledge about quality and composition is a necessity. The products and the processes to produce these must be economically viable. There is still great potential in utilisation of these fractions and a need for further investigations. There is also a need for environmental restrictions and economic incentives to increase the utilisation of marine byproducts.

1.5

Future trends

The available catch from marine fisheries is not expected to rise in the future. Increase in quantity of fish will probably come from aquaculture. The increase in aquaculture will result in a demand for more feed. This will have to come from agriculture, from harvesting at lower trophic levels (krill and plankton) and also from use of marine by-products, especially as a source of marine oil. Increasing amounts of farmed fish will be a source for fresh by-products with better possibilities for sorting and preservation and utilisation for value-added products/products for human consumption. The majority of by-products utilised today are used for feed (fishmeal and oil, fish silage). The largest potential for value addition is in increasing the amount of by-products used for human consumption, either as food ingredients or as nutraceuticals. The need to use more of the by-products for human consumption will demand that this raw material is treated as a valuable raw material starting onboard the fishing boat or at the processing plant, resulting in rapid sorting and storage/preservation or processing into bulk products for later processing. There is also a need for national and international authorities to provide stronger regulations so that wasting of this valuable raw material is not possible. Enzymes or other bioactive molecules found in by-products will usually be found in very low concentrations and production of these molecules will therefore most likely be through the use of microorganisms via recombinant technology.

1.6

Sources of further information and advice

ed (2002), Safety and quality issues in fish processing, Boca Raton, FL, CRC Press. SIKORSKI Z E, SUN PAN B and SHAHIDI F eds (1995), Seafood proteins, New York, Chapman & Hall. VANNUCCINI S (2003), Overview of fish production, utilisation, consumption and trade (based on 2001 data). FAO, Fisheries Information, Data and Statistics Unit. BREMNER A H

18

Maximising the value of marine by-products and BOTTA J R eds (1990), Advances in fisheries technology and biotechnology for increased profitability, Lancaster, PA, Technomic Publishing.

VOIGT M N

Websites: http://www.rubin.no/eng/ (visited 15 June 2005). http://www.dced.state.ak.us/oed/seafood/by_products.htm (visited 15 June 2005). http://www.Fishbase.org (visited 15 June 2005). http://www.intrafish.com/laws-and-regulations/report_bc/vol3-d.htm (visited 15 June 2005). http://www.globefish.org (visited 15 June 2005).

1.7

References

ADLER-NISSEN J

Publ.

(1986), Enzymic hydrolysis of food proteins, London, Elsevier Science

and JOHNSEN G (2002), Landing av uslùyd fisk for utnyttelse av biproduktene. Rapport 3/2002. Fiskeriforskning, Tromsù, Norway (In Norwegian). Ê S K A (1990), `Utilization of marine biomass for production of microbial growth ALMA media and biochemicals', in Voigt M N and Botta J R, Advances in Fisheries Technology and Biotechnology for increased profitability, Lancaster, PA, Technomic Publishing Co., Inc., 361±372. AMAROWICZ R and SHAHIDI F (1997), `Antioxidant activity of peptide fractions of capelin protein hydrolysates', Food Chem 58(4), 355±359. BADAL C S and KIYOSHI H (2001), `Debittering of protein hydrolysates', Biotechnol Advanc 19(5), 355±370. BLEDSOE G E, BLEDSOE C D and RASCO B (2003), `Caviars and fish roe products', Crit Rev Food Sci Nutr 43(3), 317±356. BORDERIAS J, MARTI M A and MONTERO P (1994), ` Influence of collagenous material during frozen storage when added to minced cod (Gadus morhua)', Z Lebensm Unters Forsch 199 (4), 255±261. BUKOVSKAYA O and BLOKHIN S (2004), Utilization of by-products from cod species, final report from DDL (EU-project QLKI-CT-2000-01017), June. CORCORAN T H (1963), `Roman fish sauce', Classical J 58(5), 204±210. CRAPCO C and BECHTEL P J (2003), `Utilization of Alaska's seafood processing byproducts', in Bechtel P J, Advances in Seafood Byproducts Conference Proceedings, Alaska Sea Grant College Program, University of Alaska: Fairbanks, AK. DAUKSAS E, SLIZYTE R, STORRé I and RUSTAD T (2004), `Bitterness in fish protein hydrolysates and methods for removal', J Aquatic Food Prod Technol 13(2), 101± 114. DE VECCHI S and COPPES Z (1996), `Marine fish digestive proteases ± Relevance to food industry and the south-west Atlantic region ± A review', J Food Biochem 20(3), 193±214. FALCH E, AURSAND M and RUSTAD T (2006a), `By-products from gadiform species as raw material for production of marine lipids as ingredients in food or feed', Process Biochemistry 41, 666±674. AKSE L, JOENSEN S, BARSTAD H, EILERTSEN G

FALCH E, RUSTAD T, JONSDOTTIR R, SHAW N B, DUMAY J, BERGE J P, ARASON S, KERRY J, SANDBAKK M

and

AURSAND M

(2006b), `Geographical and seasonal differences in

Physical and chemical properties of protein seafood by-products

19

lipid composition and relative weight of by-products from Gadidae species', J Food Comp Anal 18(6±7), 727±736. GILDBERG A (1993), `Enzymic processing of marine raw materials', Process Biochem 28, 1±15. GILDBERG A (1999), Value-added products from fish processing offals/by-products with special reference to developments in Norway, Proceedings IFCON 98, Mysore, India. GILDBERG A (2002), `Enhancing returns from greater utilization', in Bremner H A, Safety and Quality issues in fish processing, Cambridge, CRC Press, Woodhead Publ. Ltd., 425±449. GILDBERG A, ARNESEN J A and CARLEHOÈG M (2002), `Utilisation of cod backbone by biochemical fractionation' Process Biochem 38, 475±480. GLOBEFISH (2002), `Globefish statistical estimates', Food and Agricultural Organization of the United Nations, Rome, www.globefish.org GOMEZ-GUILLEN M C and MONTERO P (2001), `Extraction of gelatin from megrim (Lepidorhombus boscii) skins with several organic acids', J Food Sci 66(2), 213±216. GROSSMAN S and BERGMAN M (1992), `Process for the production of gelatin from fish skins', US Patent 5,093,474. GUDBJARNASON S (1999), `Bioactive marine natural products', Rit Fiskideildar 16, 107±110. GUDMUNDSSON M and HAFSTEINSSON H (1997), `Gelatin from cod skins as affected by chemical treatments', J Food Sci 62(1), 37±39, 47. HAARD N F, SIMPSON B K and SIKORSKI Z E (1994), Biotechnological applications of seafood proteins and other nitrogenous compounds, New York, Chapman & Hall, 195± 216. HAMADA Y, NAGASHIMA Y and SHIOMI K (2001), `Identification of fish collagen as a new allergen', Bioscience Biotechnology Biochemistry 65(2), 285±291. HOLZER D (1996), `Gelatin production', US Patent 5,484,888. JEON Y-J, BYUN H-G and KIM S-K (2000), `Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes', Process Biochem 35, 471±478. KIM S-K, JEON Y-J, BYUN H-G, KIM Y-T and LEE C-K (1997), `Enzymatic recovery of cod frames proteins with crude proteinase from tuna pyloric caeca', Fisheries Science 63(3), 421±427. KIM S-E, KIM Y-T, BYUN H-G, NAM K-S, JOO D-S and SHAHIDI F (2001), `Isolation and characterisation of antioxidative peptides from gelatin hydrolysate of Alaska pollock skin', J Agric Food Chem 49, 1984±1989. KINSELLA J E (1976), `Functional properties of food proteins: A review', CRC Crit Rev Food Sci Nutr 7, 219±280. KRISTINSSON H G and RASCO B A (2000), `Fish protein hydrolysates: Production, biochemical and functional properties', Crit Rev Food Sci Nutr 40(1), 43±81. LAHL W J and BRAUN S D (1994), `Enzymatic production of protein hydrolysates for food use', Food Technol October 1994, 67±71. LALL S P (1995), `Macro and trace elements in fish and shellfish', in Ruiter, A, Fish and fishery products, Wallingford, CAB International, 187±213. LIASET B, LIED E and ESPE M (2000), `Enzymatic hydrolysis of by-products from the fishfilleting industry; chemical characterisation and nutritional evaluation', J Sci Food Agric 80, 581±589. LIASET B, NORTVEDT R, LIED E and ESPE M (2002), `Studies on the nitrogen recovery in enzymic hydrolysis of Atlantic salmon (Salmo salar, L.) frames by ProtamexTM

20

Maximising the value of marine by-products

protease', Process Biochem 37, 1263±1269. and RUSTAD T (2004), `Utilisation of by-products from farmed Atlantic salmon (Salmo salar)', Presentation 34th WEFTA Meeting, Proceedings of the WEFTA Conference 2004. NAGAI T and SUZUKI N (2000), `Isolation of collagen from fish waste material ± skin, bones and fins', Food Chem 68, 277±281. NORLAND P E (1989), `Fish gelatine', in Voigt M N and Botta J R, Advances in Fisheries Technology and Biotechnology for increased profitability, Lancaster, PA, Technomic Publishing Co., Inc., 325±333. éINES S, RUSTAD T and ROSNES J T (1995), `Biprodukter fra filetproduksjon ± lagringsforsùk', Norconserv, Stavanger, Norway, rapport 13/95. OLSEN R L, JOHANSEN A and MYRNES B (1990), `Recovery of enzymes from shrimp waste', Process Biochem 25, 67±68. PEDERSEN G M, GILDBERG A and OLSEN R L (2004), `Effects of including cationic proteins from cod milt in the feed to Atlantic cod (Gadus morhua) fry during a challenge trial with Vibrio anguillarum', Aquaculture 233(1±4), 31±43. POERONOMO A and BUCKLE K A (2002), `Crude peptones from cowtail ray (Trygon sephen) viscera as microbial growth media', World J Microbiol Biotechnol 18, 333±340. QUAGLIA G B and ORBAN E (1987), `Enzymatic solubilisation of sardine (Sardinia pilchardius)', J Sci Food Agric 38(3), 263±269. RUBIN (2001), Biprodukter fra fiskerinñringen ± fra utkast til inntekt, Trondheim, in Norwegian (www.rubin.no). SAKAGUCHI M, HORI H, EBIHARA T, IRIE S, YANAGIDA M and INOUYE S (1999), `Reactivity of the immunoglobulin E in bovine gelatin-sensitive children to gelatins from various animals', Immunology 96, 286±290. Ê stoff ved hjelp av SANDNES K, PEDERSEN K and HAGEN H (2003), Prosessering av fiskera industrielle enzymer, Final report, RUBIN, July 2003. SATHIVEL S, BECHTEL P, BABBITT J, PRINYAWIWATKUL W, NEGULESCU I I and REPPOND K D (2004), `Properties of protein powders from Arrowtooth flounder (Atheresthes stomias) and herring (Clupea harengus) byproducts', J Agric Food Chem 52, 5040±5046. SHAHIDI F (1994), `Proteins from seafood processing discards', in Sikorski Z E, Sun Pan B and Shahidi F, Seafood proteins, New York, Chapman & Hall, 171±193. SHAHIDI F, HAN X-Q and SYNOWIECKI J (1995), `Production and characteristics of protein hydrolysates from capelin (Mallotus villosus)', Food Chem 53, 285±293. SHOJI Y (1990), `Creamy fish protein', in Keller S, Making profits out of seafood wastes, Alaska Sea Grant Program, Report no 90-07, Fairbanks, AK, USA, 87±93. SIKORSKI Z E (1994), `The contents of proteins and other nitrogenous compounds in marine animals', in Sikorski Z E, Sun Pan B and Shahidi F, Seafood proteins, New York, Chapman & Hall, 6±12. SIKORSKI Z E and BORDERIAS J A (1994), `Collagen in the muscle and skin of marine animals', in Sikorski Z E, Sun Pan B and Shahidi F, Seafood proteins, New York, Chapman & Hall, 58±70. SIKORSKI Z E, SCOTT D N and BUISSON D H (1984), `The role of collagen in the quality and processing of fish', CRC Crit Rev Food Sci Nutr 20(4), 301±343. SLIZYTE R, DAUKSAS E, FALCH E, STORRé I and RUSTAD T (2005), `Characteristics of protein fractions generated from hydrolysed cod (Gadus morhua) by-products', Proc Biochem 40, 2021±2033. SLIZYTE R, RUSTAD T and STORRé I (2006), `Enzymatic hydrolysis of cod (Gadus morhua) MICHELSEN H, FALCH E

Physical and chemical properties of protein seafood by-products

21

by-products. Optimization of yield and properties of lipid and protein fractions', Proc Biochem 40, 3680±3692. SOLSTAD J and MUNIZ I P (2001), Stùrlim og konservering, Utvikling av konserveringsmetoder og tradisjonelle metoder for et miljù i endring, NIKU, 104, 57±64. In Norwegian. SéVIK, S L (2005), Characterisation of enzymatic activities in by-products from cod species Effect of species, season and fishing ground, Doctoral theses at NTNU, Trondheim, Norway. SéVIK S L and RUSTAD T (2004), `Seasonal changes in trypsin and chymotrypsin activity in viscera from cod species', J Aquat Food Prod Technol 13(2), 13±30. SéVIK S L and RUSTAD T (2005a), `Proteolytic activity in byproducts from cod species caught at three different fishing grounds', J Agric Food Chem 53(2), 452±458. SéVIK S L and RUSTAD T (2005b), `Effect of season and fishing ground on the activity of cathepsin B and collagenase in byproducts from cod species', Lebensmittel Wissenschaft und Technologie 38(8), 867±876. SéVIK S L, BUKOVSKAYA O S, éSTGAARD P R and RUSTAD T (2004), `Enzymatic activity in by-products from cod species and carp', Report EU-project QLK1-CT-200001017, Dep. Biotechnol.; NTNU. Trondheim. STEFANSSON G and STEINGRIMSDOTTIR U (1990), `Application of enzymes for fish processing in Iceland ± present and future aspects', in Voigt M N and Botta J R, Advances in Fisheries Technology and Biotechnology for increased profitability, Lancaster, Pennsylvania, USA, Technomic Publishing Co., Inc., 237±250. STOKNES I S and HELLEVIK A H (2000), 'Bearbeiding og utnyttelse av fiskehoder', Rapport Ê 0002, Mùreforsking, A Ê lesund. In Norwegian. nr A THORARINSDOTTIR K, GUDMUNDSDOTTIR G and ARASON S (2004), Ratio and chemical contents of by-products from five cod species, Report EU-project QLK1-CT-200001017; IFL, Iceland. VAN DE VIS J W, LAMMERS C and DE WOLF F A (1996), `Extraction of collagen from plaice skin on a technical scale', Poster presented at the 26th WEFTA meeting, Gdynia, Poland. VENUGOPAL V (1995), `Methods for processing and utilization of low cost fishes: a critical appraisal. J Food Sci Technol 32(1), 1±12. VENUGOPAL V and SHAHIDI F (1995), `Value added products from underutilised fish species', J Food Science and Nutrition 35, 431±453.

2 Physical and chemical properties of lipid by-products from seafood waste J. P. Kerry and S. C. Murphy, National University of Ireland, Cork, Ireland

2.1

Introduction to fish lipids

Lipids constitute between 10 and 40% of the total human diet. They play a pivotal role in terms of flavour and palatability of food products and, in addition, their presence affects general physical properties of foods. Furthermore, lipids are an important source of essential fatty acids and serve as carriers of fat soluble vitamins. Renewable marine resources have always been important origins of the human food supply. In recent times, these resources have been, and continue to be, overexploited. This has resulted in global regulatory organizations restricting numerous fishing practices, while at the same time, emphasizing a requirement to do more with fishery by-products in order to maximize product utilization. Recent research on the beneficial health effects associated with the long chain polyunsaturated omega-3 fatty acids and more specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has led to an increase in seafood consumption and a renewed interest in fish lipids, particularly those present in the under-utilized by-product landing. Lipids are soluble in organic solvents such as acetone, ethanol and chloroform but insoluble in water. They include fatty acids, fats, oils, waxes, phospholipids, glycolipids, steroids and some vitamins. Lipids are defined on the basis of a specific physical property ± namely, solubility. They do not have any common structural feature (Freemantle, 1995). Naturally occurring fats and oils are the esters formed by propane-1,2,3-triol (also known as glycerol or glycerine) and fatty acids. They are known as triacylglycerols. Fatty acid is a general name for a monobasic aliphatic carboxylic acid, often abbreviated as RCOOH. Fatty acids may be loosely divided into three categories: saturated,

Physical and chemical properties of lipid by-products from seafood waste

23

unsaturated and branched or cyclic (Freemantle, 1995). Naturally occurring branched or cyclic fatty acids are rarely found in foods, whereas, saturated and unsaturated fatty acids are found in abundance (Freemantle, 1995; Gunstone and Norris, 1982). The major fatty acid classes in animal fats and fish oils are saturated and unsaturated fatty acids (Gunstone and Norris, 1982). Saturated fatty acids have the general formula CH3 (CH2)nCOOH, where n may range from 2 to over 20 carbon atoms. When n is low the acid is known as a short chain fatty acid and when n is high the acid is known as a long chain fatty acid (Freemantle, 1995). Long chain palmitic acid CH3(CH2)14COOH (C16:0) has been shown to be a predominant saturated fatty acid in fish species (Freemantle, 1995; Moreira et al., 2001; Mendez and Gonzalez, 1997; Nettleton et al., 1990; Watanabe et al., 1995). Unsaturated fatty acids contain at least one double bond in the chain and may range in length from short chain to long chain fatty acids (Freemantle, 1995). The unsaturated fatty acids may be subdivided into monounsaturated or polyunsaturated fatty acids (PUFA). Monounsaturated fatty acids (MUFA) contain one double bond and PUFA contain more than one double bond (Gunstone and Norris, 1982). One of the most abundant fatty acids in all plants and land animals is oleic acid (Freemantle, 1995; Gottenbos, 1985). Oleic acid CH3(CH2)7CH=CH(CH2)7COOH (C18:1) has been shown to be a predominant MUFA in fish species (Freemantle, 1995; Moreira et al., 2001; Mendez and Gonzalez, 1997; Nettleton et al., 1990; Watanabe et al., 1995). Most PUFA contain two or more double bonds (Gunstone and Norris, 1982). PUFA, especially the n-3 and n-6 fatty acids have been shown to be essential for healthy human development and since they cannot be synthesized by the body, they must therefore be supplemented in the diet (Bjerve et al., 1989). The essential omega-6 fatty acid, from which other omega-6 fatty acids like arachidonic acid can be synthesized, is called linolenic acid. The corresponding essential omega3 fatty acid, from which other omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are made, is referred to as -linolenic. Arachidonic acid and EPA are responsible for the further production of the metabolites called eicosanoids, which play a very important role in different reactions in the vascular system and other bodily functions such as the immune system (Leaf and Weber, 1988; Simopoulos, 1991). PUFA particularly EPA and DHA, have been shown to be abundant in fish lipids (Alasalvar et al., 2002; Aursand et al., 1994; Hardy and Keay, 1972; Mendez and Gonzalez, 1997; Nettleton et al., 1990; Njinkoue et al., 2002; Osman et al., 2001; Saglik and Imre, 2001; Trigari et al., 1992; Watanabe et al., 1995). Although, it is generally recognized that PUFA composition may vary among species of fish, when choosing fish for human consumption little attention has been paid to the PUFA composition. All fish are considered to be of similar nutritional value, and selection is chiefly based on availability, freshness, flavour, colour, odour and texture (Hearn et al., 1987). Research has shown that freshwater fish have lower contents of PUFAs than equivalent marine fish species (Vlieg and Body, 1988). Osman et al. (2001) suggested that

24

Maximising the value of marine by-products

the difference in PUFA content may be attributed to the fact that freshwater fish feed largely on vegetation and plant materials, whereas marine fish staple diets are mainly zooplankton rich in PUFAs. The omega-3 fatty acids of marine origin are formed in the chloroplast of marine plants that form part of the phytoplankton or algae consumed by fish. The main source for novo synthesis of n-3 fatty acids are marine autotrophic bacteria, algae and protozoa which constitute the phytoplankton and the zooplankton (Iwamoto and Sato, 1986; Seto et al., 1984). Fish, higher in the marine food chain, incorporate the n-3 PUFA and further elongate them to 20 and 22 carbon atom fatty acids countering four, five and up to six double bonds by the action of specific desaturases. Thus fish will concentrate EPA and DHA as triglycerides, mainly in adipose tissue and in the fat of muscle and visceral organs (Brockenhorff et al., 1963). Therefore, the greater the fat content of fish, the higher the content of n-3 fatty acids in the fish.

2.2

Health benefits associated with fish lipids

Fish lipids are known to be rich in polyunsaturated fatty acids (PUFA), especially the n-3 PUFA family of linolenic acid (C18:3) and its derivatives such as eicosapentaenoic acid (EPA/C20:5), docosapentaenoic acid (DPA/C22:5) and docosahexaenoic acid (DHA/C22:6) (Sargent et al., 1989, 1999). Interest in the high content of these long chain polyunsaturated omega-3 fatty acids (EPA and DHA) grew during the 1970s and 1980s with the observation that the mortality of coronary heart disease was low in Eskimos who consume a large amount of seafood. This was first proposed by Bang and Dyerberg (Bang et al., 1971; Dyerberg and Bang, 1979) who studied the diet of Greenland Eskimos who are known to have an exceptionally low frequency of such diseases. Medical investigations have shown that the intake of oil containing high contents of these acids have beneficial effects on human health and, more particularly, lower susceptibility to coronary heart disease (Abeywardena and Head, 2001; Baybutt et al., 2002; Chagan et al., 2002; Fantoni et al., 1996; Herrera, 2002; Love, 1997; Uauy and Valenzuela, 2000; Rafflenbeul, 2001). These fatty acids play a vital role in human nutrition, disease prevention and health promotion (Horrocks and Yeo, 1999; Kinsella, 1986; Simopoulos, 1991; Ulbricht and Southgate, 1991). Long chain n-3 PUFA cannot be synthesized by humans and must be obtained from the diet. The consumption of fish has been linked to increased health benefits, such as reduced risk of coronary heart disease attributable to the PUFAs in fish oils (Burr, 1992; Gordon and Ratliff, 1992; Hearn et al., 1987; Kinsella, 1986; Morris and Culkin, 1989; Paige et al., 1996). A preventative and/or curative effect also linked to the PUFAs in fish oils has been reported for arterial hypertension (Millar and Waal-Manning, 1992), human breast cancer (Rose and Connolly, 1993), inflammatory diseases (Belluzi et al., 1993: James and Cleland, 1996), asthma (Dry and Vincent, 1991; Hodge et al., 1996) and disorders of the immune system (Kenneth, 1986; Levine and

Physical and chemical properties of lipid by-products from seafood waste

25

Labuza, 1990). Links between these PUFAs and health benefits have stemmed the recent increased interest in fish and fish oils arising from such PUFA content. Fish liver has long been a source of oils (e.g., cod liver oil) for the prevention of disorders associated with vision and growth (Njinkoue et al., 2002). Results of clinical and epidemiological research suggest that EPA and DHA acids, found primarily in fish and seafoods, have extremely beneficial properties for the prevention of human coronary artery disease (Leaf and Weber, 1988). The beneficial effects of such lipids have been attributed to an increased ratio of omega-3 and omega-6 PUFAs in human blood lipids and cell membranes. Extracted fish fractions with high omega-3/omega-6 ratios have been shown to have the greatest health benefits (Hearn et al., 1987). Adults are recommended a daily intake of 350 mg of EPA/DHA, which corresponds to approximately 30 g fish/day of mixed fish species (The Danish Ministry of Health). Generally, marine organisms are very rich in EPA and DHA, especially oily fish like herring, mackerel and salmon. For those who prefer not to eat fish, they may use dietary supplementation of marine oil in the pure oil form, oil enriched food or nutritional products, or capsules (Andersen, 1994). White bread enriched in n-3 fatty acids in the form of gelatine-coated fish oil and marketed in Denmark since 1990 under the name of `Omega Bread', was found to be a reliable and significant source of higher n-3 PUFAs such as EPA and DHA (Nielsen, 1992). Kolanowski et al. (1999) suggested that it may be possible to increase the nutritional quality of food products by the addition of n3 PUFA, resulting in increased EPA and DHA content in the diet. More recent research indicates that a high intake of monounsaturated fatty acids from marine mammals may also contribute to protection against coronary heart diseases (Elvevoli et al., 1990).

2.3

Fatty acids found in fish muscle

Lipids are basic components of marine organisms. Although present in all tissues, they are concentrated mostly in the subcutaneous fatty layer of marine mammals and fatty fish, in the liver of lean fish, in muscle tissue and in mature gonads (Sikorski et al., 1990). Kiessling et al. (2001) measured the fatty acid composition in the red and white muscle, viscera and tissue from the abdominal wall of rainbow trout and reported that the most important factor governing fatty acid composition in fish tissue, pending changes in feeding, is total lipid content. The distribution of lipids in fish muscle has shown to vary with species, type of muscle and muscle location (Ackman, 1967). Therefore, when comparing population and samples, it is necessary to specify the exact area of sampling when determining the muscle lipid content of fish species (Bell et al., 1998). The considerable variation in lipid content among the fish population reported in many studies may be attributed to factors such as season, temperature, diet, age, size and sex (Alasalvar et al. 2002; Aursand et al., 1994; Chen et al., 1995; Jahncke et al.,

26

Maximising the value of marine by-products

1988; Kherunnisa et al., 1996; Suzuki et al., 1986; Waagbo et al., 1993). Arts et al. (2001) and Shirai et al. (2002) reported that the percentages of PUFA such as DHA and EPA in fish muscle are dependent on diet. Many authors have determined the fatty acid composition in the fillets of fish species. Palmitic acid (C16:0) and oleic acid (C18:1) have been shown to be the predominant saturated and monounsaturated fatty acids, respectively, in the fillets of southwest Atlantic hake (Mendez and Gonzalez, 1997), catfish (Nettleton et al., 1990), Brazilian brycon freshwater fish (Moreira et al., 2001) and in bonitio, caught off the Japanese coast (Watanabe et al., 1995), whereas, EPA and DHA were shown to be the predominant PUFAs. Njinkoue et al. (2002) reported C16:0 as the main fatty acid in Sardinella maderensis, Sardinella aurita and Cephalopholis taeniops, species from the Senegalese coast, and they also found high concentrations of PUFAs, including EPA and DHA. Njinkoue et al. (2002) reported that the percentages of PUFA found in such fish species were very similar to those species used commercially as sources of PUFA such as herring, sardines and menhaden. Osman et al. (2001) determined the fatty acid composition of selected marine fish caught in Malaysian waters. They reported that all fish samples showed a much higher content of n-3 PUFA when compared to standard menhaden oil. European anchovy and European pilchard fish species from Turkey were discovered to be good dietary sources of n-3 PUFA (Saglik and Imre, 2001). Commercial fish oil is usually composed of over 90% triacylglycerols, each usually containing three different fatty acids (Hjaltason, 1990). An additional 8% consists of mono- and di-acylglycerols and other lipids such as phospholipids. The unsaponifiable portion that accounts for an additional 1.0% to 2.0% consists principally of sterols, glyceryl ethers, hydrocarbons and fatty alcohols, along with the fat-soluble vitamins A, D and E (Hjaltason, 1990). Marine oils are produced from the body of fatty fish, livers of lean fish, as well as from blubber of marine mammals. Marine oils form a significant proportion (2±3%) of the world's edible oil production. The relative amount of EPA and DHA varies from 5±20% and 3±26%, respectively, of fatty acids (Kamal-Eldin and Yanishlieva, 2002).

2.4

Fatty acids found in fish by-products

Renewable marine resources are important origins of human food supply (Shahidi et al., 1991). Annually, over 100 million tonnes of fish are harvested worldwide (Venugopal, 1992), however, up to 25% of marine catches are discarded (Valdimarsson and James, 2001). Fish is a rich source of easily digestible protein that also provides polyunsaturated fatty acids, vitamins and minerals for human nutrition. Increasing concern worldwide about the use and over exploitation of natural seafood resources and in particular, the dwindling stocks of commercially important fish species has led to greater utilization of fish landings, and waste use has become an important issue for the seafood industry

Physical and chemical properties of lipid by-products from seafood waste

27

(Morrissey et al., 2000; Venugopal, 1997). Processing discards from fisheries account for as much as 70±80% of the total weight of catch after filleting, and these discards offer a myriad of possibilities in extracting valuable components and have a huge potential for further use. To date, many studies have measured the fatty acid composition of fish muscle from species such as hake (Mendez and Gonzalez, 1997), blue whiting (Dapkevicius et al., 1998), sea bass (Trigari et al., 1992), mackerel (Hardy and Keay, 1972), turbot (Ruff et al., 2002a,b), halibut (Ruff et al., 2002b,c) and salmon (Ackman and Takeuchi, 1986; Aursand et al., 1994; Katikou et al., 2001), however, little work has focused on extracting these lipids from byproducts for use as potential functional ingredients. Watanabe et al. (1995) measured the fatty acid composition in the gills, spleen, heart, pyloric caecum, stomach and gonads in bonitio, caught off the Japanese coast. They reported that C16:0, C16:1, C18:1, C20:5 and C22:6 were the predominant fatty acids determined and that DHA was the overall dominant fatty acid accounting for 25% or more of the total fatty acids. Njinkoue et al. (2002) recorded the fatty acid composition in the liver and skin of three fish from the Senegalese coast, in addition to that of the muscle. They determined that C16:0 was the dominant fatty acid in the skin of all samples and C18:1 was dominant in the liver of Sardinella maderensis and Sardinella aurita. They also reported high PUFA content in these by-products as well as in the muscle of the three fish species studied. Aursand et al. (1994) reported EPA and DHA levels were higher in liver than in the edible belly flap region of Atlantic salmon. Sun et al. (2002) reported that viscera from salmon was a good source of EPA and DHA and by using microbial lipases the concentration of these omega-3 PUFAs could be doubled. Brockenhorff et al. (1963) reported that EPA and DHA are stored as triacylglycerols, mainly in the adipose tissue and in the fat of the visceral organs. Satoh et al. (1989) suggested that catfish can accumulate n-3 fatty acids in liver lipids. Chantachum et al. (2000) recovered oil from precooked and nonprecooked skipjack tuna heads by wet reduction. The study found that oil obtained from non-precooked tuna heads gave superior yield and higher quality compared to the oil from precooked tuna heads. However, DHA content was higher in the crude oil extracted from precooked tuna heads. Dauksas et al. (2005) recovered oil fractions from enzymatic hydrolysis of combinations of viscera, backbone and digestive tract of cod. While many of the studies described above are limited in scope and magnitude, they all share the important commonality of demonstrating the potential that exists in terms of extracting valuable lipid contents, and possibly other and more significant components, from the by-product remains of fish landings. Clearly, more concerted research is required in order to fully elucidate the true potential that fish wastes, in the form of fishery by-products, have in terms of their contribution of extractable bulk and fine chemicals for commercial utilization.

28

Maximising the value of marine by-products

2.5 Factors affecting the fatty acid composition of fish and their associated by-products It has been reported that the type and amount of fatty acids in fish tissue are influenced by diet, and other factors such as size, age, reproductive status, geographical location and season have been shown to affect fat content and composition of fish muscle (Ackman, 1989; Nettleton 1985; Saito et al., 1999). Fishing region has been shown to affect the condition, diet and composition of fish species (Du Buit, 1995; Gieseg et al., 2000; Ratz and Lloret, 2002; Shirai et al., 2002). Ratz and Lloret (2002) suggested that the condition of cod stock in the North Atlantic may be affected by the different temperature regimes of their habitats. Mendez and Gonzalez (1997) reported seasonal variation in fatty acid composition in hake. Similarly, Malone et al. (2004) also reported variation in fatty acid composition of by-products liver, viscera and cut-off of cod, ling, saithe and haddock due to season. Lipid content of fish is dependent on breed, food supply and other factors beside season (Hardy and Keay, 1972). According to Viarengo et al. (1998) and Kiessling et al. (2001), dietary intake is the most important factor governing fatty acid composition and -tocopherol levels. Thererfore, it is necessary to review how fishing region influences the dietary intake of fish species. Du Buit (1995) suggested that variations in the diet of cod in the main stocks of the North Atlantic may be a function of temporal or geographic changes, and the diversity of prey increases as the cod move from northern to southern regions. Prey such as pelagic species including krill, capelin and herring are mostly dominant in the cold waters of the Arctic Ocean (Mehl, 1986; Zamarro, 1985), off Iceland (Palsson, 1983), and in the fjords of northern Norway (Dos Santos and Falk-Petersen, 1989; Klemetsen, 1982). Trisopterus, and to a lesser extent, blue whiting, are the most important prey in more temperate waters off the Faroe Isles, the British Isles and the Norwegian Deep (Bergstad, 1991; Du Buit, 1995; Rae, 1967) whereas, such species are in less abundance in the North Sea (Daan, 1973). Other prey, such as benthic species are in maximum abundance in the southern North Sea (Cramer and Daan, 1986; Daan, 1973), the Irish Sea (Armstrong, 1982; Brander, 1981) and the Celtic Sea (Du Buit, 1995). In addition to both pelagic- and benthic-based diets, all proportions of nektonic and bottom-dwelling or even burrowing prey are available for feeding by cod species, depending on location. Few authors have compared the fatty acid profiles of wild fish species caught in different sea fishing regions. Many authors have compared the fatty acid compositions of wild and cultured fish species such as catfish (Shirai et al., 2002), sea bass (Alasalvar et al., 2002; Orban et al., 2002) and yellowtail (Arakawa et al., 2002) and generally the wild fish species tended to have higher n-3 PUFA content than their cultured counterparts. Many of these authors attributed the differences in PUFA content to the dietary intake of the fish species. Trigari et al. (1992) compared the fatty acid compositions of warm and

Physical and chemical properties of lipid by-products from seafood waste

29

cold adapted sea bass and overall the fatty acid composition did not indicate any increase in unsaturation in response to temperature. Shirai et al. (2002) compared the lipid content between cultured Thai and Japanese catfish and reported higher lipid content in the dorsal muscle of Thai catfish when compared with Japanese catfish. They suggested that lipid content may be affected by species and/or dietary intake. They also suggested that, in general, high lipid contents in muscle may result in an increase in the percentage of triacylglycerol, the storage lipid. Dey et al. (1993) and Wodtke (1981) have suggested that saturated and monounsaturated fatty acids are generally abundant in fish from warm and temperate regions, whereas PUFAs are more abundant in fish from colder regions. Olsen and Skjervold (1991) studied the effect of geographical location on n-3 fatty acids in wild Atlantic salmon and suggested that the habitat and season of capture may produce broad changes in the omega-3 content of fish oil, even among the same fish species. As the temperature of the water decreased, as in the polar regions, fish showed an increase in PUFAs in their tissues, in order to compensate for the reduction in the fluidity of their membranes due to low temperature. When capture occurred in temperate regions where the seawater was over 12ëC, the oil obtained after fish processing showed significant reductions in omega-3 content. By similar mechanism, the season of capture may have influenced the omega-3 content of the same species (Olsen and Skjervold, 1991). Czesny et al. (2000) compared the fatty acid composition between wild and domestic sturgeon eggs. They showed that fatty acids C18:0 and C18:1 n-9 were origin specific rather than species specific. They reported lower levels of C18:0 in wild fish eggs than in domesticated fish ova, whereas, C18:1 n-9 showed the opposite trend. Czesny et al. (2000) demonstrated that sturgeon environment played an important role and markedly influenced fatty acid composition of their eggs. Research into measuring natural levels of -tocopherol in wild fish species and the effect fishing region has on these levels is only beginning to emerge in the literature. Ruff et al. (2002c) measured the natural -tocopherol levels in the fillets of wild turbot caught off the south coast of Ireland and in Atlantic halibut caught off the north-west coast of Iceland. They reported fillet -tocopherol content was nearly twice as high in Atlantic halibut than in turbot, and suggested this could be due to differences in the natural diet since they came from different waters. Gieseg et al. (2000) compared the -tocopherol levels in the plasma of two Antarctic and two temperate water fish species. They showed that the plasma from both Antarctic fish species had -tocopherol concentrations 5±6 times higher than those found in the two temperate water fish species. They attributed these varying levels of -tocopherol to the diet obtained by the fish species in their specific habitats. Yamamoto et al. (1999) isolated a new vitamin E, -tocomonoenol from salmon eggs having an unusual methylene unsaturation at the isoprenoid-chain terminus. This new vitamin E was designated `marinederived tocopherol' (MDT) for its exclusive occurrence in marine organisms.

30

Maximising the value of marine by-products

High concentrations of MDT were found in fish inhabiting cold-water environments suggesting a specific metabolic function in low temperature adaptation (Yamamoto et al., 2001). Dunlap et al. (2002) examined the vitamin E content of tissues from Antarctic notothenioid fish and extracts of Antarctic krill and reported very high levels of -tocopherol in these species, which were accompanied, by high levels of MDT. They suggested that these high levels of tocopherol in Antarctic fish species might be attributed to the cold-water environment. Seasonality of fish capture has been shown to affect nutritional composition including fatty acid profiles and -tocopherol levels. Mendez and Gonzalez (1997) reported seasonal variation in the condition factor, the total non-volatile nitrogen and the lipid contents in hake species. These values decreased during the spawning period for hake species and they attributed this to the use of muscle lipids and protein as energy reserves during these reproductive months. Seasonal variation in the fatty acid composition and total fat content has been reported for Cornish mackerel (Hardy and Keay, 1972), south-west Atlantic hake (Mendez and Gonzalez, 1997), and in Japanese and Thai catfish (Shirai et al., 2002). These authors have reported a decline in the total fat content and percentage total fatty acids during the spawning season of these fish species. Cordier et al. (2002) reported seasonal effects on the fatty acid composition of tissue phospholipids in farmed sea bass. They observed major changes in percentage phosphatidylethanolamine and phosphatidylcholine in all tissues between February and March, and the phosphatidylethanolamine and phosphatidylcholine ratio was drastically reduced at this time. They found that these changes corresponded to the beginning of the spawning period of sea bass. Both Refsgaard et al. (1998) and Hamre and Lie (1995) reported that the tocopherol levels of Atlantic salmon were affected by season. Hamre and Lie (1995) reported that the -tocopherol levels in the whole body of Atlantic salmon fed diets unsupplemented with vitamin E, showed significantly higher levels of -tocopherol in May compared with January (Hamre and Lie, 1995). These results were consistent with those recorded by Refsgaard et al. (1998) and they suggested seasonally varying factors such as water temperature may have influenced the -tocopherol content. Nettleton et al. (1990) reported very low levels of -tocopherol in the fillets of Channel catfish and seasonal variation was not detected, with little deviation in -tocopherol levels recorded in February, April and August. To date little research into the effect of season on the natural -tocopherol levels in wild fish species has been carried out and the seasonal variation reported above is that for cultured fish species fed diets supplemented with dietary -tocopheryl acetate.

2.6

Deterioration of fish lipids

Oxidation of lipids in biological food systems adversely affects nutritional quality, wholesomeness, safety, colour, flavour, and texture. Major negative

Physical and chemical properties of lipid by-products from seafood waste

31

quality changes that occur during processing, distribution, and final preparation of lipid-rich foods may be attributable to oxidation effects (Shahidi and Wanansudara, 1992). Fish lipids are known to be rich in PUFAs, however, these are highly vulnerable to oxidative deterioration, especially, during and after processing of fish products (Aidos et al., 2002; Kamal-Eldin and Yanishlieva, 2002; Kulas et al., 2002; Jensen et al., 1998; Sargent et al., 1989; Yanishlieva and Marinova, 2001). Lipid oxidation is a process by which molecular oxygen reacts with unsaturated lipids to form lipid peroxides (Hawrysh, 1990; Lawson, 1995), and is catalysed by factors such as temperature, water activity, pH and chemical environment (Ashie et al., 1996). Oxidation results in the production of off-flavours and off-odours, as well as colour and texture deterioration. Gu and Weng (2001) reported that oxidation of fatty acids found in oils may induce aging and carcinogenesis. Other workers reported that oxidation may also generate toxic compounds, which have the potential to affect health (Gurr, 1984). In the living animal the ingestion and regeneration of antioxidants prevents excessive oxidative deterioration of important biological components. Post mortem, the protective systems become depleted and are unable to regenerate and thus, oxygen may react with any of the biochemical components when exposed to air (Ashton, 2002). Post mortem lipid degradation proceeds mainly due to enzymatic hydrolysis. Phospholipids are hydrolyzed most readily, followed by triacylglycerols, to produce free fatty acids (lipolysis) (Sikorski et al., 1990; Ashton, 2002). Free fatty acids oxidize readily, especially in the presence of enzymes. The development of free fatty acids in different marine systems has been well documented (Harris and Tall, 1994; Hwang and Regenstein, 1993; Ingemansson et al., 1995; Kaneniwa et al., 2000; Miyashita and Takagi, 1986; Refsgaard et al., 1998). Free fatty acids are not only important from the point of view of oxidation products, but they have also been reported to have a direct sensory impact (Refsgaard et al., 1998). Lipid oxidation is an autocatalytic chain reaction, which takes place through four main stages: initiation, propagation, chain branching and termination (Allen and Foegeding, 1981; Hultin, 1992). The primary products of lipid oxidation, lipid hydroperoxides, are generally considered not to have a flavour impact. The volatile secondary oxidation compounds, aldehydes and ketones, derived from breakdown of primary oxidation compounds are responsible for rancid flavours and aromas. Some aldehydes and ketones react further with compounds containing free amino groups, resulting in oxidation products affecting fish colour and texture (Aubourg et al., 1997; Milo and Grosh, 1993). The level of lipid in fish varies depending on species, and it is well established that oily fish are particularly susceptible to lipid oxidation and spoilage due to their high content of PUFA in their lipid. As well as lipid level and fatty acid composition of the lipids, levels of endogenous antioxidants and endogenous oxidative catalysts will also affect development of rancidity. External factors, such as heat, light, processing procedures and handling will

32

Maximising the value of marine by-products

also change the equilibrium of tissue compounds and thus play a large part in the development of rancidity. Susceptibility to rancidity does not only depend on the amount of lipid present, but the lipid composition and location in the fish matrix (Khayat and Schwall, 1983; Ingemansson et al., 1993; Icekson et al., 1998). Oxidative rancidity problems appear differentially disposed among specific tissue regions. Undeland et al. (1998, 1999) reported that compositional analysis showed the highest levels of pro-oxidants in dark muscle and the highest level of polar lipids in light muscle in herring fillets. Ablett and Gould (1986) found that in cooked mussels, oxidation occurred especially in tissue from the digestive gland region yielding highest oxidative rancidity. To reduce the formation of volatile compounds associated with off-flavour, the oxidation process has to be stopped or slowed down. The most widely studied methods for reducing oxidation are the direct application of antioxidants. There are several categories of antioxidants that may be used and, in general, antioxidants must be effective at low concentrations, not have a sensory impact and must be non-toxic (Ashton, 2002). Antioxidants work by a number of methods including sequestration of catalytic metal ions preventing propagation, decreasing oxygen concentration, quenching singlet oxygen, decomposing primary oxidation products to non-volatile compounds, preventing first chain initiation by scavenging initial radicals and chain breaking or free radical interceptor antioxidants (Ashton, 2002). Vitamin E has been shown to be an effective antioxidant in fish systems (Ohshima et al., 1998) and is regarded as nature's most effective lipid soluble scavenger of free radicals.

2.7

Implications for fish fat by-product valorization

As previously stated, oxidation results in the production of off-flavours and odours, as well as colour and texture deterioration. Gu and Weng (2001) reported that oxidation of fatty acids found in oils may induce aging and carcinogenesis. Other workers reported that oxidation may also generate toxic compounds, which have the potential to damage health (Gurr, 1984). Oxidation can be controlled to an extent by limiting the factors that influence the process, such as light, temperature, heat and metals, processing procedure and oxygen. As previously outlined, season can have a large effect on properties of lipids present. The most significant seasonal effect is that of spawning when as a consequence much of the lipid is converted to either eggs or sperm. Rough handling may also cause loss of desirable properties, and bruising may increase the rate of rancidity development on frozen storage (Hedges, 2002) and thus careful handling and storage of fish onboard is essential. Diet is a pre-slaughter factor that can have a major effect on the quality of fish. From an aquacultural perspective, modification of the fish diet can significantly and positively affect the quality and stability of the final product, both at a primary and secondary product level. Dietary supplementation with

Physical and chemical properties of lipid by-products from seafood waste

33

-tocopherol has been reported to improve the stability of tissue lipids to oxidation in trout (Frigg et al., 1990), Atlantic Salmon (Waagbo et al., 1993), turbot (Ruff et al., 2002a; Stephan et al., 1995), sea bass (Messager et al., 1992; Pirini et al., 2000) and halibut (Ruff et al., 2002b, 2004a,b). A study by Ruff et al. (2002a) examined the effect of slaughtering method as well as dietary supplementation on the quality of turbot fillets. The authors showed that fish fed supplemented diets of -tocopheryl acetate had reduced levels of lipid oxidation post mortem in comparison to fish fed unsupplemented diets. Slaughtering method was also shown to have an impact on the final quality of the fish. To date, many studies have measured the fatty acid composition of fish fillets such as hake (Mendez and Gonzalez, 1997) blue whiting (Dapkevicius et al., 1998), sea bass (Trigari et al., 1992), mackerel (Hardy and Keay, 1972) turbot (Ruff et al., 2002a,b), halibut (Ruff et al., 2002b,c, 2004a,b) and salmon (Ackman and Takeuchi, 1986; Aursand et al., 1994; Katikou et al., 2001), but little work has focused on extracting these lipids from by-products for use as potential functional ingredients. Increasing research has been conducted on extraction, concentration and stability of fish oil. Fish oil can be produced by several methods, including physical fractionation (Hirata et al., 1993), low temperature solvent fractionation (Moffat et al., 1993), supercritical fluid extraction (Dunford et al., 1997) and wet reduction (Bimbo and Crowther, 1990; Chantachum et al., 2000). Many approaches using enzymatic methods have also been carried out to increase the concentration of n-3 fatty acids in fish oil (Moore and McNeill, 1996; Shimada et al., 1997). Optimum freezing practices may reduce rancidity during storage by reducing ice crystal damage, particularly to membrane lipids where the initial onset of lipid oxidation may occur. It is possible that the formation and even distribution of small ice crystals via a rapid freezing process could reduce initial damage generally associated with freezing. The use of cryoprotectants to reduce freezing damage could also be a potential route to prevention of off-flavour development (Ashton, 2002; Toama, 1990). Cryoprotectants are compounds that have the ability to extend the shelf life of frozen foods. A wide variety of compounds will cryoprotect during freezing, including sugars, amino acids, polyols, methyl amines, carbohydrate polymers, synthetic polymers and inorganic salts (MacDonald and Lanier, 1991; Matsumoto and Noguchi, 1992; Toyoda et al., 1992; Park, 1994; Park et al, 1997; MacDonald et al., 2000). Modified atmosphere and vacuum packaging are designed to extend shelf life by reducing or eliminating oxygen and thereby reducing oxidative deterioration during storage. Modified atmosphere packaging (MAP) has been used to extend the shelf life and maintain high quality of salmon (Amanatidou et al., 2000: Stier et al., 1981), whiting and mackerel (Fagan et al., 2004; Hong et al., 1996), channel catfish (Silva et al., 1993; Silva and White, 1994), haddock and herring (Dhananjaya and Stroud, 1994; Ozogul et al., 2000), tilapia (Reedy et al., 1994) and halibut (Ruff et al., 2004a). Vacuum packaging may also be used effectively to inhibit lipid oxidation, but should be accompanied by low chill temperatures,

34

Maximising the value of marine by-products

as the growth of anaerobic bacteria is favoured under these conditions (Sikorski et al., 1990). Antioxidants are compounds which have been shown to maintain acceptable levels of food quality by neutralizing the free radicals produced during the oxidative process (Haard and Simpson, 2000) and subsequently delaying the deterioration of food quality. Traditional methods to reduce lipid oxidation utilize synthetic phenolic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (Shahidi and Wanasundara, 2001). Ito et al. (1986) reported a possible link between the dose of synthetic antioxidants used in food products and the development of cancer in rats. This study and other research (Ito and Hirose, 1989; Clayson et al., 1993; Botterweck et al., 2000) have resulted in pressure from consumer groups to reduce the amount of synthetic additives in foods and substitute with natural alternatives (Marshell, 1974). Many authors (Chang et al. 1977; Stoick et al. 1991; Frankel et al. 1997) have highlighted the antioxidant potential of both tea and rosemary in fish oil systems. Tsimidou et al. (1995) reported a decline in oxidation when rosemary was added to mackerel oil at 0.5% (w/w) concentration and Roedig-Penman and Gordon (1997) further reported that tea extract (0.03%, w/w) produced an equal antioxidant activity to that of BHT (0.02%, w/w) in an oil-in-water emulsion. Tang et al. (2001) also reported a significant decrease in oxidation when tea catechins were incorporated into cooked fish patties. -Tocopherol (Ohshima et al., 1998; Kulas et al., 2002), tea (Wanasundara and Shahidi, 1998), rosemary (Xin and Shun, 1993; Frankel et al., 1996) and oregano (Tsimidou et al., 1995) have been shown to possess antioxidant properties in fish systems. Other natural alternatives such as clove (Beddows et al., 2000) and mustard (McCarthy et al., 2001a,b) have exhibited antioxidative effects when added to food systems. A recent study by O'Sullivan et al. (2005) examined the effect of a variety of antioxidants in oil extracted from cod and pollack. The authors found that cod liver oil samples containing rosemary and tea catechins had lower levels of lipid oxidation in comparison to the samples containing the other test antioxidants (BHT, mustard, carvacrol, white clove oil, black clove oil, natural vitamin E and synthetic vitamin E). White pollack liver oil samples containing tea catechins had lower levels of lipid oxidation in comparison to the oil samples containing the other test antioxidants (rosemary, BHT, mustard, carvacrol, white clove oil, black clove oil, natural vitamin E and synthetic vitamin E). Black clove oil, white clove oil, mustard and carvacol had no significant effect whereas the addition of synthetic or natural vitamin E had a negative effect on the oxidative stability of the extracted oil samples.

2.8

Future trends

No growth is expected in marine fisheries in the foreseeable future. Any increase in total quantity of raw fish materials will come as a result of increased

Physical and chemical properties of lipid by-products from seafood waste

35

aquaculture production. In the by-product sector, however, there is still a large potential for improved recovery both from fisheries and aquaculture. With an increasing awareness among consumers of marine foods as a source of healthy polyunsaturated omega fatty acids, the fisheries industry must focus on the huge potential of recovering valuable marine lipids from by-products. By-products could provide us with a constant supply of valuable marine lipids and by applying appropriate measures and technologies for lipid recovery, it may be possible to achieve more efficient utilization of total catch as well as a potential ingredient source for the ever increasing demand for omega fatty acids. This would utilize both fishery by-products and secondary raw materials and, in addition, underutilized species that would otherwise be discarded. Rancidity is a clear and evident problem associated with marine lipids. Measures must be taken to prevent or slow the oxidation process and prevent the deterioration of flavour and aroma. Therefore, the control of lipid oxidation in such products is of vital importance. The use of dietary antioxidants or antioxidants used as processing aids are important areas of research which warrant further investigation, particularly, in relation to the use of natural and potent forms of antioxidants to stabilize fish oil systems.

2.9

References

and HEAD, R. (2001). Long chain fatty acids and blood vessel function. Cardiovascular Research, 52, 361±371. ABLETT, R.F. and GOULD, S.P. (1986). Comparison and sensory quality and oxidative rancidity status of frozen cooked mussels (Mytilus edulis). Journal of Food Science, 51, 809±811. ACKMAN, R.G. (1967). The influence of lipids on fish quality. Journal of Food Technology, 2, 169±181. ACKMAN, R.G. (1989). Nutritional composition of fats in seafoods. Prog. Food and Nutrition Science, 13, 161±241. ACKMAN, R.G. and TAKEUCHI, T. (1986). Comparison of fatty acids and lipids of smolting hatchery-fed and wild Atlantic salmon Salmo salar. Lipids, 21, 117±120. AIDOS, A., JACOBSEN, C., JENSEN, B., LUTEN, J.B., VAN DER PADT, A. and BOOM, R.M. (2002). Volatile oxidation products formed in crude herring oil under accelerated oxidative conditions. European Journal of Lipid Science Technology, 104 (12), 808±818. ALASALVAR, C., TAYLOR, K.D.A., ZUBCOV, E., SHAHIDI, F. and ALEXIS, M. (2002). Differentiation of cultured and wild sea bass (Dicentrarchus labrax): total lipid content, fatty acid and trace mineral composition. Food Chemistry, 79, 145±150. ALLEN, C.E. and FOEGEDING, E.A. (1981). Some lipid characteristics and interactions in muscle foods ± a review. Food Technology, 35, 253±257. È TER, O., LEMKAU, K., GORRIS, L., SMID, E. and KNORR, D. (2000). AMANATIDOU, A., SCHLU Effect of combined application of high pressure treatment and modified atmospheres on the shelf-life of fresh Atlantic salmon. Innovative Food Science and Emerging Technologies, 1, 87±98. ANDERSEN, S. (1994). Microencapsulated marine omega-3 fatty acids for use in the food industry. Food Technology Europe, Dec 1994/Jan 1995, 104±106. ABEYWARDENA, M.

36

Maximising the value of marine by-products

and TAKEUCHI, T. (2002). Comparison of lipid classes and fatty acid compositions between hatchery reared and wild caught yellowtail Seriola quinqueradiata juvenile. Japanese Society of Fisheries Science, 68, 374±381. ARMSTRONG, M.J. (1982). The predator prey relationships of the Irish Sea poor cod (Trisopterus minutus L.), pouting (Trisopterus luscus L.), and cod (Gadus morhua L.). Journal of Conservation and International Exploration de la Mer, 40, 135± 152. ARTS, M.T., ACKMAN, R.G. and HOLUB, B.J. (2001). Essential fatty acids in aquatic ecosystems: a crucial link between diet and human health and evolution. Canadian Journal of Fisheries Aquaculture Science, 58, 122±137. ASHIE, I.N.A., SMITH, J.P. and SIMPSON, B.K. (1996). Spoilage and shelf-life extension of fresh fish and shellfish. Critical Reviews in Food Science and Nutrition, 36, 87±121. ASHTON, I.P. (2002). Understanding lipid oxidation in fish. In: Safety and Quality Issues in Fish Processing (Ed. Bremner, H.A.), Woodhead Publishing Ltd, Cambridge. AUBOURG, S.P., SOTELO, C.G. and GALLARDO, J.M. (1997). Quality assessment of sardines during storage by measurement of fluorescent compounds. Journal of Food Science, 62, 295±298, 304. AURSAND, M., BLEIVIK, B., RAINUZZO, J.R., JORGENSEN, L. and MOHR, V. (1994). Lipid distribution and composition of commercially farmed Atlantic Salmon (Salmo salar). Journal of Science of Food and Agriculture, 64, 239±248. BANG, H.O., DYERBERG, J. and NIELSEN, A.B. (1971). Plasma lipid and lipoprotein pattern in Greenlandic west-coast Eskimos. Lancet, 1, 1143±1146. BAYBUTT, C., ROSALES, C., BRADY, H. and MOLTENI, A. (2002). Dietary fish oil protects against lung and liver inflammation and fibrosis in monocrotaline treated rats. Atherosclerosis, 162, 335±344. BEDDOWS, C., JAGAIT, C. and KELLY, M. (2000). Preservation of alpha-tocopherol in sunflower oil by herbs and spices. International Journal of Food Science and Nutrition, 51, 327±339. BELL, J.G., MCEVOY, J., WEBSTER, J.L., MCGHEE, F., MILLAR, R.M. and SARGENT, J.R. (1998). Flesh lipid and carotenoid composition of Scottish farmed Atlantic salmon (Salmo salar). Journal of Agriculture and Food Chemistry, 46, 239±248. BELLUZI, A., CAMPIERI, M., BRIGNOLA, C., GIONCHETTI, P., MILLIOLI, M. and BARBARA, L. (1993). Polyunsaturated fatty acid pattern and oil treatment in inflammatory bowel disease. Gut, 34, 1289±1290. BERGSTAD, O.A. (1991). Distribution and trophic ecology of some gadoid fish of the Norwegian deep. Sarsia, 75: 269±313. BIMBO, A. and CROWTHER, J.B. (1991). Fish oils: processing beyond crude oil. Infofish International, 6, 20±25. BJERVE, K.S., FISCHER, S., WAMMER, F. and EGELAND, T. (1989). -Linolenic acid and long chain X-3 fatty acid supplementation in three patients with X-3 fatty acid deficiency: Effect of lymphocyte function plasma and red cell lipids, and prostanoid formation. American Journal of Clinical Nutrition, 49, 290±300. BOTTERWECK, A., VERHAGEN, H., GOLDBOHM, R., KLEINJANS, J. and VAN DEN BRANDT, P. (2000). Intake of butylated hydroxyanisole and butylated hydroxytoluene and stomach cancer risk: results from analyses in the Netherlands cohort study. Food and Chemical Toxicology, 38, 599±605. BRANDER, K. (1981). On the application of models incorporating predation in the Irish Sea. ICES, CM G:29, p11. ARAKAWA, T., ISHIZAKI, Y., CHUDA, H., SHIMIZU, K., ARIMOTO, M.

Physical and chemical properties of lipid by-products from seafood waste

37

and HOYLE, R.J. (1963). Specific distribution of fatty acids in membrane lipids. Archives of Biochemistry and Biophysics, 100, 93±100. BURR, M.L. (1992). Fish food, fish oil and cardiovascular disease. Clinical and Experimental Hypertension, 14, 181±192. CHAGAN, L., IOSELOVICH, A., ASHEROVA, L. and CHENG, J. (2002). Use of alternative pharmacotherapy in management of cardiovascular diseases. The American Journal of Managed Care, 8, 270±285. CHANG, S., MATIJASEVIC, B., HSIEH, O. and HUANG, C. (1977). Natural antioxidants from rosemary and sage. Journal of Food Science, 42, 1102±1106. CHANTACHUM, S., BENJAKUL, S. and SRIWIRAT, N. (2000). Separation and quality of fish oil from precooked and non-precooked tuna heads. Food Chemistry, 69, 289±294 CHEN, I.C., CHAPMAN, F.A., WEI, C.I., PORTIER, K.M. and O'KEEFE, S.F. (1995). Differentiation of cultured and wild sturgeon (Acipenser oxyrinchus desotoi) based on fatty acid composition. Journal of Food Science, 60, 631±635. CLAYSON, D., IVERSON, F., NERA, E. and LOK, E. (1993). The importance of cellular proliferation induced by BHA and BHT. Toxicology and Industrial Health, 9, 231±242. CORDIER, M., BRICHON, G., WEBER, J.M. and ZWINGELSTEIN, G. (2002). Changes in the fatty acid composition of phospholipids in tissues of farmed sea bass (Dicentrarchus labrax) during an annual cycle. Roles of environmental temperature and salinity. Journal of Comparative Biochemistry and Physiology, 133, 281±288. CRAMER, S. and DAAN, N. (1986). Consumption of benthos by North Sea cod and haddock in 1981. ICES, CM G:56, p14. CZESNY, S., DABROWSKI, K., CHRISTENSEN, J.E., VAN EENENNAAM, J. and DOROSHOV, S. (2000). Discrimination of wild and domestic origin of sturgeon ova based on lipids and fatty acid analysis. Journal of Aquaculture, 189, 145±153. DAAN, N. (1973). A quantitative analysis of the food intake of North Sea cod. Netherlands Journal of Sea Research, 6, 479±517. DAPKEVICIUS, M.E., BATISTA, I., NOUT, M.J.R. and ROMBOUTS, F.M. (1998). Lipid and protein changes during the ensilage of blue whiting (Micromesistius poutassou Risso) by acid and biological methods. Journal of Food Chemistry, 63, 97±102. DAUKSAS, E., FALCH, E., SLIZYTE, R. and RUSTAD, T. (2005). Composition of fatty acid and lipid classes in bulk products generated during enzymic hydrolysis of cod (Gadus morhua) by-products. Process Biochemistry, 40, 2659±2670. DEY. I., BUDA, C., WIIK, H., HALVER, J.E. and FARKAS, T. (1993). Molecular and structural composition of phospholipid membranes in livers of marine and freshwater fish in relation to temperature. Proceedings of the National Academy of Science USA, 90, 7498±7502. DHANANJAYA, S. and STROUD, G.D. (1994). Chemical and sensory changes in haddock and herring stored under modified atmosphere. International Journal of Food Science and Technology, 29, 575±583. DOS SANTOS, J. and FALK-PETERSON, S. (1989). Feeding ecology of cod (Gadus morhua) in Balsfjord and Ullsfjord, northern Norway, 1982±1983. Journal of Conservation and International Exploration de la Mer, 45, 190±199. DRY, J. and VINCENT, D. (1991). Effects of fish oil diet on asthma: results of a year double blind study. International Archives of Allergy and Applied Immunology, 95, 156± 157. DU BUIT, M.H. (1995). Food and feeding of cod (Gadus morhua L.) in the Celtic Sea. Fisheries Research, 22, 227±241. DUNFORD, N.T., TEMELLI, F. and LEBLANC, E. (1997). Supercritical CO2 extraction of oil and BROCKENHORFF, H., ACKMAN, R.G.

38

Maximising the value of marine by-products

residual proteins from Atlantic mackerel (Scomber scombrus) as affected by moisture content. Journal of Food Science, 62, 289±294. DUNLAP, W.C., FUJISAWA, A., YAMAMOTO, Y., MOYLAN, T.J. and SIDELL, B.D. (2002). Notothenioid fish, krill and phytoplankton from Antarctica contain a vitamin E constituent (-tocomonoenol) functionally associated with cold water adaptation. Journal of Comparative Biochemistry and Physiology, 133B, 299±305. DYERBERG, J. and BANG, H.O. (1979). Haemostatic function and platelet polyunsaturated fatty acids in Eskimos, Lancet 2, 433±435. ELVEVOLI, E.O., MOEN, P., OLSEN, R.L. and BROX, J. (1990). Some possible effects of dietary monounsaturated fatty acids on cardiovascular disease. Atheriosclerosis, 81, 71±74. FAGAN, J.D., GORMLEY, T.R. and MHUIRCHEARTAIGH, M.M. (2004). Effect of modified atmosphere packaging with freeze-chilling on some quality parameters of raw whiting, mackerel and salmon portions. Innovative Food Science and Emerging Technologies, 5, 205±214. FANTONI, C.M., CUCCIO, A.P. and BARRERA-ARELLANO, D. (1996). Brazilian encapsulated fish oils: oxidative stability and fatty acid composition. Journal of American Oil Chemists Society, 73, 251±253. FRANKEL, E.N., HUANG, S.W., PRIOR, E. and AESCHBACH, R. (1996). Evaluation of antioxidant activity of rosemary extracts, carnosol and carnosic acid in bulk vegetable oils and fish oil and their emulsions. Journal of the Science of Food and Agriculture, 72, 201±208. FRANKEL, E.N., HUANG, S.W. and AESCHBACH, R. (1997). Antioxidant activity of green tea in different lipid systems. Journal of the American Oil Chemists Society, 74, 1309± 1315. FREEMANTLE, M. (1995). Natural products and polymers. In: Chemistry in Action (Ed. Freemantle, M.), Macmillan Press Ltd, London. FRIGG, M., PRABUCKI, A.L. and RUHDEL, E.U. (1990). Effect of dietary vitamin E levels on oxidative stability of trout fillets. Aquaculture, 84, 145±158. GIESEG, S.P., CUDDIHY, S., HILL, J.H. and DAVISON, W. (2000). A comparison of plasma vitamin C and E levels in two Antarctic and two temperate water fish species. Journal of Comparative Biochemistry and Physiology, 125, 371±378. GORDON, D.T. and RATLIFF, V. (1992). The implications of Omega 3 fatty acids in Human Health. In: Advances in Seafood Biochemistry: Composition and Quality (Eds. Flick Jr., G.J. and Martin R.E.), Techomic Publishing Company, Pennsylvania. GOTTENBOS, J.J. (1985). In: The Role of Fats in Human Nutrition (Eds. Padley, F.B. and Podmore, J.), Ellis Horwood, Chichester. GU, L. and WENG, X. (2001). Antioxidant activity and components of Salvia plebeian R.Br. ± a Chinese herb. Food Chemistry, 73, 299±305. GUNSTONE, F.D. and NORRIS, F.A. (1982). The structure of fatty acids and lipids. In: Lipids in Foods Chemistry, Biochemistry and Technology. Pergamon Press, New York. GURR, M.I. (1984). Determination of the amounts and types of fats in foods. In: Role of fats in Food and Nutrition (Ed. Gurr, M.I.). Elsevier Applied Science Publishers, London. HAARD, N.F. and SIMPSON, B.K. (2000). Seafood Enzymes ± Utilisation and Influence on Postharvest Seafood Quality. Marcel Dekker, New York. HAMRE, K. and LIE, O. (1995). -Tocopherol levels in different organs of Atlantic salmon (Salmo salar L.) ± effect of smoltification, dietary levels of n-3 polyunsaturated fatty acids and vitamin E. Journal of Comparative Biochemistry and Physiology, 111, 547±554.

Physical and chemical properties of lipid by-products from seafood waste

39

and KEAY, J.N. (1972). Seasonal variations in the chemical composition of Cornish mackerel, Scomber scombrus (L), with detailed reference to the lipids. Journal of Food Technology, 7, 125±137. HARRIS, P. and TALL, J. (1994). Substrate specificity of mackerel flesh lipoxygenase. Journal of Food Science, 59, 504±506. HAWRYSH, Z.J. (1990). Stability of Canola Oil. In: Canola and Rapeseed ± Production, Chemistry, Nutrition and Processing Technology (Ed. Shahidi, F.). Van Nostrand Reinhold, New York. HEARN, T.L., SGOUTAS, S.A., HEARN, J. and SGOUTAS, D.S. (1987). Polyunsaturated fatty acids and fat in fish flesh for selecting species for health benefits. Journal of Food Science, 52, 1209±1211. HEDGES, N. (2002). Maintaining the quality of frozen fish. In: Safety and Quality Issues in Fish Processing (Ed. Bremner, H.A.). Woodhead Publishing Ltd, Cambridge. HERRERA, E. (2002). Implications of dietary fatty acids during pregnanacy on placental, fetal and postnatal development ± a review. Placenta, 23, S9±S19. HIRATA, F., SAEKI, N.M, KAWASAKI, K., OOIZUMI, T. and MOTOE, K. (1993). Recovery of fish oil from the manufacturing process of highly nutritional fish meat for foodstuffs from sardines. Nippon Suisan Gakkaishi, 59, 111±116. HJALTASON, B. (1990). New products, processing possibilities and markets for fish oil. In: Proceedings of the International By-products Conference, Anchorage, Alaska, USA, p131. HODGE, L., SALOME, C.M., PEAT, J.K., HABY, M.M., XUAN, W. and WOOLCOCK, A.J. (1996). Consumption of fish oil and childhood asthma risk. Medical Journal of Australia, 164, 137±140. HONG, L.C., LEBLANC, E.L., HAWRYSH, Z.J. and HARDIN, R.T. (1996). Quality of Atlantic mackerel (Scomber scrombus L.) fillets during modified storage. Journal of Food Science, 61, 646. HORROCKS, L.A. and YEO, Y.K. (1999). Health benefits of docosahexaenoic acid (DHA). Pharmacologial Research, 40, 211±225. HULTIN, H.O. (1992). Lipid oxidation in fish muscle. In: Advances in Seafood Biochemistry: Composition and Quality (Eds. Flick, Jr., G.J. and Martin, R.E.), Techomic Publishing Company, Pennsylvania. HWANG, K.T. and REGENSTEIN, J.M. (1993). Characteristics of mackerel mince lipid hydrolysis. Journal of Food Science, 58, 79±83. ICEKSON, I., DRABIN, V., AIZENDORF, S. and GELMAN, A. (1998). Lipid oxidation levels in different parts of the mackerel, Scomber scombrus. Journal of Aquatic Food Product Technology, 7, 17±29. INGEMANSSON, T., PETTERSSON, A. and KAUFMANN, P. (1993). Lipid hydrolysis and oxidation related to astaxanthin content in light and dark muscle of frozen stored rainbow trout (Oncorhynchus mykiss). Journal of Food Science, 58, 513±518. INGEMANSSON, T., KAUFMANN, P. and EKSTRAND, B. (1995). Multivariant evaluation of lipid hydrolysis and oxidation data from light and dark muscle of frozen stored rainbow trout. Journal of Agricultural Food Chemistry, 43, 2046±2052. ITO, N. and HIROSE, N. (1989). Antioxidants ± carcinogenic chemopreventative properties. Advances in Cancer Research, 53, 247±302. ITO, N., HIROSE, M., FUKSHIMA, S., TSUDA, H., SHIRAI, T. and TATEMATSU, M. (1986). Studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogens. Food Chemical Toxicology, 24, 1099±1102. IWAMOTO, H. and SATO, G. (1986). Production of EPA by freshwater unicellular algae. HARDY, R.

40

Maximising the value of marine by-products

Journal of American Oil Chemists' Society, 63, 434±438. and HOPKINS, J.S. (1988). Comparison of pond raised and wild red drum (Sciaenops ocellatus) with respect to proximate composition, fatty acid profiles, and sensory evaluations. Journal of Food Science, 53, 286±287. JAMES, M.J. and CLELAND, L.G. (1996). Dietary polyunsaturated fats and inflammation. Proceedings of the Nutrition Society of Australia, 20, 71±77. JENSEN, C., BIRK, E., JOKUMSEN, A., SKIBSTED, L. and BERTELSEN, G. (1998). Effect of dietary levels of fat, -tocopherol and astaxanthin on colour and lipid oxidation during storage of frozen rainbow trout (Oncorhynchus mykiss) and during chill storage of smoked trout. Z Lebensm Unters Forsch, 207, 189±196. KAMAL-ELDIN, A. and YANISHLIEVA, N.V. (2002). N-3 fatty acids for human nutrition: stability considerations. European Journal of Lipid Science Technology, 104(12), 825±836. KANENIWA, M., MIAO, S., YUAN, C.H., ILDA, H. and FUKUDA, Y. (2000). Lipid components and enzymatic hydrolysis of lipids in muscle of Chinese freshwater fish. Journal of American oil Chemists' Society, 77, 825±830. KATIKOU, P., HUGHES, S.I. and ROBB, D.H.F. (2001). Lipid distribution within Atlantic salmon (Salmo salar) fillets. Journal of Aquaculture, 202, 89±99. KENNETH, C. (1986). Biological effects of fish oil in relation to chronic diseases. Lipids, 21, 731±732. KHAYAT, A. and SCHWALL, D. (1983). Lipid oxidation in seafood. Food Technology, 37, 130±140. KHERUNNISA, R.B., QADRI, S., TOUHEED, A. and VIQARUDDIN, A. (1996). Fatty acid profile of four marine fish species from Karachi coastal waters. Journal of Chemistry Society of Pakistan, 18, 44±47. KIESSLING, A., PICKOVA, J., JOHANSSON, L., ASGARD, T., STOREBAKKEN, T. and KIESSLING, K.H. (2001). Changes in fatty acid composition in muscle and adipose tissue of farmed rainbow trout (Oncorhynchus mykiss) in relation to ration and age. Journal of Food Chemistry, 73, 271±284. KINSELLA, J.E. (1986). Food components with potential therapeutic benefits: The n-3 polyunsaturated fatty acids of fish oils. Food Technology, 40, 89±97. KLEMETSEN, A. (1982). Food and feeding habits of cod (Gadus morhua) in Balsfjorden, northern Norway, during a one year period. Journal of Conservation and International Exploration of the Sea, 38, 18±27. KOLANOWSKI, W., SWIDERSKI, F. and BERGER, S. (1999). Possibilities of fish oil application for food products enrichment with omega-3 PUFA. International Journal of Food Science and Nutrition, 50, 39±49. KULAS, E., OLSEN, E. and ACKMAN, R.G. (2002). Effect of -, - and -tocopherol on the distribution of volatile secondary oxidation products in fish oil. European Journal of Lipid Science Technology, 104, 520±529. LAWSON, H. (1995). Food Oils and Fats: Technology, Utilisation and Nutrition. Chapman and Hall, London. LEAF, A. and WEBER, P.C. (1988). Cardiovascular effects on n-3 fatty acids. The New England Journal of Medicine, 318, 549±557. LEVINE, A.S. and LABUZA, T.P. (1990). Food systems: the relationship between health and food science/technology. Environmental Health Perspectives, 86, 233±238. LOVE, R. (1997). Biochemical dynamics and the quality of frozen fish. In: Fish Processing Technology (Ed. Hall, G.M.). Blackie Academic and Professional, London. JAHNCKE, M.L., HALE, M.B., GOOCH, J.A.

Physical and chemical properties of lipid by-products from seafood waste

41

and LANIER, T.C. (1991). Carbohydrates as cryoprotectants for meat and surimi. Food Technology, 45, 150±159. MACDONALD, G.A., LANIER, T.C. and CARVAJAL, P.A. (2000). Stabilisation of Proteins. In: Surimi and Surimi Seafood (Ed. Park, J.W.), Marcel Dekker, New York. MALONE, C., SHAW, N.B. and KERRY, J.P. (2004). Effect of season on vitamin E, fatty acid profile, and nutritional value of fish by-products from cod, saithe, ling and haddock species caught in southern Irish coastal waters. Journal of Aquatic Food Product Technology, 13, 127±149. MARSHELL, W. (1974). Health foods, organic foods, natural foods. Food Technology, 28, 50±56. MATSUMOTO, J.J. and NOGUCHI, S.F. (1992). Cryostabilisation of protein in surimi. Surimi Technology (Eds. Lanier, T.C. and Lee, C.M.), Marcel Dekker, New York. MCCARTHY, T., KERRY, J.P., KERRY, J.F., LYNCH, P.B. and BUCKLEY, D.J. (2001a). Assessment of the antioxidant potential of natural food and plant extracts in fresh and previously frozen pork patties. Meat Science, 57, 177±184. MCCARTHY, T., KERRY, J.P., KERRY, J.F., LYNCH, P.B. and BUCKLEY, D.J. (2001b). Evaluation of the antioxidant potential of natural food/plant extracts as compared with synthetic antioxidants and vitamin E in raw and cooked pork patties. Meat Science, 57, 45± 52. MEHL, S. (1986). Stomach contents of North East Arctic cod and possible changes in the diet the last years. ICES, CM G:29, p24. MENDEZ, E. and GONZALEZ, R. (1997). Seasonal changes in chemical and lipid composition of fillets of Southwest Atlantic hake (Merluccius hubbsi). Journal of Food Chemistry, 59, 213±217. MESSAGER, J.L., STEPHAN, G., QUENTEL, C. and BAUDIN LAURENCIN, F. (1992). Effects of dietary oxidized fish oil and antioxidant deficiency on histopathology haematology, tissue and plasma biochemistry of sea bass (Dicentrarchus labrax). Aquatic Living Resources, 5, 205±214. MILLAR, J.A. and WAAL-MANNING, H.J. (1992). Fish oil in treatment of hypertension. New Zealand Medical Journal, 105, 155. MILO, C. and GROSH, W. (1993). Changes in the odorants of boiled trout as affected by the storage of the raw material. Journal of Agriculture and Food Chemistry, 41, 2076± 2081. MIYASHITA, K. and TAKAGI, T. (1986). Study on the oxidative rate and pro-oxidant activity of free fatty acids. Journal of American Oil Chemists' Society, 63, 1380±1384. MOFFAT, C.F., MCGILL, A.S., HARDY, R. and ANDERSON, R.S. (1993). The production of fish oils enriched in polyunsaturated fatty acids containing triglycerides. Journal of American Oil Chemists' Society, 70, 133±138. MOORE, S.R. and MCNEILL, G.P. (1996). Production of triglycerides enriched in long chain n3 polyunsaturated fatty acids from fish oil. Journal of American Oil Chemists' Society, 73, 1409±1414. MOREIRA, A.B., VISENTAINER, J.V., DE SOUZA, N.E. and MATSUSHITA, M. (2001). Fatty acids profile and cholesterol of three Brazilian Brycon freshwater fish. Journal of Food Composition and Analysis, 14, 565±574. MORRIS, R.J. and CULKIN, F. (1989). Fish. In: Marine Biogenic Lipids, Fats and Oils (Ed. Ackman, R.G.). CRC Press, Boca Raton, FL. MORRISSEY, M.T., PARK, J.W. and HUANG, L. (2000). Surimi processing waste, its control and utilisation. In: Surimi and Surimi Seafood (Ed. Park, J.W.), Marcel Dekker, New York. MACDONALD, G.A.

42

Maximising the value of marine by-products

(1985). Seafood Nutrition. Van Nostrand Reinhold, New York. and ACKMAN, R.G. (1990). Nutrients and chemical residues in one to two pound Mississippi farm-raised channel catfish (Ictalurus punctatus). Journal of Food Science, 55, 954±958. NIELSEN, H. (1992). N-3 polyunsaturated fish fatty acids in a fish oil supplemented bread. Journal of Science and Food Agriculture, 59, 559±562. NJINKOUE, J.M., BARNATHAN, G., MIRALLES, J., GAYDOU, E.M. and SAMB, A. (2002). Lipids and fatty acids in muscle, liver and skin of three edible fish from the Senegalese coast: Sardinella maderensis, Sardinella aurita and Cephalopholis taeniops. Journal of Comparative Biochemistry and Physiology. 131, 395±402. O'SULLIVAN, A., MAYR, A., SHAW, N.B., MURPHY, S.C. and KERRY, J.P. (2005). Use of natural antioxidants to stabilize fish oil systems. Journal of Aquatic Food Product Technology, 14, 75±94. OHSHIMA, T., YANKAH, V., USHIO, H. and KIOZUMI, C. (1998). Antioxidising potentials of BHA, BHT, TBHQ, Tocopherol, and oxygen absorber incorporation in a Ghanaian fermented fish product. Process-induced Chemical Changes in Food, 15, 181±188. OLSEN, Y. and SKJERVOLD, H. (1991). Impact of latitude on n-3 fatty acids in wild Atlantic salmon. Omega-3 news, VI, 1±4. ORBAN, E., LENA, G.D., NEVIGATO, T., CASINI, I., SANTARONI, G., MARZETTI, A. and CAPRONI, R. (2002). Quality characteristics of Sea Bass intensively reared and from lagoon as affected by growth conditions and the aquatic environment. Journal of Food Science, 67, 542±545. OSMAN, H., SURIAH, A.R. and LAW, E.C. (2001). Fatty acid composition and cholesterol content of selected marine fish in Malaysian waters. Journal of Food Chemistry, 73, 55±60. OZOGUL, F., TAYLOR, K.D.A., QUANTICK, P. and OZOGUL, Y. (2000). Chemical, microbiologial and sensory evaluation of Atlantic herring (Clupea harengus) stored in ice, modified atmosphere and vacuum pack. Food Chemistry, 71, 267 PAIGE, J.A., LIAO, R., HAJJAR, R.J., FOISY, R.L., CORY, C.R., O'BRIEN, P.J. and GWATHMEY, J.K. (1996). Effects of high omega-3 fatty acid diet on cardiac contractile performance in Oncorhynchus mykyss. Cardiovascular Research, 31, 249±262. PALSSON, O.K. (1983). The feeding habitats of demersal fish species in Icelandic waters. Rit Fiskideilar, 7, 60. PARK, J.W. (1994). Cryoprotection of muscle proteins by carbohydrates and polyalcohols ± a review. Journal of Aquatic Food Product Technology, 3, 23±41. PARK, J.W., LIN, T.M. and YONGSAWATDIGUL, J. (1997). New developments in surimi and surimi seafood. Food Review International, 13, 577±610. PIRINI, M., GATTA, P.P., TESTI, S., TRIGARI, G. and MONETTI, P.G. (2000). Effect of refrigerated storage on muscle lipid quality of sea bass (Dicentrarchus labax) fed on diets containing different levels of vitamin E. Food Chemistry, 68, 289±293. RAE, B.B. (1967). The food of cod in the North Sea and on the West of Scotland grounds. Marine Research, 1, 67. RAFFLENBEUL, W. (2001). Fish for a healthy heart. European Journal of Lipid Science and Technology, 103, 315±317. RATZ, H.J. and LLORET, J. (2002). Variation in fish condition between Atlantic cod (Gadus morhua) stocks, the effect on their productivity and management implications. Fisheries Research, 1436, 1±12. REEDY, N.R., SCHREIBER, C.L., BUZARD, K.S., SKINNER, G.E. and ARMSTRONG, D.J. (1994). Shelf NETTLTEON, J.A.

NETTLETON, J.A., ALLEN, W.H., KLATT, L.V., RATNAYAKE, W.M.N.

Physical and chemical properties of lipid by-products from seafood waste

43

life of fresh tilapia fillets packaged in high barrier film with modified atmospheres. Journal of Food Science, 59, 260±264. REFSGAARD, H.F., BROCKHOFF, P.B. and JENSEN, B. (1998). Biological variation of lipid constituents and distribution of tocopherols and astaxanthin in farmed Atlantic salmon (Salmo salar). Journal of Agriculture and Food Chemistry, 46, 808±812. ROEDIG-PENMAN, A. and GORDON, M. (1997). Antioxidant properties of catechins and green tea extracts in model food emulsions. Journal of Agricultural and Food Chemistry, 45, 4267±4270. ROSE, D.P. and CONNOLLY, J.M. (1993). Effects of dietary omega-3 fatty acids on human breast cancer growth and metastases in nude mice. Journal of the National CancerInstitute, 85, 1743±1747. RUFF, N., FITZGERALD, R.D., CROSS, T.F., TEURTRIE, G. and KERRY, J.P. (2002a). Slaughtering method and dietary -tocopheryl acetate supplementation affect rigor mortis and fillet shelf-life of turbot Scophthalmus maximus L. Aquaculture Research, 33, 703± 714. RUFF, N., FITZGERALD, R.D., CROSS, T.F. and KERRY, J.P. (2002b). Fillet shelf-life of Atlantic halibut Hippoglossus hippoglossus L. fed elevated levels of -tocopheryl acetate. Aquaculture Research, 33, 1059±1071. RUFF, N., FITZGERALD, R.D., CROSS, T.F. and KERRY, J.P. (2002c). Comparative composition and shelf-life of wild and cultured turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus). Aquaculture International, 10, 241±256. RUFF, N., FITZGERALD, R.D., CROSS, T.F. and KERRY, J.P. (2004a). Shelf life evaluation of modified atmosphere and vacuum packaging fillets of Atlantic halibut (Hippoglossus hippoglossus), following dietary -tocopheryl acetate supplementation. Journal of Aquatic Food Product Technology, 12, 23±37. RUFF, N., FITZGERALD, R.D., CROSS, T.F., LYNCH, A. and KERRY, J.P. (2004b). Distribution of tocopheryl in fillets of turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus) following dietary -tocopheryl acetate supplementation. Aquaculture Nutrition, 10, 75±81. SAGLIK, S. and IMRE, S. (2001). Omega-3 fatty acids in some fish species from Turkey. Journal of Food Science. 66, 210±212. SAITO, H., YAMASHIRO, R., ALASALVAR, C. and KONNO, T. (1999). Influence of diet on fatty acids of three subtropical fish, subfamily caesioninae (Caesio diagramma and C.tile) and family siganidae (Siganus canaliculatus). Lipids, 34, 1073±1082. SARGENT, J., HENDERSON, R.J. and TOCHER, D.R. (1989). The lipids. In: Fish Nutrition (Ed. Halver, J.E.), Academic Press, San Diego. SARGENT, J., BELL, G., MCEVOY, L., TOCHER, D. and ESTEVEZ, A. (1999). Recent developments in the essential fatty acid nutrition of fish. Journal of Aquaculture, 177, 191±199. SATOH, S., POE, W.E. and WILSON, R.P. (1989). Effect of dietary n-3 fatty acids on weight gain and liver polar fatty acid composition of channel catfish. Journal of Nutrition, 119, 23±28. SETO, A., WANG, L. and HESSELITIME, C.W. (1984). Culture conditions affect eicosapentaenoic acid content of Chlorella minutissma. Journal of American Oil Chemists' Society, 61, 892±894. SHAHIDI, F. and WANASUNDARA, P.D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32, 67±103. SHAHIDI, F. and WANASUNDARA, U.N. (2001). Stability of canola oil and its stabilisation by natural antioxidants. Canola Council of Canada, 12th project report, 92±102.

44

Maximising the value of marine by-products

and SYNOWIECKI, J. (1991). Chemical composition and nutritional value of processing discards (Gadus morhua). Food Chemistry, 42, 145±151. SHIMADA, Y., MARUYAMA, K., SUGIHARA, A., MORIYAMA, S. and TOMINAGA, Y. (1997). Purification of docosahexaenoic acid from tuna oil by a two step enzymatic method; hydrolysis and selective esterification. Journal of American Oil Chemists' Society, 74, 1441±1446. SHIRAI, N., SUZUKI, H., SHIGERU, T., EHARA, H. and WADA, S. (2002). Dietary and seasonal effects on the dorsal meat lipid composition of Japanese (Silurus asotus) and Thai catfish (Clarias macrocephalus and hybrid Clarias macrocephalus and Clarias galipinus). Journal of Comparative Biochemistry and Physiology, 132, 609±619. SIKORSKI, Z.E., KOLAKOWSKA, A. and PAN, B.S. (1990). The nutritive compostion of the major groups of marine food organisms. In: Seafood: Resources, Nutritional Composition and Preservation (Ed. Sikorski, Z.E.). CRC Press, Boca Raton, FL. SILVA, J.L. and WHITE, T.D. (1994). Bacteriological and colour changes in modified atmosphere packaged refrigerated channel catfish. Journal of Food Protection, 57, 715±719. SILVA, J.L., HARKNESS, E. and WHITE, T.D. (1993). Residual effect of CO2 on bacterial counts and surface pH of channel catfish. Journal of Food Protection, 56, 1051±1053. SIMOPOULOS, A.P. (1991). Omega-3 fatty acids in health and disease and in growth and development, a review. Americam Journal of Clinical Nutrition, 54, 438±463. STEPHAN, G., GUILLAUME, J. and LAMOUR, F. (1995). Lipid peroxidation in turbot (Scophthalmus maximus) tissue: effect of dietary vitamin E and dietary n-6 or n3 polyunsaturated fatty acids. Aquaculture, 130, 251±268. SHAHIDI, F., NACZK, M., PEGG, R.B.

STIER, R.F., BELL, L., ITO, K.A., SHAFER, B.D., BROWN, L.A., SEEGER, M.L., ALLEN, B.H., PORCUNA,

M.N. and LERKE, P.A. (1981). Effect of modified atmosphere storage on C. botulinum toxigenesis and the spoilage microflora of salmon fillets. Journal of Food Science, 46, 1639±1642. STOICK, S., GRAY, J., BOOREN, A. and BUCKLEY, D. (1991). Oxidative stability of restructured beef steaks processed with oleoresin rosemary, tertiary butylhydroquinone, and sodium tripolyphosphate. Journal of Food Science, 56, 597±600. SUN, T., PIGOTT, G.M. and HERWIG, R.P. (2002). Lipase assisted concentration of n-3 polyunsaturated fatty acids from viscera of farmed Atlantic salmon (Salmo salar L.). Journal of Food Science, 67, 130±136. SUZUKI, H., OKAZAKI, K., HAYAKAWA, S., WADA, S. and TAMURA, S. (1986). Influence of commercial dietary fatty acids on polyunsaturated fatty acids of cultured freshwater fish and comparison with those of wild fish of the same species. Journal of Agricultural and Food Chemistry, 34, 58±60. TANG, S., KERRY, J.P., SHEEHAN, D., BUCKLEY, D.J. and MORRISSEY, P.A. (2001). Antioxidative effect of added tea catechins on susceptibility of cooked red meat, poultry and fish patties to lipid oxidation. Food Research International, 34, 651±657. THE DANISH MINISTRY OF HEALTH. `General Publication on Guidelines on General Nutrition for the Danish Population' National Food Agency. TOAMA, M. (1990). Study on the influence of freezing rate on lipid oxidation in fish (salmon) and chicken breast muscles. International Journal of Food Science and Technology, 25, 718±721. TOYODA, K., KIMURA, I., FUJITA, T., NOGUCHI, S.F. and LEE, C.M. (1992). Surimi Manufacturing

Physical and chemical properties of lipid by-products from seafood waste

45

from whitefish. Surimi Technology (Eds. Lanier, T.C. and Lee, C.M.), Marcel Dekker, New York. TRIGARI, G., PIRINI, M., VENTRELLA, V., PAGLIARANI, A. and TROMBETTI, F. (1992). Lipid composition and mitochondrial respiration in warm and cold adapted sea bass. Lipids, 27, 371±377. TSIMIDOU, M., PAPAVERGOU, E. and BOSKOU, D. (1995). Evaluation of oregano antioxidant activity in mackerel oil. Food Research International, 28, 431±433. UAUY, R. and VALENZUELA, A. (2000). Marine oils: the health benefits of n-3 fatty acids. Nutrition, 16(7/8), 680±684. ULBRICHT, T.L.V. and SOUTHGATE, D.A.T. (1991). Coronary heart disease: seven dietary factors. The Lancet, 338, 985±994. UNDELAND, I., EKSTRAND, B. and LINGNERT, H. (1998). Lipid oxidation in herring (Clupea harengus) light muscle, dark muscle and skin stored separately or as intact fillets. Journal of American Oil Chemists' Society, 75, 581±589. UNDELAND, I., HALL, G. and LINGNERT, H. (1999). Lipid oxidation in fillets of herring (Clupea harengus) during ice storage. Journal of Agricultural and Food Chemistry, 47, 524±532 VALDIMARSSON, G. and JAMES, D. (2001). World fisheries-utilisation of catches. Ocean and Coastal Management, 44, 619±633. VENUGOPAL, V. (1992). Mince from low-cost fish species. Trends in Food Science and Technology, 3, 2±5. VENUGOPAL, V. (1997). Functionality and potential applications of thermostable water dispersions of fish meat. Trends in Food Science and Technology, 8, 271±276. VIARENGO, A., ABELE-OESCHGER, D. and BURLANDO, B. (1998). Effects of low temperature on pro-oxidant processes and antioxidant defence systems in marine organisms. In: Cold Ocean Physiology (Eds. Portner, H.Q. and Playle, R.C.), Cambridge University Press, Cambridge. VLIEG, P. and BODY, D.B. (1988). Lipid contents and fatty acid composition of some New Zealand freshwater finfish and marine finfish, shellfish and roes. New Zealand Journal of Marine Freshwater Research, 22, 151. WAAGBO, R., SANDNES, K., TORRISSEN, O.J., SANDVIN, A. and LIE, O. (1993). Chemical and sensory evaluation of fillets from Atlantic salmon (Salmo salar) fed three levels of n-3 polyunsaturated fatty acids at two levels of Vitamin E. Food Chemistry, 46, 361±366. WANASUNDARA, U.N. and SHAHIDI, F. (1998). Antioxidant and pro-oxidant activity of green tea extracts in marine oils. Food Chemistry, 63, 335±342. WATANABE, T., MURASE, T. and SAITO, H. (1995). Specificity of fatty acid composition of highly migratory fish. A comparison of docosahexaenoic acid content in total lipids extracted in various organs of bonitio (Euthynnus pelamis). Journal of Comparative Biochemistry and Physiology, 111, 691±695. WODTKE, E. (1981). Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (Ciprinus carpio L.). Biochemistry and Biophysics. Acta 640, 698±709. XIN, F. and SHUN, W. (1993). Enhancing the antioxidant effect of -tocopherol with rosemary in inhibiting catalyzed oxidation caused by iron (II) and hemoprotein. Food Research International, 26, 405±411. YAMAMOTO, Y., MAITA, N., FUJISAWA, A., TAKAHIMA, J., ISHII, Y. and DUNLAP, W.C. (1999). A new vitamin E (alpha-tocomonoenol) from eggs of the Pacific salmon

46

Maximising the value of marine by-products

Oncorhynchusketa. Journal of Natural Products, 62, 1685±1687. and DUNLAP, W.C. (2001). An unusual vitamin E constituent provides antioxidant protection in marine organisms adapted to coldwater environments. Proceedings of the National Academy of Science, USA, 98. 13144±13148. YANISHLIEVA, N.V. and MARINOVA, E.M. (2001). Stabilisation of edible oils with natural antioxidants. European Journal of Lipid Science Technology, 103, 752±767. ZAMARRO, J. (1985). On food of Gadus morhua in the Arctic Ocean. ICES CM G:3, p.12. YAMAMOTO, Y., FUJISAWA, A., HARA, A.

3 On-board handling of marine byproducts to prevent microbial spoilage, enzymatic reactions and lipid oxidation E. Falch, M. Sandbakk and M. Aursand, SINTEF Fisheries and Aquaculture, Norway

3.1

Introduction

Globally, more than 100 million tonnes of fish and shellfish are caught annually with 75% being utilised for human consumption (FAO, 2004). Deterioration of the biomass begins immediately post mortem leading to quality loss and limitations in the possible end products generated from the raw material. Optimal handling procedures, from the catching to the product being conserved, are necessary for reducing the rate of the spoilage and thereby maintaining the fresh quality that is harvested from the sea. The quality of the raw material will be crucial for pricing of the end products. Fish are caught by different fishing vessels operating in different areas of the sea, such as ocean and coastal trawlers, Danish seine, and net fishing vessels. While the coast fleet generally deliver on shore on a daily basis, the ocean trawlers may be out for weeks before unloading the catch. These vessels are generally freezing the fish round or gutted; however, some of the vessels contain an on-board processing plant producing fish fillet as the main product. These two groups of vessels, divided into the coastal fleet and the ocean trawlers, will bring up different possibilities and require different solutions.

3.2

Deterioration of marine biomass

Microbial safety is crucial for all fish product intended for human or animal consumption. Fish as a raw material is highly perishable and the action of

48

Maximising the value of marine by-products

microbes might spoil and make it into waste, if it is not satisfactorily preserved. Deterioration of the biomass is also due to lipid oxidation and reactions catalysed by enzymes. The acceptable levels of lipid and protein hydrolysis are highly product specific, depending on the end use of the products. 3.2.1 Microbial activity The fish biomass is generally a good substrate for microbial growth. While the muscle tissue of fresh and healthy fish is sterile (Liston, 1980), some of the byproducts such as intestines and gills contain large numbers of bacteria (Sikorski et al., 1990). It is therefore important to take some precautions such as to inactivate these microbes or to separate these fractions from the more valuable parts. The raw materials and processed products might also be contaminated by the microbial flora from people, equipment or other environmental conditions which also involve a risk of introducing harmful pathogens. By-products for consumption should be produced hygienically after a gentle first handling of the fish. Bacteria may produce degradation enzymes such as lipases, proteases, peptidases and reductases (e.g. TMAO reductase) resulting in spoilage of the byproducts. Furthermore, microbes are responsible for the formation of certain volatile bases and biogenic amines. Microbial spoilage of fish is excellently reviewed by Gram and Huss (1996). 3.2.2 Enzymatic spoilage In live fish the enzymes are regulated by different biochemical systems, but immediately post mortem, these systems are inactivated and the deterioration begins, leading to the development of off-flavour, off-taste and release of nutrients enabling microbial growth. The hydrolysis of fish by-products is mainly caused by proteases, peptidases and lipases and the activity of these enzymes is reported to be organ- and species-specific (Hidalgo et al., 1999). The activity also varies among different organs with higher activity generally found in the viscera and the intestinal tract in particular (Hildago et al., 1999; Sùvik and Rustad, 2005a,b). The digestive processes in fish are less known than in mammals, however, the digestive enzymes are reported to be qualitatively similar (Hildago et al., 1999). Post-mortem enzymatic reactions can be catalysed by both endogenous and bacterial enzymes, however, it is difficult to precisely distinguish between the origin of the enzymes (Sikorski et al., 1990). The major enzymes of fish gut are proteases (pepsin, trypsin and chymotrypsin), lipases and carbohydrases (Mukundan et al., 1986). The proteolysis degrades proteins into smaller peptides and amino acids. The molecular weight is important for proteins to act as functional ingredients in food or feed applications. Minimising or controlling the proteolysis is therefore important whether the end products are intended for food or feed (Falch et al., 2000). The uncontrolled proteolysis of fish viscera may also lead to the formation of bitter peptides that affect the sensory properties of the end products (Dauksas et al., 2004). Lipolytic activity

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

49

leads to formation of free fatty acids which affect the sensory attributes and the oxidative shelf life of the lipids. Phospholipids are hydrolysed most rapidly followed by triacylglycerols, cholesterol esters and wax esters (Sikorski et al., 1990). Fish lipases are also shown to specifically hydrolyse polyunsaturated fatty acids (Lie et al., 1987) and, since free fatty acids are shown to oxidise at a faster rate compared to their esterified counterparts (Labuza, 1971; Shewfelt, 1981) it also affects the nutritional value of the marine lipids. The level of free fatty acids is one of the main parameters classifying the quality of marine oils. Other lipolytic activity in fish by-products caused by hydrolases (wax ester hydrolases and cholesteryl ester hydrolases) and esterases are not widely studied; however, such activity has been demonstrated in different fish species (Tocher, 2003). Esterification of cholesterol into cholesterol esters has been reported during chilled storage of cod gonads (Falch et al., 2005). Lipases in teleost fishes are discussed in a comprehensive review on lipid metabolism in fish in general by Tocher (2003) while the literature describing the proteolytic activity in fish is generally divided into papers on individual species or families of fish. 3.2.3 Lipid oxidation The rate of lipid oxidation in fish biomass is generally high due to the levels of polyunsaturated fatty acids. Lipid oxidation leads to the formation of secondary products such as aldehydes, ketones and acids that affect the sensory and nutritional properties of lipids. Reaction products from oxidation of fish oil are reported (Olsen et al., 2005) and some of these reaction products are detectable by sensory analysis at levels as low as 10ÿ5±10ÿ2 g/g oil (KulaÊs et al., 2003). With no conservation, fish by-products will generally be rejected based on spoilage by bacteria or enzymes long before lipid oxidation reaches an unacceptable level. Lipid oxidation is, however, the main cause of shelf-life limitation of marine oil, so the lipid oxidation should therefore be taken into account when producing marine lipids and products with high lipid content.

3.3

Handling and sorting

The coast fleet generally deliver the catch with or without refrigeration onboard. The fishermen deliver the fish round or gutted, depending on factors such as fish species, statutory regulations, temperature and season and whether the by-products should be utilised or not. The limited area on-board vessels, is in many cases reducing the possibilities for handling and utilisation of by-products. A system has been developed for small coastal vessels in order to easily sort the individual by-products. This recently commercialised system is illustrated in Fig. 3.1 and enables sorting of head, liver and roe, among others, from whitefish into thick plastic bags placed in slurry ice. Loading of round fish, both fresh and frozen, has received growing interest and is under investigation as a possible

50

Maximising the value of marine by-products

Fig. 3.1 A system for sorting and refrigerating by-products in a coastal vessel. The fishermen (one or two) are handling cod by-products and sort them into head, liver, roe, milt, stomachs, etc. Source: Reprinted with the permission from Rubin (www.rubin.no).

solution (Akse, 2002), where the whole processing can be done on-shore, under controlled conditions. Furthermore, it will be possible to process raw material from several vessels at the same place, and thereby enable a more economic and better utilised production facility. The ocean trawlers are generally freezing the fish round or gutted; however, some of the vessels contain on-board processing plants producing fish fillet as the main product. By-products are generated during a typical filleting operation and suggested preservation technology is shown in Fig. 3.2. Whitefish such as gadiform species deliver a wide range of possible products that can be sorted out, depending on the processing; these are head, liver, roe, milt, stomach, skin, bones, and trimmings. The main processing steps are bleeding, head cutting, gutting and filleting and the technology chosen to preserve the by-products is highly dependent on the end product. Some of the fractions are more valuable than others and need particular care. These are, for example, liver, roe and milt from white fish since they may be used non-processed as consumer products. In vessels utilising specific by-products such as liver, roe, milt, and stomach, the gutting operation has traditionally been done manually in order to keep the inner fractions intact. Focus has recently been put into the development of more gentle automatic processing machines (Fig, 3.3) and the visceral fractions are now successfully recovered automatically from whitefish (Baader 444) and

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

Fig. 3.2

51

By-products generated during filleting of gadiform species with suggested methods of preservation and bulk production.

salmon (Baader 142) (Einarsson, 2004). The new and gentle gutting operations now available enable differentiation between quality levels and end uses and thereby yield individual optimal handling and preservation of the different fractions. Gentle and non-destructive gutting also makes it possible to get reasonable prices for the most valuable parts. An automatic process to utilise individual by-products should satisfy the following three stages: 1) gentle removal of viscera enabling preservation of each fraction undamaged; 2) recognition and automatic separation of specific fractions; and 3) automatic sorting of the separated fractions.

Fig. 3.3 A recently developed gutting machine with gentle removal of visceral components. Source: Reprinted with permission from BAADER.

52

Maximising the value of marine by-products

The visceral fractions obtained from the gentle gutting machine might be automatically sorted using imaging technology. This technology is under development. Tests on images from cod by-products indicate that the proposed method has the potential to automatically recognise cod viscera components. The combination of automatic visual recognition and robotic technology to facilitate subsequent separation and sorting, have a lot of possibilities for use in the fish processing industry (Mathiassen et al., 2002). The system has to perform the separation at a speed that satisfies the need of processing. This work is brought forward in new projects with industrial partners, with an objective of a commercialised solution. However, it is necessary to get profitability in such concepts and the possibilities for obtaining relatively high prices for the end products are essential to defend this still expensive technology. Independent of fishing vessels, gentle handling during and after catch will retain the biological membranes and act as a physical barrier to biochemical deterioration until the products are conserved. Furthermore, the time of bleeding will affect the quality since the blood, in fish that are not bled, will diffuse into the muscle and visceral fractions and promote biochemical degradation such as lipid oxidation.

3.4

Conservation and stabilisation

Traditional methods of preservation might be divided into four different categories: refrigeration technology, heat preservation, dehydration (drying) and chemical preservation (Table 3.1). Based on the end purpose of the raw material; human consumption or feed, several different techniques of conservation and stabilisation can be employed, alone or in combination. Quality loss is an irreversible process and is also cumulative. This means that a quality loss from one stage of the line cannot be regained at later stages. It is crucial to reduce the possible quality losses to a minimum in all stages, from the very beginning. 3.4.1 Refrigeration technology Refrigeration technology including freezing and chilling are basic technologies offering stabilisation of the products, particularly on the matter of microbial growth but also autolysis, and lipid oxidation processes are reduced at lower temperatures. As early as 1797, natural ice was used for chilling of fish that was transported to the UK (Dellacasa, 1987). Later, various mechanical systems were developed, using several different approaches. For raw material that can be used directly as consumer products, such as roe, liver and stomach, proper chilling and freezing capacity are essential. Chilling is an operation where the temperature is lowered down to values between ÿ1 and ‡8ëC. The lower the storage temperature one can achieve, the longer the possible storage time. For fresh fish products the suitable temperature range is ÿ1 to ‡1ëC (Fellows, 2000). It is appropriate to consider these numbers as

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

53

Table 3.1 Effect of different preservation techniques on the main deterioration processes in by-products. A positive effect of the technique is marked Main deterioration processes

REFRIGERATION Chilling and freezing

Microbial growth

Autolysis

Lipid oxidation

X

X

X

1

X

2

HEAT PROCESSING

X

DEHYDRATION Drying

X

X

CHEMICAL PRESERVATION Lowering of pH Use of antioxidants Salting

X X

X

PROCESSING Sorting (prevent contamination) Separation of oil Use of inert gaseous or replacement of O2 Physical membranes intact

X X3 X X

X X3 X

X X X3 X X

1

Reduced growth. Inactivation suggested for cod by-products: 10 min, 70ëC (Sùvik, 2005). 3 Production of oil as soon as possible after catch will prevent microbial growth in the oil fraction (there is generally no microbial growth in pure oil). 2

relevant for the by-products as well. Storage life of fish products stored at different chilling temperatures has shown an increase in shelf life of up to several days if temperature is reduced by 2ëC (Rùyrvik, 1979), and the storage temperature during the first 24 hours has a major influence on the subsequent shelf life. As for freezing, a temperature of ÿ18ëC is considered to be the normal value (Fellows, 2000), but lower temperatures will add shelf life to the product. Some products are more demanding than others. In a Norwegian report from 1995 (Hardarson, 1995) frozen storage of cod liver, a product with extremely high lipid content, has been reported to be of little use to prevent lipolysis unless the temperature could be reduced to ÿ45ëC (Fig. 3.4). Normal storage temperatures, like ÿ18ëC gave the same level of free fatty acids as chilled storage. It is crucial that the catch is adequately chilled from when it is taken on board and that backbones and/or trimmings are chilled and kept under hygienic conditions throughout the processing, if the by-products are sources for minced products. For bulk products, like silage, oil or meal, the correct combination of temperature treatment (chilling of the catch, preheating of the raw material to the correct temperature) and processing aid (e.g., organic acid, commercial proteases, etc.) will give the possible directions for the end product and decide

54

Maximising the value of marine by-products

Fig. 3.4 Free fatty acids development during frozen storage of cod liver at different temperatures. Source: Reprinted with permission from Hardarson (1995).

whether the quality is acceptable for applications for human consumption, or used in low value applications (feed). There are several possibilities for chilling onboard the vessels: use of ice, RSW (mechanically refrigerated sea water), CSW (ice-chilled sea water), iceslurry, cold air and others. Achieving refrigeration demands electricity and this is usually produced with diesel engines. The horsepower of the engine will be the limitation for the possibility to produce refrigerating capacity. A major effort has been put into developing refrigeration technology for fishing vessels that demands less space and power consumption than traditional solutions (Wang and Wang, 2005). For smaller vessels in the coastal fleet, carrying ice produced on shore with them out to the fishing grounds is a possibility but for the trawlers, suitable equipment has to be placed on board. Using chilled sea water for the chilling of the catch offers several advantages over the use of ice (Kelman, 2001): 1) due to better heat transfer, the catch is cooled more rapidly; 2) less effort is required to stow and unload it; 3) less likelihood of fish being crushed or losing weight; and 4) in addition, sea water can be safely reduced in temperature to about ÿ1ëC without freezing the fish contained in it. This method is commonly in use within the aquaculture slaughtering industry as a suitable pre-chilling before further processing, and will offer good possibilities also for vessels that process on board.

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

55

A cold chain on board can include an initial stage of RSW or CSW for chilling of the catch (round fish), then the different products will follow different lines through and after sorting and processing. Chilled water may not be adequate for the sorted fractions of by-products. Unless the fractions are packed in water-resistant packages (e.g., vacuum-packed), direct contact with water is not recommended. Use of ice and storage in chilled rooms is the best choice. Freezing and frozen storage is a possible choice for some products. 3.4.2 Heat preservation High temperature treatment is commonly used in processing of fish by-products. Both enzymes and microbes might be inactivated at high temperatures. Heat treatment reduces the number of microbes and prolongs the shelf life of the product. Microbes generally do not grow at temperatures above 90ëC; however, a full heat sterilisation requires stronger thermal treatment. Enzymatic deterioration is arrested by inactivation of enzymes as soon as possible post mortem and might be obtained by heat treatment. Unfortunately, high temperatures lead to unwanted side reactions such as denaturation of proteins, which will affect the physical properties of the proteins generated. The inactivation conditions for enzymes are temperature and time dependent; however, factors such as pH and type of enzyme are important influences (Sùvik, 2005). Overall, a temperature of 85ëC for 10 minutes is found to inactivate proteases and lipases in visceral fractions of cod (unpublished data). In some products, such as silage, the enzyme activity is wanted and high temperature treatment should be avoided. 3.4.3 Dehydration (drying) The shelf life of fish by-products is increased by reducing the moisture content and thereby the water activity to levels (aw < 0.6) preventing growth of bacteria and moulds. Also, enzymes are affected by water activity and most enzymatic reactions are reduced at water activities below 0.8. The rate of lipid oxidation, however, is reduced at aw levels between 0.2 and 0.6, but is higher above and below these levels (Labuza, 1971). aw in oils is generally too low to permit any microbial growth. 3.4.4 Chemical treatment It has been reported that chemicals are, in some countries, used to add shelf life to fresh fish, but these applications are usually subject to local or national restrictions (FAO). Addition of acid, however, is widely used to reduce the pH and liquefy by-products. In order to protect the by-product from microbial spoilage, hurdle technology might be used. This technology is based on the fact that a combination of different preservation methods results in a synergistic effect and therefore rough

56

Maximising the value of marine by-products

treatment might be avoided. The principles and uses of hurdle technology have been reviewed by Leistner (1995). The major factors necessary to control microbial activity, enzymatic activity and lipid oxidation are explained below (see also Table 3.1). Microbial activity is reduced by refrigeration and heat treatment, low water activity, adjustment of pH and gentle and hygienic handling. To prevent formation of microbial enzymes, such as TMAO-reductase, the recommended practice is to reduce the amount of microbes and handle the by-products with sufficient hygiene practice. Enzyme activity is reduced by refrigeration and heat treatment, low water activity, adjustment of pH, chemicals which can inhibit enzyme action, alteration of substrates, alteration of products and pre-processing control. Lipid oxidation is reduced by: reduction of temperature, minimising oxygen availability and light and retaining the biological membranes intact. Lipid oxidation may also be reduced by antioxidants and it has become more common to use antioxidants in combination with an appropriate acid during bulk processing such as silage production.

3.5

On-board processing

Fresh raw material will always be an advantage, and offers a wider range of possibilities compared to frozen-thawed raw material or material that has been stored for several days or weeks under varying conditions. On board the fishing vessels there is a unique possibility for supply of completely fresh raw material, and, it is an advantage to be able to utilise as much as possible of the catch on board. This adds quality to the end product that cannot be achieved otherwise. 3.5.1 Process technology Fish processing traditionally takes place at on-shore processing facilities, and the processing of by-products on-board fishing vessels has declined during the last decades. This is especially due to the decrease of on-board fishmeal and fish-oil processing plants. Three of the most important bulk processes utilising by-products are shown in Fig. 3.5, these are fish silage, fish-oil and fish protein hydrolysate (FPH). Fish silage (A) is produced by acidification of fish material (offal and viscera) using an organic acid such as formic acid to reduce the pH to around 4. This pH will prevent microbial growth, but endogenous enzymes achieve optimal conditions to hydrolyse proteins and thereby liquefy the material. An excellent review of the technology used to produce silage is written by Raa and Gildberg (1992). Lipid oxidation in the silage during storage might be prevented by using antioxidants along with the acid. Much research has stated the fact that the acidic treatment deteriorates the lipids by increasing the level of free fatty acids (Johnsen and Skrede, 1981; Tatterson and Windsor, 1974). Fish protein hydrolysates (FPH). In recent years, the fishing industry and research, has addressed the use of commercial proteases (Fig. 3.5(B)) to produce

Fig. 3.5 Illustration of three alternative production lines for on-board processing of by-products. (A) Silage production where an organic acid is added to preserve the raw material by lowering the pH. (B) Controlled hydrolysis of proteins by a reaction catalysed by added enzymes. Bulk products are oil, fish protein hydrolysates (FPH) or fish powder. The FPH are perishable and need to be preserved by reducing pH, for example. (C) Traditional production of cod liver oil. Steaming and decanting. Rest material from oil production has a water activity > 0.6 and therefore needs to be conserved (Beuchat, 1981). This illustration is made in Microsoft Visio 2000 SR1 by E. Falch.

58

Maximising the value of marine by-products

fish protein hydrolysate from marine raw material (Gildberg, 1992, 1994; Liaset et al., 2002; Mohr, 1977, 1980; Slizyte et al., 2003, 2005a,b; Dauksas et al., 2005) under controlled conditions. During this process the stick water, and alternatively parts of the sludge, are evaporated. This pumpable liquid demands conservation, such as acid addition (lowering of pH), in order to be storage stable on-board. Alternatively, the evaporated FPH can be dried to storage stable powders. Fish oil and fish meal. Process technology where the marine lipids are the main product generated from the raw material, has traditionally been based on thermal treatment followed by centrifugal separation (Aure, 1967) for liver oils (Fig. 3.5(C)) and cooking, pressing and centrifugal separation (mechanical expression) (Windsor and Barlow, 1981) of fish raw material. This mechanical expression is the traditional process converting anchovies, menhaden, sardines and Atlantic herring into fishmeal and oil. Today, these species are diverted for human consumption and the resulting reduced availability of raw material for feed applications has led to the use of other raw material in the processes, such as fish by-products. Fishmeal is produced through thermal treatment. Focus on increased protein digestibility of feed has led to the development of gentle heat treatment (LT quality) in order to prevent protein denaturation. The digestibility of fishmeal is also affected by the freshness of the raw material (Aksnes and Mundheim, 1997) and effort has been put into the early conservation and production of meal. Fishmeal plants are offered to be individually designed for on-board operation for fishmeal (BAADER Food Processing Machinery). In Norway, less than 5% of the fishmeal is currently produced on on-board processing facilities. Drying the raw material on-board is a solution that has an answer to two different challenges: 1) Dry matter weighs less than wet matter; this reduces the weight of the vessel and thereby also reduces the fuel-costs and 2) Dry matter takes less space/volume than wet matter. Other possibilities. The cut-offs and trimmings, mainly generated during the filleting process of white fish might be used in fish mince or washed to produce surimi. The surimi process is currently located on-board some fishing trawlers. Fish sauce, which is a popular product in south-east Asia (Huang and Huang, 1999) is another product possible to produce from fish by-products (Gildberg, 2001). The raw material is conserved by using large amounts of salt followed by a fermentation period. Both of these process steps might be done on-board the vessel. Gelatine and dried heads are usually not produced on-board, but heads, skin and bones are, in some vessels, frozen for later processing. In Iceland, dried cod heads are a major export product (Arason, 2002). Heads are generally dried at on shore processing facilities. One of the main challenges on-board is the possibility to handle, store and transport the bulk products ± which are factors limiting the processing and utilisation. This is a matter of available space and also a matter of weight. Figure 3.6 illustrates parts of an on-board processing plant for producing hydrolysed

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

59

Fig. 3.6 Parts of an on-board processing plant for hydrolysation of by-products followed by a three phase separation into oil, glue water and sludge. Photo: Eva Falch (Falch et al., 2000).

by-products. Compact and efficient process equipment is necessary due to the limited available area on board.

3.6

Utilisation of by-products from gadiform species

It is a great potential for the fishing industry to land and utilise a greater part of the total catch of gadiform species for higher value products. Utilisation of byproducts requires a predictable delivery quantity and also raw material with potential for producing relatively standardised products to be able to satisfy the customers. To utilise more than the fish fillet for consumable products, data on weight and composition of different by-products are needed (Froese and Pauly, 2000). Fishbase (www.fishbase.org), which is a comprehensive database, has included a processing table for each species. These tables show the weight distribution of by-products along with some approximate composition data. Such data on different gadiform species are not completely established at this stage. However, a major study on the available by-products, available lipids and their composition has been reported for five different gadiform species (Falch et al., 2006a,b). These data show that, from an average daily production of fillet (10 tonnes), the by-products contain approximately one tonne of the health beneficial marine lipids with 30% n-3 fatty acids. The data show that, regardless of species and fraction, the n-3 content is high and close to the specifications for medicinal oil (European Pharmacopoeia, 1999). However, the lipid classes such as phospholipids and triacylglycerols are unequally distributed among different fractions

60

Maximising the value of marine by-products

and processing technology such as the enzymatic processing (Fig. 3.5, process (B)) generally differentiate among the lipid classes (Dauksas et al., 2005). These experiments showed that the phospholipids are generally following the proteinrich fractions during centrifugal separation. Gadiform species such as cod, saithe, haddock, tusk and ling, are also reported to be good sources for production of fish protein hydrolysates and protein-rich fractions (Slizyte et al., 2003, 2004a,b) with specific functional properties applicable in food. The processing challenges are discussed in the above-mentioned papers. In order to explore the possible profit from processing by-products and to decide on where to do the processing; on the vessel or on shore, profitability analyses are needed. As assistance to such an analysis, a program called MaxFish has been developed to help calculate the possible output and profitability from a given catch. For the time being, the database is built on data from cod species and the weights and fractions from these. Chemical composition data and their variations may be added and increase the usefulness even more. It can also easily be extended with any species of interest. An example of the use of the program is shown in Fig. 3.7. Basically it utilises knowledge of the different end products and the possible income these can give, and the program enables calculation of the investments that can be justified. Studies of biochemical reactions in the visceral fractions of cod show that these organs are highly perishable during chilled storage. One week of storage (4ëC) shows minor lipid oxidation, but major changes due to lipolysis (unpublished data). In our studies of roe and milt from cod we found limitations

Fig. 3.7 An example showing applications for the MaxFish program to plan the utilisation of by-products.

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

61

in using free fatty acid assessment as a measure of lipolysis in such raw material since fatty acids hydrolysed from phospholipids and triacylglycerols are esterified to cholesterol and thereby not detected by using free fatty acids as an indicator of lipolysis (Falch et al., 2005).

3.7

Future trends

The growing awareness of the health benefits of marine products is turning the focus from waste or bulk products into higher value end products such as food ingredients and bioactive compounds. This will be a challenge for the fishing vessels, demanding more advanced equipment on board and more competence from the people handling the raw material and the equipment. The end product will not necessarily be produced on the vessel, but preservation or alternative preprocessing will be recommended on board to retain the quality. There will be a need for technology and knowledge which necessitate investment in hardware and in education of the personnel. The consumer trends show preference for more freshness and higher quality levels, but also milder processing, minimal preservation and replacement of synthetic additives with naturally occurring compounds. Knowledge about the available biomass and its chemical composition will be required in order to secure a delivery of a specified quality and also use such data to find optimal combinations of raw material available. Several vessels have quotas for different fish species and finding optimal combinations of raw material will therefore be an advantage. Furthermore, the sorting operation, which is now a manual operation, will have to be more efficient and more automated solutions are currently being investigated. New technologies are currently put into more gentle catching methods. Catching the fish into lock bay is one of these new technologies, which prevent physical damage of the fish and thereby a more gentle treatment of the byproducts. It will try to ensure that the fish is alive when reaching the slaughter line. Overall, the health aspect is becoming more important and fish species traditionally used for production of fishmeal into aquaculture feed are now turned into consumer products. As global fish farming increases, the aquaculture industry will continue to request marine feed ingredients, and higher utilisation of by-products is therefore an alternative. The marine resources, however, are limited and an optimal utilisation of all the available raw material is essential.

3.8

Acknowledgements

Norwegian Research Council (project: Increased value adding from by-products and by-catches and project: Technology for production of ultra stable marine oils and functional food applications) and EU (QLK1-CT2000-01017) Utilisation and stabilisation of by-products from cod species are thanked for funding the majority of the research presented from our research group.

62

Maximising the value of marine by-products

3.9

References

AKSE L,

`Kvalitet av Fisk og biprodukter ved ilandfùring av fisken uslùyd', Rubin report (4202/99), 2002. AKSNES A and MUNDHEIM H, `The impact of raw material freshness and processing temperature for fish meal on growth, feed efficiency and chemical composition of Atlantic halibut (Hippoglossus hippoglossus)', Aquaculture 1997 149(1/2) 87±106. ARASON S, `Utilization of fish byproducts in Iceland' 2nd International Seafood Byproduct Conference, Anchorage, Alaska, 2002. AURE L, `Manufacture of fish liver oils', in Fish Oils ± Their Chemistry, Technology, Stability, Nutritional Properties and Uses, Stansby M E, ed., AVI , Westport, CN, 1967 193 (14). BAADER FOOD PROCESSING MACHINERY, Fish meal plants. Received from World Wide Web April 2005. http://www.baader.de/Fish_Meal_Plants.82.0.html BEUCHAT L R, `Microbial stability as affected by water activity', Cereal Foods World, 1981 26 345±349. DAUKSAS E, SLIZYTE R, RUSTAD T and STORRé, `Bitterness in fish protein hydrolysates and methods for removal', J Aquatic Food Prod Technol, 2004 13 101±114. DAUKSAS E, FALCH E, SLIZYTE R and RUSTAD T, `Composition of fatty acids and lipid classes distribution after enzymatic hydrolysis of cod (Gadus morhua) byproducts', Proc Biochem, 2005 40 2659±2670. DELLACASA A, `Refrigerated transport by sea', Int J Refrig, 1987 10 349±352. EINARSSON T (Baader Norge), `SkaÊnsomme slùyemaskiner for laks og hvitfisk', RUBIN conference, Marine biprodukter ± Fremtidens Fiskerinñring, Stjùrdal, Norway, 2004. EUROPEAN PHARMACOPOEIA (3rd edn, supplement, 1999), Omega-3-acid Ethyl Esters (1998: 1250) and Omega-3-acid Triglycerides (1999: 1352), Council of Europe, Strasbourg, 1999, pp. 723±730. FALCH E, AURSAND M and RéSSVIK H, `Gjennomgang av ensilasjeprosessen ombord i M/S Tenor' 2000B, SINTEF report (STF80 F00039) and `Videreforedling av ensilasje ombord i Tenor. Dokumentasjon av kvalitet, prosess og ùkonomi' 2000A (RUBIN report 4301). FALCH E, STéRSETH T R and AURSAND M, `HR NMR to study quality changes in marine byproducts', in Magnetic Resonance in Food Science. The multivariate challenge, Engelsen S B, Belton P S and Jakobsen H J (eds), The Royal Society of Chemistry, Cambridge, 2005, 11±19. FALCH E, RUSTAD T and AURSAND M, `By-products from cod species (Gadiforms) as a raw material for production of marine lipids as an ingredient for food and feed', Proc Biochem, 2006a 41 666±674. FALCH E, RUSTAD T, JONSDOTTIR R, SHAW N B, DUMAY J, BERGE J P, ARASON S, KERRY J,

and AURSAND M, `Geographical and seasonal differences in lipid composition and relative weight of by-products from Gadiform species', J Food Comp Anal, 2006b 19 727±736. FAO FISHERIES DEPARTMENT, FOOD AND AGRICULTURE ORGANIZATIONS OF UNITED NATIONS, The state of world fisheries and aquaculture 2004, Rome, Italy. FELLOWS P J, Food Processing Technology ± Principles and Practice, Woodhead Publishing Limited, Cambridge, 2000. FROESE R and PAULY D (2000) FishBase 2000 (www.fishbase.org), Concepts, design and data sources, retrieved April 2005 from the World Wide Web: http:// SANDBAKK M

Preventing microbial spoilage, enzymatic reactions and lipid oxidation

63

www.fishbase.org/manual/english/contents.htm `Recovery of proteinases and protein hydrolysates from fish viscera', Bioresource Technol, 1992 39 271±276. GILDBERG A, `Enzymic processing of marine raw materials', Proc Biochem, 1994 28(1) 1± 15. GILDBERG A, `Utilisation of male Arctic capelin and Atlantic cod intestines for fish sauce production ± evaluation of fermentation conditions', Bioresource Technol, 2001 76 119±123. GRAM L and HUSS H H, `Microbial spoilage of fish and fish products', Int J Food Microb, 1996 33 121±137. HARDARSON V, `Metoder for konservering av biprodukter i fangstleddet ± lever', SINTEF Report STF11 A95088 Trondheim 1995 (in Norwegian). HIDALGO M C, UREA E and SANZ A, `Comparative study of digestive enzymes in fish with different nutritional habits. Proteolytic and amylase activities', Aquaculture, 1999 170 267±283. HUANG Y-W and HUANG CY, `Traditional Oriental Seafood products', Asian Foods ± Science and Technology, Technomic Publishing Co. Inc, Lancaster, PA, 1999. JOHNSEN F and SKREDE A, `Evaluation of fish viscera silage as a feed resource ± Chemical characterisation', Acta Agric Scandinavica 1981 31 21±28. KELMAN J H, `Stowage of fish in chilled sea water', www.fao.org, Torry advice note no 73 2001. KULAÊS E, OLSEN E and ACKMAN R G, `Oxidation of fish lipids and its inhibition with tocopherols', in Lipid Oxidation Pathways, Kamal-Eldin A (ed.), AOCS Press, Champaign, IL, 2003. LABUZA T P, `Kinetics of lipid oxidation in food', Crit Rev Food Technol, 1971 2 355±405. LEISTNER L, `Principles and applications of hurdler technology', in New Methods of Food Preservation, Gould G W (ed.), Blackie Academic & Professional, London, 1995. LIASET B, NORTVEDT R, LIED E and ESPE M, `Studies on the nitrogen recovery in enzymic hydrolysis of Atlantic salmon (Salmo salar, L.) frames by ProtamexMT protease', Proc Biochem, 2002 37 1263±1269. LIE é, LIED, E and LAMBERTSEN G, `Lipid digestion on cod (Gadus morhua)', Comp. Biochem. Physiol, 1987 88B 697±700. LISTON J, `Microbiology in fisheries science', Advances in Fish Science and Technology, Connell J (ed.), Fishing News Books, Farnham, 1980. MATHIASSEN J R, SKAVHAUG A and Bé K, Texture Similarity Measure Using KullbackLeibler Divergence between Gamma Distributions, 7th European Conf Computer Vision (ECCV 2002) Springer Verlag, Copenhagen, Denmark, 2002. MOHR V, `Fish protein concentrate production by enzymic hydrolysis', in Biochemical Aspects of New Protein Food, Adler-Nissen J (ed.), Vol. 44 Symposium A3, FEBS Federation of European Biochemical Societies 11th Meeting, Copenhagen, Denmark, 1977. MOHR V, `Enzymes technology in the meat and fish industries'. Proc Biochem. 1980 15(6) 18±21, 32. MUKUNDAN M K, ANTHONY P D and NAIR M R, `A review of autolysis in fish', Fisheries Res, 1986 4 259±269. OLSEN E, VOGT G, SAAREM K, GREIBROKK T and NILSEN A, `Autoxidation of cod liver oil with tocopherol and ascorbylpalmitate', J Am Oil Chem Soc, 2005 82 97±103. RAA J and GILDBERG A, `Fish silage: a review', CRC Crit. Rev Food Sci Nutr, 1992 16 383±419. GILDBERG A,

64

Maximising the value of marine by-products

RéYRVIK J,

`Transport og lagring av fisk' Doctoral thesis at the Norwegian University of Science and Technology, Trondheim 1979 (in Norwegian). SHEWFELT R L, `Fish muscle lipolysis ± a review', J Food Biochem, 1981 5 79±100. SIKORSKI Z E, KOLAKOWSKA A and BURT J R, `Postharvest biochemical and microbial changes', in Seafood Resources, Nutritional Composition and Preservation, Sikorski Z E (ed.), CRC Press, Florida, 1990. SLIZYTE R, ALVES-FILHO O, FALCH E and RUSTAD T, `The influence of drying processes on functional properties of fish protein hydrolysates from cod (Gadus morhua) byproducts', Proceedings from 2nd Nordic Drying Conference, Copenhagen, Denmark, 2003. SLIZYTE R, DAUKSAS E, FALCH E, STORRé I and RUSTAD T, `Characteristics of protein fractions generated from hydrolysed cod (Gadus morhua) by-products', Proc Biochem, 2005a 40 2021±2033. SLIZYTE R, DAUKSAS E, FALCH E, STORRé I and RUSTAD T, `Yield and composition of different fractions obtained after enzymatic hydrolysis of cod (Gadus morhua) byproducts', Proc Biochem, 2005b 40 1415±1424. SéVIK S L, Characterisation of Enzymatic Activities in By-products From Cod Species, Effect of Species, Season and Fishing Ground, Doctoral thesis at The Norwegian University of Science and Technology, Trondheim, Norway, 2005, 138. SéVIK S L and RUSTAD T, `Effect of season and fishing ground on the activity of lipases in byproducts from cod (Gadus morhua)', Lebensm Wiss und -Technol 2005a 38 867±876. SéVIK S L and RUSTAD T, `Proteolytic activity in by-products from cod species caught at three different fishing grounds', J Agric Food Chem, 2005b 53 452±458. TATTERSON I N and WINDSOR M L, `Fish silage', J Sci Food Aquaculture, 1974 25 369±379. TOCHER D R, `Metabolism and function of lipids and fatty acids in teleost fish', Rev Fisheries Sci, 2003 11 107±184. WANG S G and WANG R Z, `Recent developments of refrigeration technology in fishing vessels', Renewable Energy, 2005 30 589±600. WINDSOR M and BARLOW S, `Fish meal production', in Introduction to Fishery ByProducts, Farnham, Surrey, 1981.

4 Recovery of by-products from seafood processing streams J. A. Torres, Oregon State University, USA, Y.-C. Chen, Chung Shan Medical University, Taiwan, J. Rodrigo-GarcõÂa, Universidad AutoÂnoma de Ciudad JuaÂrez, Mexico and J. Jaczynski, West Virginia University, USA

4.1

Introduction

The transformation of raw materials into foods inevitably generates some type of by-products and the processing of aquatic foods is no exception. Therefore, developing new technologies for the full utilization of these by-products is of critical importance to the future economic viability of this industry (Gildberg, 2002). In traditional and non-industrialized fisheries, where most of the labour is provided by personnel with skills often passed down by generations, the fish is almost completely utilized for human consumption, animal feed, or as plant fertilizer. The economy-driven industrialization of fisheries brought incredible advances, but at the same time, the amounts of by-products generated during harvesting and processing increased dramatically (Gildberg, 2002). Typical examples are commercial shrimp trawling, krill processing and mechanized fish filleting. In shrimp trawling, sometimes 90% of the total catch volume corresponds to species with no commercial value and this by-catch is therefore most often discarded (Raa and Gildberg, 1982). The meat recovery yield during the commercial processing of whole krill (Euphausia superba) is extremely low, fluctuating between 10 and 15% by weight (Suzuki, 1981). Finally, when fish are mechanically processed for fillets, the recovery yields are typically 30±40% of fillets and the by-products account for 60±70% by weight of the whole fish (Gildberg, 2002). While it is not uncommon to just grind-and-discard this 60± 70% of fish by-products, this practice should be considered an irresponsible utilization of natural resources and should be used instead to fulfil human nutritional needs.

66

Maximising the value of marine by-products

In the fisheries industry and the scientific literature, there is a common misunderstanding regarding how processing `by-products' are defined. Three terms are frequently and interchangeably used to describe the same materials: (1) `offal', (2) `waste', and (3) `by-products'. The first two terms imply that these materials must be discarded, because they cannot be used for any application, and therefore, have a negative connotation. The third term suggests that they may be valuable if an appropriate technology is available, and is therefore positive. Currently, the preferred and most common definition of the term `by-products' is to refer to all edible or inedible materials remaining following processing of the main product. A typical example is fish filleting where the fillets are the main product and the frames, heads, and guts are the `by-products'. It would be misleading to name these by-products as `waste', because the fish meat and oil left on the frame and heads have value and could be recovered if their quality is not compromised by poor handling (Strom and Eggum, 1981). With an appropriate technology, the meat reclaimed represents additional revenue for the processor, and at the same time, its recovery decreases environmental pollution. By-products can be converted into four major product groups: (1) plant fertilizers, (2) livestock feeds, (3) value-added foods, and (4) specialty ingredients. In general, conversion of by-products into fertilizers results in the lowest value addition, while it is highest for value-added foods and specialty ingredients. The future is promising as it has been estimated that as a result of new technologies, the value addition to by-products will increase fivefold within the next decade (Gildberg, 2002).

4.2

State of global fisheries and by-products

Global fish catch and aquaculture production information is readily available from the Food and Agricultural Organization (FAO) Fisheries Department (Anonymous, 2004). However, the amounts of by-products generated by these activities can only be estimated (Table 4.1). While the global capture fisheries have remained fairly stable for the past twenty years and have been forecasted as unlikely to increase in the future, the aquaculture sector increased its production by 27% for the same period and currently contributes nearly 50% of the global annual catch (Vannuccini, 2004). The Food and Agriculture Organization (FAO) predicts that the future demand for aquatic foods will have to be met by increasing aquaculture production. Then again, in 2002, about 76% of world fisheries production was used for direct human consumption and the remaining 24% was converted into fishmeal and oil (i.e., reduction fisheries), yielding essentially no by-products. However, while on a weight basis this industry accounted for a quarter of total fish utilization, it contributed a low 3.8% to the fisheries total economic value (Anonymous, 2004). About 100 million tonnes of the global fisheries production is processed for direct human consumption. Commercial processing of fish such as cod, salmon,

Recovery of by-products from seafood processing streams Table 4.1

67

Global fisheries production and utilization in million tonnes

Capture Aquaculture Total fisheries Human consumption Non-food uses Population (billions) Per capita fish food supply

1998

1999

2000

2001

2002

2003

87.7 30.6 118.2 93.6 24.6 5.9 15.8

93.8 33.4 127.2 95.4 31.8 6.0 15.9

95.5 35.5 131.0 96.8 34.2 6.1 15.9

92.9 37.8 130.7 99.5 31.1 6.1 16.2

93.2 39.8 133.0 100.7 32.2 6.2 16.2

90.3 41.9 132.2 103.0 29.2 6.3 16.3

Source: Adapted from Anonymous (2004). The state of world fisheries and aquaculture. Food and Agriculture Organization. Rome (Italy).

trout, tilapia, seabream and pollack typically yields about 30±40% of fillets as products, and while meat and oil left on the remaining by-products range widely, they account typically for 20±30% and 5±15% of whole fish weight, respectively. Fish oil is highly polyunsaturated (omega-3 fatty acids), and therefore, very susceptible to lipid oxidation, resulting in rancidity development. If fish oil were to be efficiently recovered from by-products, the use of fat-free fish byproducts for animal feed and pet food could be expanded. The 100 million tonnes processed for direct human consumption do not include the fish by-catch and discards estimated to account for an additional 30 million tonnes of available catch not yet utilized (Zugarramurdi et al., 1995). Another aquatic resource that is scarcely utilized for human consumption is krill (Fig. 4.1). At present, krill is commercially utilized mostly by the reduction fisheries to manufacture fish feed. The development of an appropriate technology to efficiently convert this resource into food could contribute significantly to fulfil nutritional needs for proteins and help alleviate over-fishing and stock depletion problems affecting several aquatic species. This vast resource has been estimated at 400±1550 million tonnes with a sustainable annual harvest of about 70±200 million tonnes (Suzuki and Shibata, 1990). The krill biomass potentially available for human food is comparable to that of all of the other aquatic species currently under commercial exploitation and is probably the largest of any multicellular animal species on the planet (Nicol and Endo, 1999). Krill, small crustaceans that resemble shrimp, are not fully utilized for human consumption owing to the lack of efficient meat recovery technology. Krill meat is literally liquefied at high rates by extremely active proteolytic enzymes released during harvest (Kolakowski and Lachowicz, 1982). The biomass of aquatic by-products and underutilized species is staggering. At the same time, over-fishing, stock depletion, and other environmental issues associated with aquatic food production are increasingly more emphasized in popular media. Also, the world population is increasing and it is becoming more difficult to meet nutritional needs for proteins and lipids from aquatic resources. Although aquaculture can alleviate these problems, technologies that increase recovery yields and reduce the amount of by-products will have to be developed and

68

Maximising the value of marine by-products

Fig. 4.1 Body composition of krill whose annual sustainable biomass for human consumption has been estimated as larger than the global consumption of all other aquatic species combined.

implemented. A challenge will be to find applications for the materials recovered, to ensure that the process is not only environmentally sustainable but also economically viable. This industrial development effort must be based on a full understanding of fundamental properties of the raw materials. Only then, will it be possible to manipulate the behaviour of the aquatic biomolecules and in turn increase recovery yields. By definition, meat is the edible portion of aquatic animals derived from muscle tissue and is mostly a mixture of water, proteins, and intramuscular lipids. Therefore, to develop and implement new recovery technologies, it will be necessary to understand the behaviour of these three components.

4.3 Basic properties of water, proteins and lipids in aquatic foods 4.3.1 Water properties The moisture content of meat depends on the species and may range widely, reaching values up to 90%. In a final food product, not only does water control product weight, and therefore, the revenue, but also the sensory attributes perceived by customers. From a nutrition standpoint, water does not contribute calories and therefore, the higher the water content, the lower the caloric contribution of the product. However, when water is added to the product, waterbinding compounds may need to be used to prevent `drip' losses. Meat (muscle) proteins have good water holding capacity (WHC) if they have not been abused,

Recovery of by-products from seafood processing streams

69

Fig. 4.2 Electrostatically charged water dipoles are the basis for the solubility of proteins in water. In the water dipole, the oxygen atom is negatively charged (2ÿ) with respect to the two positive (‡) hydrogens. In a meat system, dipoles form hydrogen bonds between each other and with charged molecules such as proteins.

particularly from a thermal point of view. For example, krill meat proteins have good WHC; however, they are particularly sensitive to temperature abuse (Suzuki, 1981). In meat systems, water provides a reaction medium in which all other compounds such as proteins and lipids may be dissolved or suspended. Therefore, it is critical to be familiar with how water interacts with them. A water molecule is a dipole due to the slight negative and positive charge of its oxygen and hydrogen atoms, respectively. These charges allow an interaction called hydrogen bond between water dipoles and also with other charged molecules (Fig. 4.2). This property is very important in food technology, because important molecules such as proteins may also be charged, and therefore, have an ability to interact with water dipoles via electrostatic interactions. 4.3.2 Fish proteins The biological value (BV) of a dietary protein measures its efficiency in supporting physiological needs reflecting its digestibility and content of essential amino acids. Egg proteins are used as a reference protein source and have a BV of 100, while milk, beef, corn, and rice proteins have BV of 93, 75, 72, and 59, respectively. The BV of fish meat is 75 while the value for krill meat proteins is slightly higher than that of milk proteins (Whitney and Rolfes, 2005; Murano, 2003; Suzuki and Shibata, 1990). Two muscle types are present in aquatic foods, striated muscle characterized by transverse stripes and smooth muscle that lacks them. Striated muscle is the

70

Maximising the value of marine by-products

main component in fish meat while smooth muscle is typical of meat from molluscans. Fish muscle is divided further into white and dark muscle. The dark muscle lies alongside the fish body and under the skin. White muscle proteins can be further classified into (www.fao.org/documents/show_cdr.asp?url_file=/ docrep/v7180e/v7180e05.htm): (1) Myofibrillar proteins. These are proteins organized into myofibrils that are soluble in concentrated saline solutions. The myofibrils are made of two major ultramicroscopic myofilaments, a thick and a thin filament containing the major muscle proteins myosin and actin, respectively, the former being largely responsible for the functional properties of meat. In the process of muscle contraction, tropomyosin and troponin act as regulatory proteins, initiating and terminating the contraction process of myosin and actin. These contractile agents are arranged to form the myofilaments of a sarcomere, continuing with the formation of myofibrils from many myofilaments kept together by connective tissue or stroma proteins. However, the amount of connective tissue in fish meat (3±5%) is low when compared to other meat sources (e.g., 16±28% in beef) (Suzuki, 1981). (2) Sarcoplasmic proteins. These proteins are soluble in dilute saline solutions and correspond to myoalbumins, globulins and various enzymes. In the muscle, myoglobin has a role similar to that of blood haemoglobin. Both form a complex with oxygen, but myoglobin is essentially an oxygen storage mechanism in the cell. It is a conjugated protein with a peptide chain bound to the heme containing an iron atom and a large heterocyclic organic ring called a porphyrin. (3) Connective tissue or stroma proteins. These proteins are insoluble in concentrated saline solutions, and when heated to 60 or 70ëC the collagen fibres contract to one third or one quarter of the original length. At 80ëC, the collagen becomes water soluble gelatine. Ionic strength (IS) is an important factor in the water solubility of meat proteins. The IS represents the status of electrolytes (i.e., ionized salts) present in a system. The two major myofibrillar proteins, myosin and actin are water insoluble at the fish physiological pH (about 0.05 for rainbow trout), but they are soluble at extremely low IS or above 0.6. While sarcoplasmic proteins are quite water soluble, stroma proteins are completely water insoluble. The water solubility of sarcoplasmic proteins decreases with the increase of IS (Lanier et al., 2005; Suzuki, 1981). In an aqueous protein solution, the side chain and the amino and acid (carboxyl) groups bound to the central carbon atom of an amino acid can be electrostatically charged, and therefore, participate in protein-water interactions via weak hydrogen bonds (Fig. 4.3). Although the bonding energy of the hydrogen bond is low, however when present in high numbers, these bonds efficiently stabilize the complex three dimensional structure of food proteins. Depending on the side chain attached to the central carbon atom of an amino acid, a protein will have different properties. While hydrophobic side chains

Recovery of by-products from seafood processing streams

71

Fig. 4.3 Proteins are chains of amino acids containing an amino (‡NH3) group, an acid (COO±) group and a side chain (R) all bonded to a central carbon atom. The side chain allows proteins to participate in protein-protein hydrophobic interactions and can also form protein-water hydrogen bonds.

limit its water solubility, polar (charged) side chains may result in considerable protein-water interaction via the hydrogen bonds (Fig. 4.3). The protein-water interaction is essential for protein solubility as well as water holding capacity (WHC). On the other hand, the weak hydrophobic interactions may result in protein-protein interactions causing their aggregation and subsequent precipitation. Finally, the side chain of cysteine contains a sulphydryl group (±SH) that when oxidized (i.e., during heat treatment) will create a covalent disulphide bond (±SS±). This strong bond combined with weak interactions between hydrophobic side chains are important in the gelation of fish proteins and are

Fig. 4.4 A protein at its isoelectric point (pI) has a zero net electrostatic charge. At its pI, protein-water interactions are at its minimum, while protein-protein interactions via weak hydrophobic bonds are at its maximum, causing protein precipitation. Protein-water interactions prevail under acidic or basic conditions far from the pI, resulting in protein solubility.

Recovery of by-products from seafood processing streams

73

essential for the proper texture development of value-added foods. The side chains of amino acids can be chemically modified, and even though chemically modified proteins may be unsuitable for human consumption, unconventional applications may be developed for proteins recovered from by-products. The side chains of the amino acids can assume different electrostatic charges depending on the conditions of the fish protein solution (Fig. 4.4). Acid added to a protein solution dissociates forming the positive hydronium ion (H3O+) neutralizing negative charges on the protein and making it more positively charged. By adding a base to a solution, the protein will increase negative charges on its surface. As the protein becomes more positively or negatively charged, electrostatic interactions with water dipoles increase while hydrophobic protein-protein interactions decrease. Therefore, as a protein becomes more charged (polar), its solubility increases and the protein becomes water soluble. The pH at which the overall electrostatic charge of a protein is zero is called the isoelectric point (pI). 4.3.3 Fish lipids The fat content in fish muscle is highly variable, varying with species, age, spawning season, fish diet, and body part. While the protein content in fish muscle is relatively stable, the fat content is in general inversely correlated to the moisture content. Although, fat deposits are found over the entire muscle mass, the concentration of fatty cells seems to be higher near the myocommata and in the region between the white and dark muscle (Kiesling et al., 1991). Dark muscle contains some triacylglycerols in the muscle cells, even in lean fish, because this muscle can metabolize lipids directly to generate energy. This is why the dark muscle contains more fat and more blood supply to metabolize lipids; as well as a higher concentration of pro-oxidative myoglobin and mitochondria (organelle that oxidize energy-yielding substrates) than white muscle. Therefore, the dark muscle is more prone to lipid oxidation and the development of rancidity (Hultin et al., 2005). For these reasons, the white flesh fish is preferred by customers, and hence, it would be economically beneficial to develop a technology capable of fish oil removal from the dark fish meat to add value to lower grade products. The properties of the triacylglycerols in fish oils depend on the composition of the fatty acid (FA) chains attached to glycerol (Fig. 4.5). Lipids interact with the hydrophobic side chains of amino acids forming weak lipid-protein hydrophobic bonds. Probably more important to fish technologists than triacylglycerols are phospholipids, an integral component of cell membranes. Phospholipids are amphiphilic compounds, that is, they have lipo- and hydrophilic properties. Phospholipids can create hydrophobic bonds with other apolar substances (for example, fish oil) and also interact with water and charged proteins (Fig. 4.6). Therefore, when processing aquatic foods such as minced fish meat, the separation of fish oil from the water and proteins is difficult and requires the use of additives that can act as emulsion breakers.

74

Maximising the value of marine by-products

Fig. 4.5

Fig. 4.6

Triglycerides are a major component of meat lipids.

Phospholipids, another major component of meat lipids, are water soluble and can interact with charged proteins and water dipoles.

Recovery of by-products from seafood processing streams

75

Fish oil is liquid at ambient temperature, but becomes significantly more viscous at the colder temperatures used in its production. To facilitate the separation of fish oil from the fish meat, producers could elevate slightly the processing temperature so as to solve this viscosity problem. This strategy is used in the dairy industry to separate cream from milk, although it should be pointed out that fatty acids in milk are more saturated, and therefore, less susceptible to lipid oxidation. Membrane phospholipids have a larger surface area than muscle triacylglycerols, tend to include fatty acid side chains with a higher level of unsaturation, and are often associated with pro-oxidative processes such as those occurring in the mitochondria. They are also important components in semipermeable membranes, which require certain fluidity for proper functioning, and this property is a direct function of the unsaturation degree of the fatty acid side chains of phospholipids. For all these reasons, membrane phospholipids are more susceptible to oxidation than intramuscular triacylglycerols, and even though their content is lower than that of triacylglycerols, they contribute more to rancidity development in fish meat (Hultin et al., 2005). The problem is complex, because phospholipids are difficult to separate from minced fish owing to their amphiphilic characteristics. Although the higher levels of polyunsaturated fatty acids (PUFA) in fish oil have been correlated with cardiovascular health improvement, they are highly susceptible to lipid oxidation. Omega-3 (!-3) fatty acids are considered `essential' diet components, because they cannot be synthesized by humans. The !-3 nomenclature refers to the third carbon atom in the fatty acid chain where the first double bond (C=C) occurs counting from the methyl end (Fig. 4.5). The health benefits of fish oils have been ascribed to their eicosapentaenoic (EPA, 20:5!-3) and docosahexaenoic (DHA, 22:6!-3) content. Although these oils are highly valued by consumers, if fish products are not properly processed and stored, their contribution to rapid rancidity can be offensive.

4.4 Recovery of functional proteins and lipids from by-products 4.4.1 Isoelectric precipitation and solubilization of fish muscle proteins The pH adjustment of a protein solution to its isoelectric point (pI) is used in manufacturing cheese and soy protein isolates. In cheese making, pH is lowered to pH 4.6 by direct acidification or the action of lactic acid bacteria, which precipitates casein at its pI and forms the curd. The pI of fish muscle proteins is at pH 5.5 and the proteins precipitate at this pH while becoming gradually soluble as the pH is more acidic or basic than this value. The isoelectric precipitation and solubilization of fish muscle proteins with concurrent separation of fish oil was recently patented (Hultin and Kelleher 1999, 2000, 2001, 2002) by food scientists from the University of Massachusetts and is under investigation in several laboratories. The temperature during the

76

Maximising the value of marine by-products

Fig. 4.7 Diagram of the isoelectric precipitation and solubilization technology with concurrent oil separation proposed for processing fish by-products. Materials in boxes are fractions to be further processed into food and other applications.

following five processing steps is controlled at 1±8ëC to minimize protein and lipid degradation (Fig. 4.7): (1) Homogenization of by-products in a meat homogenizer (for example MCH10, Stephan Machinery, Columbus, OH) reducing particle size to below 0.2 mm to increase surface area for the protein solubilization in the next step. (2) Solubilization of fish muscle proteins at either acidic or basic pH. This pH shift results in weaker protein-protein hydrophobic interactions, increased electrostatic protein-protein repulsion and more predominant protein-water interaction. As proteins start interacting with water, viscosity increases drastically, but drops when the proteins become water soluble. This viscosity increase may cause mixing problems, poor pH control and foam formation unless the solution is maintained at the desired pH, which is facilitated by a recovery system working in a continuous mode instead of batch. (3) Centrifugation to separate the solution into a light, medium and heavy fraction corresponding to fish oil, solubilized muscle proteins, and fat-free impurities (bones, skin, scale, skin and insoluble proteins), respectively. While the hydrophobic triacylglycerols are relatively easy to separate from the solution, the membrane phospholipids are relatively persistent due to their amphiphilic characteristics. The crude fish oil, rich in !-3 PUFA such as DHA, EPA, and ALA, can be further processed into numerous food and other applications. Although the relatively extreme pH values used in this technology could cause degradation of fatty acids, it appears to be minimal (Fig. 4.8). This is likely due

Fig. 4.8

Effect of the solubilization pH used in the solubilization and isoelectric precipitation technology on the recovery !-3 and !-6 fatty acids.

78

Maximising the value of marine by-products

Table 4.2 Ash contents (dry basis) of fractions recovered from krill and trout frames using the isoelectric solubilization/precipitation technology compared to krill tail meat and boneless skinless trout fillets, respectively

Protein solubilization pH

Krill tail meat Whole krill

% Ash 11.1 17.4

2.0 2.5 3.0 12.0 12.5 13.0

6.0 4.3 4.0 4.9 5.7 5.7

% Ash Boneless skinless trout fillet 5.5 Trout frames (by-products) 13.9 Recovered fat-free impurities 41.1 Protein solubilization pH

2.5 3.0 12.0 12.5 13.0

2.1 1.6 0.9 1.4 2.1

to the low temperature and short exposure time during the solubilization and first separation (Steps 2 and 3). The heavy fraction is essentially fat-free and rich in minerals and therefore, can be used in formulating animal feed and pet foods (Table 4.2). Since the fish oil has been removed from this fraction, unlike typical fishmeal, it should not impart a fishy odour to the meat of animals fed this fraction. (4) Precipitation of proteins in the medium fraction by pH adjustment to the pI of the fish muscle proteins (5.5). Similarly to the pH adjustment in the second step, as proteins gradually stop interacting with water dipoles, the viscosity increases significantly. This viscosity problem can be solved by maintaining the pH at 5.5, which is again facilitated by a continuous instead of a batch recovery system. (5) Centrifugation to recover the precipitated fish muscle proteins, which can be used as a food ingredient. In continuous systems, the water recovered is reused in Step 1. The solubility curves of fish muscle proteins show that myofibrillar proteins are soluble at acidic and basic pH (Fig. 4.9). Minimum water solubility, and therefore, maximum protein precipitation, occurs at pH 5±6. However, when IS is adjusted to 0.2, the minimum solubility shifts by ~1 pH unit and myofibrillar proteins precipitate at more acidic conditions. If acid or base is used in Step 2, then a base or acid, respectively, is required to adjust pH to 5.5. In either case, the salt formed increases the IS, and if the water is recycled as in a continuous recovery system, salt build up will occur, changing the pH at which myofibrillar proteins precipitate. Finally, sarcoplasmic proteins are soluble at both acidic and basic pH and do not precipitate with myofibrillar proteins (Fig. 4.8). However, as the IS increases, they begin to precipitate at pH 5.5. Therefore, sarcoplasmic proteins will be recovered in continuous systems, due to salt build up. The isoelectric solubilization and precipitation technology results in relatively high protein recovery even when using a batch mode system consisting of beakers and a typical laboratory centrifuge (Table 4.3). It is likely that a

Recovery of by-products from seafood processing streams

Fig. 4.9

79

Solubility of myofibrillar and sarcoplasmic fish proteins as a function of pH and ionic strength (IS).

continuous process would result in even higher recovery yields. In general, recovery yields are slightly higher for acidic solubilization (Table 4.3); however, protein gels made from proteins recovered at basic pH have higher texture quality (Fig. 4.10) and are described as firmer and whiter when compared to those obtained from proteins recovered at acidic pH (Hultin et al., 2005). Table 4.3 Protein recovery yield from processing trout by-products by the isoelectric solubilization/precipitation technology pH (solubilization/precipitation)

% protein recovery1

2.5/5.5 2.5/5.0 2.5/6.0 2.0/5.5 3.0/5.5 12.5/5.5 12.5/5.0 12.5/6.0 12.0/5.5 13.0/5.5

89.0 81.9 85.9 91.3 86.2 84.4 77.7 83.4 82.9 88.1

1 Calculated as (protein concentration in by-products/protein concentration in the recovered proteins)  100, all on a dry basis.

80

Maximising the value of marine by-products

Fig. 4.10

Effect of solubilization pH on the texture of gels prepared from proteins recovered from fish by-products.

The materials recovered from fish by-products in Step 3 include fish-oil rich in !-3 PUFA and the fat-free fraction rich in minerals such as Ca, Mg and P, and in Step 5 proteins recovered along with water to be reused in Step 1 (Fig. 4.11). If the proteins recovered are for use in foods, knowing their nutritional value is essential. The proteins recovered from trout frames and whole krill contain all essential amino acids (EAA), and even though they fall short when compared to the ideal pattern established by the Food and Nutrition Board, they are an

Fig. 4.11 Major materials recovered from processing fish by-products using the new isoelectric precipitation and solubilization of proteins technology: (a) Fish oil recovered in Step 3. (b) Fat-free fraction containing bones, skin, scale, fin, insoluble proteins and others recovered in Step 3. (c) Proteins recovered in Step 5. (d) Gel-forming ability of recovered proteins.

Table 4.4 Essential amino acid composition of the proteins recovered from krill and trout frames using the isoelectric solubilization/precipitation technology compared with that of a soy protein isolate and the high-quality protein pattern established by the Food and Nutrition Board Research Council Essential amino acids

Trout proteins solubilized at pH

Krill proteins solubilized at pH

Thr

Val

Met

Ile

Leu

Phe

His

Lys

Trp

Total

Average

trout frames

1.8

2.2

1.4

1.8

3.1

1.6

1.2

3.5

0.5

17.2

17.2

2.0 2.5 3.0

3.7 3.4 3.7

4.6 4.3 4.7

2.6 2.2 2.6

3.9 3.6 4.0

6.6 6.0 6.6

3.4 3.1 3.4

2.1 1.9 2.1

7.4 6.7 7.3

1.0 0.9 0.9

35.3 32.3 35.2

34.3

12.0 12.5 13.0

3.8 3.9 4.1

5.0 4.9 5.1

2.6 2.6 2.6

4.2 4.1 4.3

6.9 6.9 7.1

3.5 3.5 3.7

2.3 2.2 2.3

7.6 7.6 7.8

1.1 1.1 1.2

37.2 36.9 38.2

37.4

whole krill

2.2

2.6

1.5

2.5

4.0

2.2

1.1

4.4

0.7

21.2

21.2

2.0 2.5 3.0

4.8 4.5 4.8

6.0 5.8 5.9

2.9 3.2 3.3

5.5 5.7 5.9

9.0 8.9 9.2

5.0 4.9 5.2

2.6 2.5 2.6

9.2 9.2 9.6

1.5 1.6 1.6

46.6 46.3 48.1

47.0

12.0 12.5 13.0

4.6 4.5 4.4

5.8 5.6 5.5

3.4 3.2 3.1

5.7 5.5 5.5

8.8 8.6 8.4

5.1 5.0 4.8

2.7 2.5 2.5

9.2 8.9 8.7

1.7 1.5 1.5

47.0 45.3 44.3

45.5

soybean

3.9

4.6

1.1

4.6

7.8

5.0

2.6

6.4

1.4

37.4

37.4

FNB

3.5

4.8

2.6

4.2

7.0

7.3

1.7

5.1

1.1

37.3

37.3

Source: Adapted from Hui, Y.H. 1999. Soybean and soybean processing. In: Wiley Encyclopedia of Food Science and Technology, 2nd edn. Francis, F.J. (ed.). John Wiley & Sons. Hoboken (United States). Abbreviations: Thr threonine, Val valine, Met methionine, Ile isoleucine, Leu leucine, Phe phenylalanine, His histidine, Lys lysine, Trp tryptophan.

82

Maximising the value of marine by-products

Fig. 4.12 Improvement of the viscoelastic properties of krill protein gels using beef plasma proteins (BPP).

excellent source of methionine and lysine (Table 4.4). Lysine concentration is also critical for some non-food applications. For example, lysine-rich proteins can be chemically modified to make biodegradable super-absorbent hydrogel (SAH) trapping 400 g water/g SAH and are potential substitute for nonbiodegradable hydrocarbon-based SAH used in diapers, paper towels and others (Damodaran, 2004). Krill has extremely potent endogenous proteolytic enzymes limiting the development of food products from krill (Suzuki, 1999; Kolakowski and Lachowicz, 1982). Beef plasma protein (BPP) has been used as a protease inhibitor for surimi produced from fish species prone to enzymatic proteolysis such as Pacific whiting (Choi et al., 2005). When krill protein paste was formulated without BPP and slowly heated in a dynamic rheometer, extensive proteolysis occurred up to 60ëC and the proteins failed to form a gel. However, when BPP was added to the krill protein paste and subjected to the same heat treatment, the recovered proteins gelled, suggesting that krill proteases are present in the proteins recovered (Fig. 4.12). There are several protease inhibitors commercially available besides BPP. 4.4.2 Some equipment considerations Following homogenization (Step 1), the homogenate is pumped to the first bioreactor for a 10-min solubilization reaction (Step 2). The bioreactor is equipped for continuous pH control, and because the incoming homogenate pH is close to neutrality (~6.6±7.0), a base will be rapidly pumped into the vessel to adjust its pH to 11. Bioreactors are also equipped with mixing baffles to prevent pH

Recovery of by-products from seafood processing streams

83

Fig. 4.13 Bioreactors equipped with automatic pH and temperature controls, continuous pumping of feed and treated stream, and dosing of food-grade additives such as emulsion breakers, protein flocculants and antifoaming agents. Bioreactor A is used for protein solubilization (Step 2) while Bioreactor B is used for isoelectric precipitation (Step 4). A control box is placed between both bioreactors. This configuration is working in a continuous mode at flow rate of 300 L/h.

gradients and excessive foaming. A refrigerant is used to maintain constant temperature while small pumps are used to inject food-grade emulsion breakers and antifoam agents. The experimental recovery system shown works at 300 L/h and can process ~43 kg/h of fish by-products (Fig. 4.13). Although these small scale bioreactors are manufactured from glass and stainless steel components, industrial strength polyethylene can be used in a fish processing plant. Based on the experimental system (Fig. 4.7), a modular 600-L bioreactor has been designed to process 12 tonne/day of fish by-products. Following the 10-min pH adjustment in Step 2, the solution is pumped to a decanter centrifuge (Fig. 4.14) working typically below 4000 g and commonly used in surimi processing plants. However, surimi technology does not work under acidic or basic pH, therefore, the assessment of an available decanter should be performed prior to use with this protein recovery technology. There are no pH issues when separating proteins (Step 5); however, this can be relatively slow unless the particle size of the precipitated muscle proteins is increased by promoting protein-protein hydrophobic interactions using an extended precipitation time (~24 h) in Step 4. The particle settling velocity under the centrifugal force (g) depends on the density differential between phases (
), viscosity (), and particle size

84

Maximising the value of marine by-products

Fig. 4.14 Decanter centrifuges, typical separating equipment used in fish processing, could be utilized in the new isoelectric solubilization/precipitation technology. (a) Commercial unit shown by courtesy of Alfa Laval. (b) Cross-sectional view of the decanter centrifuge bowl.

expressed as equivalent diameter (D), which is the only variable that a processor can modify in this protein recovery technology: Sˆ


  g  D2 

…4:1†

Using only 10-min in Step 4, protein particle size can be increased by adding commercially available flocculants approved by local authorities (Figs 4.15 and 4.16).

4.5

Protein recovery from surimi processing water

Surimi is minced fish meat repeatedly washed and dewatered that is used as raw material to produce seafood analogues such as crabmeat substitutes. In the year 2002, the annual frozen surimi production in the USA was over 95 thousand tonnes. In the Pacific Northwest, the most utilized fish species are Pacific whiting and Alaska Pollock. However, processing fish using surimi technology recovers only myofibrillar proteins (Lee, 1999), while the isoelectric solubilization/precipitation allows the recovery of 78 to 91% of all by-product proteins (Table 4.3). The relatively low protein recovery by the surimi technology means that proteins accumulate in the surimi wash water. The effluent water is high in biological oxygen demand (BOD), and therefore, should be treated before

Fig. 4.15 Reduction of the supernatant optical density by 10-min incubation of the pH precipitated fish proteins with an anionic flocculant of high molecular weight.

86

Maximising the value of marine by-products

Fig. 4.16 Supernatant appearance after 10-min incubation of the pH precipitated fish proteins with an anionic flocculant of high molecular weight (b) as compared to an untreated control (a).

discharging it into local watercourses. Even more important than the low recovery yield is the use of large amounts of freshwater, about 20 times the weight of the deboned meat (Lee, 1999). The low process efficiency, high freshwater consumption, and deleterious environmental impact of surimi plants are creating in some regions political pressures for their shutdown. At present, there is a pending court case filed by the National Environmental Law Center (NELC) against owners and operators of a seafood plant in Oregon. NELC claims that `the plant has been routinely violating the Clean Water Act, degrading local waterways and threatening endangered salmon and steelhead' (NELC, 2003). A chitosan-alginate treatment has been proposed recently at Oregon State University as a new technology alternative to lower the biological oxygen demand (BOD) of water discharged from surimi processing plants. Surimi wash water (SWW) can be treated effectively with a chitosan-alginate complex prepared at the optimum chitosan to alginate mixing ratio of 0.2 (w/w) and used at a complex concentration of 0.1 kg/ton of SWW (Fig. 4.17) (Savant and Torres, 2000, 2003; Wibowo, 2003; Wibowo et al., 2005a,b, 2006a,b). Chitosan, the deacetylated derivative of chitin, is recovered from crustacean processing, particularly shrimp. After the meat is extracted, the shells are demineralized using hydrochloric acid, washed and dewatered to obtain chitin which is then deacetylated chemically or enzymatically. Alginate, on the other hand, is a polysaccharide extracted from the cell walls of brown seaweeds and used in the food industry as a thickener, stabilizer or gelling agent. The crude protein content of the insoluble solids recovered by the chitosanalginate complex technology is over 70% (Wibowo et al., 2005b, 2006a), while the amounts of recovered proteins vary with the concentration present in SWW, which ranges from 0.5±2.3% (Lin and Park, 1996; Morrissey et al., 2000). After

Recovery of by-products from seafood processing streams

87

Fig. 4.17 Surimi wash water (SWW) treated with a chitosan-alginate polymeric complex prepared at a 0.2 weight ratio and used at rates lower and higher than the recommended value (0.1 kg/ton SWW). (a) Untreated SWW, (b) SWW adjusted to pH 6, (c±f) SWW adjusted to pH 6 and after addition of 0.05, 0.1, 0.2 and 0.3 kg/ton SWW.

treatment with the polymeric complex, the proteins are recovered by centrifugation and can be sent to a disposal site, incorporated into surimi, or sold as a feed ingredient for feeds. Recovering protein from SWW not only produces protein for food and feed production, but also generates treated water for potential reuse in the plant. Proteins recovered from SWW have high concentrations of essential amino acids. Animal studies have demonstrated the superior nutritional value and the safety of SWW solids recovered by Chi-Alg when tested at the levels recommended by commercial producers of animal feeds, i.e., under 15% (Wibowo et al., 2005a). These studies showed no difference in feed consumption and growth rate, while post-mortem examination of internal organs showed no visible signs of damage caused by feeding the experimental diet. Blood analysis using 20 indicators confirmed the superior nutritional value and safety of SWW solids recovered by Chi-Alg. Subsequent studies demonstrated that 100% substitution of dietary protein by these SWW proteins was also safe and nutritionally equivalent or superior to other protein sources showing higher protein efficiency ratio (PER) and net protein ratio (NPR) than the casein control (Wibowo et al., 2006b). This outcome has economic implications for the region where a surimi plant is located. For example, in Oregon, not a major poultry producer, an estimated 100 thousand tonnes of feed are needed to sustain broiler production representing an excellent market opportunity for recovered SWW proteins. Many protein sources have been employed to improve the mechanical properties of surimi gels. The most frequently used are egg white and whey protein concentrates; other sources such as leguminous extracts and porcine plasma protein have been proposed, too. These proteins are added to inhibit the Modori phenomenon, i.e., the proteolytic degradation of fish myosin when gels are incubated at about 60ëC, or to improve the setting phenomenon associated with improved mechanical properties by the action of endogenous and added transglutaminase enzymes (An et al., 1996; GarcõÂa-CarrenÄo, 1996; SaÂnchez et al., 1998; Benjakul et al., 2001). It appears that when added to surimi, low concentrations of SWW proteins can improve their mechanical properties with minimum impact on colour (RamõÂrez et al., 2006).

88

4.6

Maximising the value of marine by-products

Conclusions

The amounts of by-products generated from processing aquatic foods and the volumes of underutilized by-catch, discards, and low-value fish are staggering. At the same time, overfishing of several species is a common problem. Aquaculture has experienced great growth during the past 25 years, filling the supply gap that cannot be provided using natural resources. However, the environmental impact of this industry is beginning to slow its growth. It appears that the only great opportunity is to improve fish processing so as to better utilize existing resources. The new technology of isoelectric precipitation and solubilization of fish muscle proteins with concurrent separation of fish oil for the processing of by-product provides a great opportunity on a large scale. In the particular case of the surimi industry, the new technology based on using natural polymeric complexes for the treatment of process water, offers an opportunity to alleviate its environmental impact, freshwater use and low yield limitations. In addition to basic research on recovering valuable fractions, it is necessary to develop applications for the recovered products. Government agencies, industry and academia must collaborate in this effort.

4.7

References

1996. Roles of endogenous enzymes in surimi gelation. Trends in Food Science and Technology 7: 321±326. ANONYMOUS. 2004. The state of world fisheries and aquaculture. Food and Agriculture Organization, Rome. BENJAKUL, S., VISESSANGUAN, W., SRIVILAI, C. 2001. Porcine plasma protein as proteinase inhibitor in bigeye snapper (Priacanthus tayenus) muscle and surimi. Journal of the Science of Food and Agriculture 81(10): 1039±1046. CHOI, Y.J., KANG, I.S., LANIER, T.C. 2005. Proteolytic enzymes and control in surimi. In: Park, J.W. (ed.), Surimi and Surimi Seafood, 2nd edn. CRC Press, Boca Raton, FL. DAMODARAN, S. 2004. Protein-polysaccharide hybrid hydrogels. US Patent No. 6,821,331. Ä O, F.L. 1996. Proteinase inhibitors. Trends in Food Science and GARCIÂA-CARREN Technology 7: 197±203. GILDBERG, A. 2002. Enhancing returns from greater utilization. In: Bremner, H.A. (ed.), Safety and Quality Issues in Fish Processing. Woodhead Publishing Limited, Cambridge. HUI, Y.H. 1999. Soybean and soybean processing. In: Francis, F.J. (ed.), Wiley Encyclopedia of Food Science and Technology, 2nd edn. John Wiley and Sons, Hoboken. HULTIN, H.O., KELLEHER, S.D. 1999. Process for isolating a protein composition from a muscle source and protein composition. US Patent No. 6,005,073. HULTIN, H.O., KELLEHER, S.D. 2000. High efficiency alkaline protein extraction. US Patent No. 6,136,959. HULTIN, H.O., KELLEHER, S.D. 2001. Process for isolating a protein composition from a muscle source and protein composition. US Patent No. 6,288,216. HULTIN, H.O., KELLEHER, S.D. 2002. Protein composition and process for isolating a protein AN, H., MARGO, Y.P., SEYMOUR, T.A.

Recovery of by-products from seafood processing streams

89

composition from a muscle source. US Patent No. 6,451,975. 2005. Process for recovery of functional proteins by pH shift. In: Park, J.W. (ed.), Surimi and Surimi Seafood, 2nd edn. CRC Press, Boca Raton, FL. KIESSLING, A., AASGAARD, T., STOREBAKKEN, T., JOHANSSON, L., KIESSLING, K.H. 1991. Change in the structure and function of the epaxial muscle of rainbow trout (Oncorhynchus mykiss) in relation to the age. III. Chemical composition. Aquaculture 93: 373±387. KOLAKOWSKI, E., LACHOWICZ, K. 1982. Application of partial autoproteolysis to extraction of protein from Antarctic krill (Euphausia superba). Part 3. Changes in and yield of nitrogen substances during autoproteolysis of fresh and frozen krill. Die Nahrung 26: 933±939. LANIER, T.C., CARVAJAL, P., YONGSAWATDIGUL, J. 2005. Surimi gelation chemistry. In: Park, J.W. (ed.), Surimi and Surimi Seafood, 2nd edn. CRC Press, Boca Raton, FL. LEE, C.M. 1999. Surimi: Science and technology. In: Francis, F.J. (ed.), Wiley Encyclopedia of Food Science and Technology, 2nd edn. John Wiley and Sons, Hoboken. LIN, T.M., PARK, J.W. 1996. Extraction of proteins from Pacific whiting mince at various washing conditions. Journal of Food Science 61: 432±438. MORRISSEY, M.T., PARK, J.W., HUANG, L. 2000. Surimi processing waste: its control and utilization. In: Park J.W. (ed.), Surimi and Surimi Seafood, pp. 127±165. Marcel Dekker Inc., New York. MURANO, P.S. 2003. Understanding Food Science and Technology. Wadsworth/Thomson Learning, Belmont. NELC. 2003. Current Litigation: Suit Challenges Seafood Facility's Pollution of Columbia Tributary (http://nelconline.org/nelc.asp?id2=8687&id3=NELC&). Visited 12 April 2004. NICOL, S., ENDO, Y. 1999. Krill fisheries: Development, management and ecosystem implications. Aquatic Living Resources 12(2): 105±120. RAA, J., GILDBERG, A. 1982. Fish Silage: A Review. CRC Press, Boca Raton, FL. Â PEZ ECHEVARRIÂA, G., TORRES, J.A. 2006. Effect of adding RAMIREZ, J.A., VELAZQUEZ, G., LO insoluble solids from surimi wash water on the functional and mechanical properties of Pacific whiting Grade A surimi. Bioresource Technology (in review). Â NCHEZ, A., RAMIÂREZ, J.A., MORALES, O.G., MONTEJANO, J.G. 1998. Detection of protease SA inhibitors in leguminose extracts and its effect on endogenous proteases from fish muscle. Ciencia y Tecnologia Alimentaria 2(1): 12±19. SAVANT, V.D., TORRES J.A. 2000. Chitosan based coagulating agents for treatment of Cheddar cheese whey. Biotechnology Progress 16: 1091±1097. SAVANT, V.D., TORRES J.A. 2003. Fourier transform infrared analysis of chitosan based coagulating agents for treatment of surimi waste water. Journal of Food Technology 1(2): 23±28. STROM, T., EGGUM, B.O. 1981. Nutritional value of fish viscera silage. Journal of the Science of Food and Agriculture 32: 115±120. SUZUKI, T. 1981. Fish and Krill Protein: Processing Technology. Applied Science Publishers, London. SUZUKI, T. 1999. Krill protein processing. In: Francis, F.J. (ed.), Wiley Encyclopedia of Food Science and Technology, 2nd edn. John Wiley and Sons, Hoboken. SUZUKU, T., SHIBATA, N. 1990. The utilization of Antarctic krill for human food. Food Reviews International 6(1): 119±147. HULTIN, H.O., KRISTINSSON, H.G., LANIER, T.C., PARK, J.W.

90

Maximising the value of marine by-products

2004. Overview of fish production, utilization, consumption and trade. Food and Agriculture Organization Fisheries Information, Data and Statistics Unit, Rome. WHITNEY, E., ROLFES, S.R. 2005. Understanding Nutrition, 10th edn. Wadsworth/Thomson Learning, Belmont. WIBOWO, S. 2003. Effect of the molecular weight and degree of deacetylation of chitosan and nutritional evaluation of solid recovered from surimi processing plant. PhD dissertation, Corvallis, OR: Oregon State Univ. WIBOWO, S., SAVANT, V., CHERIAN, G., SAVAGE, T.F., TORRES, J.A. 2005a. Evaluation as a feed ingredient of surimi wash water protein recovered using a chitosan-alginate complex. Journal of Aquatic Food Products Technology 14(1): 55±72. WIBOWO, S., VELAZQUEZ, G., SAVANT, V., TORRES, J.A. 2005b. Surimi wash water treatment for protein recovery: effect of chitosan-alginate complex concentration and treatment time on protein adsorption. Bioresource Technology 96: 665±671. WIBOWO, S., VELAZQUEZ, G., SAVANT, V., TORRES, J.A. 2006a. Effect of the chitosan type on the protein and water recovery efficiency from surimi wash water treated with chitosan-alginate complexes. Bioresource Technology (in press). WIBOWO, S., SAVANT, V., CHERIAN, G., SAVAGE, T.F., VELAZQUEZ, G., TORRES, J.A. 2006b. A rat feeding study to determine the safety and nutritional value of surimi wash water proteins (SWWP) recovered using a chitosan-alginate complex as a food or feed ingredient. Journal of Food Science (in review). ZUGARRAMURDI, A., PARIN, M.A., LUPIN, H.M. 1995. Economic engineering applied to the fishery industry. Food and Agriculture Organization Fisheries Technical Paper 351, Rome. VANNUCCINI, S.

5 Increased processed flesh yield by recovery from marine by-products K. D. A. Taylor and A. Himonides, University of Lincoln, UK and C. Alasalvar, TUBITAK, Turkey

5.1

Introduction

5.1.1 Waste from primary processing Filleting is a primary method of processing fish in many countries around the world. In the preparation of fish for today's consumer up to 50% of the whole fish is commonly discarded as waste (Windsor and Barlow, 1981). This comprises skeletal structure, intestinal organs, and also a large amount of edible fish muscle that cannot easily be removed from the bone structure by conventional fish filleting processes. There is also additional wastage arising as a consequence of further processing of fish fillets, e.g. skinning, incorrect weights, and visual appearance problems such as blood spots. Notable exceptions to this are trout and herring, which are usually sold or processed whole. Archer et al. (2001) estimated the quantity of on-shore processing waste in the UK from demersal fish processing as 154 143 tonnes per year. The principle use for the waste has been for fishmeal production (for which the processor will receive about £20/tonne waste). Fishmeal is primarily used as animal feed. Hence for the primary fish processors the result is a large difference in income between the fillets and the waste (Fig. 5.1). Some specific types of waste, however, are utilised for human consumption, e.g. cod livers for production of cod liver oil. Conversion of constituents of these wastes into food for human consumption, useful products, or extraction of valuable components prior to use for fishmeal would be both an improved utilisation of the resource and could be financially beneficial to the primary process. This chapter details the increased processing yield by flesh recovery from by-products.

92

Maximising the value of marine by-products

Fig. 5.1

Price for fish and fish waste.

5.1.2 Physical processing methods and by-product recovery from filleting waste The remaining flesh attached to the filleting waste of fish, such as cod and haddock, that cannot be removed by the filleting process, can be up to 60% of the waste's weight (Ravichander and Keay, 1976). The recovery of this flesh is a much studied field of waste utilisation. A variety of methods have been developed for the separation and recovery of fish flesh. Generally, these can be divided into mechanical and non-mechanical techniques (Grantham, 1981). Non-mechanical separation techniques, consist of chemical and biochemical methods, which normally involve the enzymatic, or acid proteolysis of waste material and the recovery of the hydrolysed (solubilised) flesh, by means of centrifugation, or filtration. The application of heat and the separation of the denatured protein (flesh) from the skeletal bones, using pressurised water, has also been reported (Grantham, 1981). Although the yields of recovery are generally high with these methods, protein functionality is often irreversibly changed. Mechanical separation techniques normally result in products that retain most of the nutritional value and organoleptic characteristics of fish flesh. Simple physical removal of tongues and cheeks can provide a nutritional and valued by-product. The application and further development of the mechanical recovery of fish flesh is desirable because it provides maximum yields at a reasonable processing cost and produces a product (minced fish) that offers scope for further improvement. As long as there is a demand for such products, the mechanical separation of fish flesh from filleting waste offers a profitable alternative to the production of fishmeal or fish silage.

5.2

Recovery of flesh from filleting waste

5.2.1 Fish cheeks and tongues The cheeks and tongues of large fish, such as cod and haddock, are often removed manually. Equipment to remove the cheek is commercially available,

Increased processed flesh yield by recovery from marine by-products

93

but has not been widely adopted (Regenstein, 2004). The meat from tongues and cheeks can be used in pies, fishcakes or retextured products. However, fried cod cheeks and tongues are considered as delicacies in different parts of the world (e.g., Italy, Canada, and Spain). Hence they can command similar or higher prices to fillets. 5.2.2 Flesh-bone separators The development of flesh-bone separators started in Europe and North America and evolved rapidly from the first machines available, taken from other industries (fruit and meat industry), to the later specialised equipment of the fish industry (Drews, 1976). Most of the mechanical separation techniques are based on the physical screening of flesh from non-flesh components through a perforated filter. Such devices should have the ability to separate and recover as much of the remaining flesh from the waste material as possible, without destroying the fibroid structure of the flesh. This is normally achieved by pressing the raw material through perforations small enough to retain the bones, but big enough to allow the flesh to pass through. The smaller the perforations, the smaller the chances are for a bone to pass through. However, very small perforations also means unwanted reduction of the flesh particles, with serious results on the products texture (Drews, 1976). Thus, there has to be a compromise between quality (in terms of texture, number of bones, etc.) and also quantity (yields) of recovered fish flesh. Flesh-bone separators can be divided into three main groups depending on the operation principles used (Grantham, 1981). · A belt and a drum is used by Baader, Pibun, Prince, Seffelaar and Looyen, Yanigiya, and Yiedmaster. Many modifications of that system have been developed. · A screw feed and perforated cylinder system is used by Beehive. · Two concentric cylinders (the inner perforated and rotating) which break up the bones and separate flesh by a micro-groove principle are used by Paoli. All systems have their own advantages and disadvantages. The belt and drum systems benefit from readily adjustable pressure although belt wear can be high when using raw materials with large, or hard bone particles. Most separation equipment was designed to be quick to dismantle, so that cleaning can be easy and thorough. It is absolutely essential that all parts of the machinery and equipment that may come in contact with the product are hygienically clean in order to minimise bacterial contamination. The screw cylinder systems do not have the same wear problems, but generate higher shear rates and consequent textural damage. In general, these systems are more expensive, but all give similar yields ranging from 60±80% for whole fish to 30±70% for filleting waste. Despite the apparently simple operating principles, the relationships between pressure, perforation size, and perforation area with yield, contaminant levels, and shear damage, are complex (Grantham, 1981).

94

Maximising the value of marine by-products

The Baader flesh-bone separator The Baader separation technique is based on the physical screening of flesh from non-flesh components through a perforated filter (Drews, 1976; Grantham, 1981). This is performed by a thick elastic conveyor belt and a metallic perforated drum (Figs 5.2 and 5.3). Both belt and drum are driven by a gear motor at the same speed. The raw material goes through an opening in a feed hopper onto the belt, which partly encompasses the drum and is pressed tightly against it by an adjustable roller. The material is squeezed through the perforation, to the interior of the drum and then it is discharged from the end of the drum by a stationary screw. Thicker bones get embedded in the elastic bed which squeezes off all the flesh around the bone. Bones and pieces of skin remain on the outside of the drum and are removed by a scraper blade. The rotation of the belt and drum are designed to minimise grinding or tearing of the material which could destroy the fibroid structure of the flesh. A small perforation size could also damage the flaky texture of the flesh, so even though there is a range of drum hole sizes (from 1 to 10 mm), perforations of 3 or 5 mm give the best results in terms of compromising an acceptable texture with a low percentage of bones escaping in to the mince and a high yield (Drews, 1976; Grantham, 1981).

Fig. 5.2

Baader flesh-bone separator.

Increased processed flesh yield by recovery from marine by-products

95

Fig. 5.3 Baader flesh-bone separator showing the perforated drum and mince production.

5.2.3 Minced fish The term minced fish is very often used to describe the mechanically recovered fish flesh. This term is one of convenience rather than accuracy since mincing occurs naturally during separation rather than deliberately as if it were passed through a mincer. It is indeed a product that retains most of its textural characteristics compared with fish which has been minced (Ravichander and Keay, 1976). Mincing accelerates the deconformation, aggregation and cross linking of the myofibrillar proteins. This results in a loss of the extractability and contractility of the actomyosins, with a consequent increase in the objective toughness, granularity and drip loss and a decrease in water binding capacity, emulsification capacity, gel-forming ability, and rheological properties. These reactions are exacerbated through the cross linking of the proteins with formaldehyde, which is derived from the breakdown of trimethylamine oxide (TMAO). This is an enzyme related reaction which is accelerated by mincing and the mixing of blood and other organs with the flesh and predominantly occurs during frozen storage. The gadoid fish species and particularly hake and cod are very susceptible to this reaction (Babbitt et al., 1974). Denaturation in frozen mince products is also accelerated by the rapid pH drop resulting from accelerated glycolysis during mincing and by temperature cycling during frozen storage (Grantham, 1981). Fat degradation can be accelerated by mincing, due to dispersion of fat degrading enzymes and oxidation catalysts and the increase of the surface area.

96

Maximising the value of marine by-products

Pelagic species are more susceptible to fat degradation, especially when processed whole, due to the high lipid content and particularly the high levels of unstable polyunsaturated lipids located on the skin, tissues, viscera and brain that may contaminate the flesh during separation. Processing of filleting waste (frames and flaps) from lean demersal species such as cod and haddock is thus less susceptible to fat oxidation, due to negligible lipid content and absence of contaminating materials such as skins, viscera, and brains. One of the primary reasons for low acceptance of minced fish products is due to the aesthetic deterioration that occurs during mechanical separation of the flesh. Mincing normally results in a product that is darker in colour and more heterogeneous than the actual flesh in the raw material. This is due to the natural mixing of components such as blood, pigments, skin, and membrane particles, bones, and other material that can pass through the perforations of the drum. Consequently, this aesthetic deterioration is not so marked in mince produced fish with coloured flesh such as salmon and salmon mince which can have a value as high as £2000/tonne. Salmon mince can be used in fishcakes, minced salmon nuggets, pastes, pates, and low cost ready meals. Although there is an enormous number of minced fish products that have been studied, reported, and developed (Bligh and Regier, 1976; King, 1976; Regenstein, 1980), only a few of these are successfully established in the world market. According to the same authors, poor recognition of the true value and potential of fish mince is often associated with the trend to develop products that mimic existing fish products, rather than creating something new that exploits the natural properties of fish mince. The world market of fish mince is dominated by products derived from frozen fish mince blocks and surimi-derived products (Grantham, 1981; Suzuki, 1981). Frozen mince blocks are usually produced from fish mince, derived from white fish such as cod and haddock. Fish mince is also incorporated into fillet blocks. These blocks are intermediate material for the production of a range of products such as coated fingers, steaks, cakes, sausages, patties, loaves, and burgers. Some of these are simply formed and coated products whilst other are totally transformed, reformed, and textured products (Grantham, 1981). The manufacture of surimi initiated in Japan where it is still believed to be the primary source of the population's protein intake. Surimi-based products have also been introduced to North America and Europe. This market is now facing a rapid growth. Surimi is basically frozen water-washed fish mince with cryoprotecting substances (Suzuki, 1981). It is primarily manufactured from Alaska pollack and other gadoid fish minces, although recent studies have shown that even pelagic species can give acceptable products (Grantham, 1981; Suzuki, 1981). Surimi is an intermediate processed seafood product, used in the formulation/fabrication of a variety of products (Hall and Ahmad, 1997), such as kamaboko and chikuwa (Suzuki, 1981). Fish mince is also used for the production of canned products (Regenstein, 1980; Grantham, 1981), dried and intermediate moisture products (IMP), fermented products, and for the production of animal feed (Taylor and Alasalvar, 2002).

Increased processed flesh yield by recovery from marine by-products

97

5.2.4 Problems of fish mince Mechanical separation of flesh from demersal species such as cod and haddock can accelerate the degradation of lipids, proteins and bacteriological quality and also results in a product that is often aesthetically unacceptable. However, one of the great advantages that minced fish offers is the possibility of great control over product flavouring, texture, appearance, and storage properties. Minced fish can be mixed with a variety of foodstuffs, including other fish. In subsequent processing, a range of textural variations can be induced by mechanical mixing, often in the presence of salt and other additives, and furthermore the addition of flavour and other desirable additives is more effectively accomplished (Ravichander and Keay, 1976). With respect to lipid oxidation, the use of antioxidants is the most widely studied and applied method of stabilisation. However, other techniques such as glazing and oxygen impermeable packaging were found to inhibit the oxidative deterioration of mince blocks. Protein stability and functionality enhancement of de-boned flesh is a widely studied subject, mainly due to the particular susceptibility to protein degradation and also to the high inherent functionality of mince. With respect to protein stabilisation during frozen storage, a wide range of stabilisers and cryoprotecting agents, such as polyphosphates, sugars, polysaccharides, and peptides, have been studied (Grantham, 1981). The study of protein functionality involves both the preservation of the inherent functionality of fish mince and/or the enhancement of certain functional properties depending on the uses of the material. For example, many cryoprotecting agents, such as phosphates and salts, which are used for the stabilisation of mince during frozen storage and thus maintain the inherent functionality of mince, are also used as functionality enhancers for their solubilisation, complex forming, and gel-forming effects. Hydrocolloids are used extensively to increase viscosity, water binding, and gel-forming capacities. A wide range of proteins are used to improve binding properties. Sugars are used in dried mince products to reduce denaturation, whilst starches are used as thickeners and gelling agents. Furthermore, fats and oils are used to improve succulence in combination with added proteins as emulsifiers (Grantham, 1981). Whiteness and colour homogeneity of fish mince derived from white flesh fish are prerequisites that determine retail price and also consumer acceptance, with the whiter mince attracting a much higher price (Meacock et al., 1997). Methods of improving mince appearance involve treatment of the fish material prior to mechanical separation, whitening of the recovered flesh, or masking of the mince colour with coloured additives. The addition of sawdust waste from band sawing of frozen blocks of white fish to fish mince can also improve the whiteness. When processing whole fish, the removal of heads and guts is essential prior to separation. This minimises the contamination of the recovered mince from coloured, non-flesh components such as blood, skin, and brain fragments, and improves the appearance and also storage life of mince (Ravichander and Keay, 1976; Connell and Hardy, 1982). Contamination of mince also occurs during the mechanical separation of cod and haddock frames, due to the crushing of the

98

Maximising the value of marine by-products

backbone section and the subsequent mixing of the blood with the separated flesh. Thus, the removal of this section prior to separation improves the appearance of the recovered mince (Ravichander and Keay, 1976; Connell and Hardy, 1982).

5.3

Recovery of flesh from demersal species

The mechanical recovery of the remaining flesh attached to the frame and flap, which cannot be removed by the filleting process is a much studied field of waste utilisation and is a potential alternative to fishmeal production. With the advent of drum flesh bone separators, fish mince production from the filleted fish frames and trimmings began, with up to 10% of the whole fish weight or 25% of the gutted weight of an averaged sized cod being recovered (Connell and Hardy, 1982). Table 5.1 shows the recovery of minced flesh from various parts of cod waste. This recovered flesh is nutritionally similar to the fish fillet. However, the visual appearance of mince which is derived from the filleting waste (flaps and frames) of white fish such as cod or haddock, is poor, principally due to the presence of pigments such as haemoglobin from blood and other haem proteins, mixed with the flesh during separation. This discolouration does not present a major problem in the case of non-white fish such as salmon. There are also associated quality problems, i.e. the lack of texture and the poor microbial profile that can result from poor handling and storage of the waste, and the additional processing required for the production of fish mince. Consequently, the amounts paid for the mince are generally mediocre, in the region of £800/tonne for white fish mince. Fish mince colour partly determines retail prices and consumer acceptance, with the whiter minces attracting a higher price than those contaminated with traces of blood. In the UK, fish mince is classified as Class A (little discolouration), Class B (discoloured with blood and haem proteins), and Class C (discoloured), and the value ranges from £400 to £1000/tonne. For these reasons, development of a method of producing a white or whiter fish mince is commercially desirable. The use of recovered flesh is primarily for human consumption in products such as fish cakes or fish fingers. As a major factor in consumer unacceptability for mince from white fish is the fish mince colour, several approaches have been investigated to overcome this. Table 5.1

Recovery of minced cod fish using Baader 694 flesh-bone separator

Whole frame Frame with back bone removed Cod flaps

Recovery (%)

Weight of fish (%)

72±82 55±65 (of whole frame) 60±67

8±10 6±8 3±4

Increased processed flesh yield by recovery from marine by-products

5.4

99

Quality and improvement of fish mince

5.4.1 Washing Washing or soaking of waste with water, prior to separation, removes part of the superficial contamination of the waste (blood, clots, and impurities) and has a small effect in improving the colouration (i.e. in reducing the red pigmentation) of the recovered mince (Ravichander and Keay, 1976; Grantham, 1981). Direct water washing of fish mince can elute part of the blood and improve the colour of mince to an off-white colouration (Ravichander and Keay, 1976; Grantham, 1981; Connell and Hardy, 1982). Alkali or acid washing is believed to improve the separation of blood (Steinberg et al., 1977; Grantham 1981). Common problems generated from the washing of the mince include the removal of the excess water and the significant loss of soluble proteins, unless a method of recovering the proteins is applied (reverse osmosis, ultrafiltration, or coagulation). 5.4.2 Use of high pressure Possible decolouration of haemoglobin in fish mince via the use of high pressure has been postulated by Hoover et al. (1989), but textural changes are a possible side-effect of such a process. 5.4.3 Removal of back bone prior to flesh bone separation Most of the blood and discoloration is in/along the spinal cord. Thus, during mechanical separation of mince from cod and haddock frames, the crushing of the backbone section results in mixing of this blood with the separated flesh. The removal of this section prior to separation improves the appearance of the recovered mince (Ravichander and Keay, 1976; Connell and Hardy, 1982). Himonides (2001) investigated the effect of removal of the backbone by a V cut prior to flesh bone separation. This process provided a much better quality mince than that of the whole frames with only about 25% reduction in recovery of flesh from the frame (Table 5.1). The recovered minced flesh accounted for 6±8% of the weight of the original cod. Whilst this was shown to work well in the laboratory, it is uneconomic on a commercial scale (until an effective machine is available for the removal of the backbone) owing to the labour required. 5.4.4 Whitening the mince with hydrogen peroxide (H2O2) Hydrogen peroxide has been used to bleach white fish mince such as that derived from cod (James and McCrudden, 1976; Young et al., 1980; Connell and Hardy, 1982), but problems have been reported concerning textural changes, and loss of proteins and amino acids through dissolution and oxidation. The study of Young et al. (1980) revealed that the application of 0.75% H2O2 at alkaline pH has a remarkable bleaching effect on mince derived from cod waste. James and McCrudden (1976) found that 1% sodium polyphosphate

100

Maximising the value of marine by-products

added into the whitening solution has a synergetic effect on both whitening and texture. They also believed that the nutritional value of treated fish was retained. The effect of the treatment on the oxidation state of the lipids, measured by peroxide and epoxide values appeared to be negligible. The same authors, however, found a significant loss of protein due to dissolution, increase in insolubilisation of protein and loss of sulphur-containing amino acids, even under relatively mild treatment conditions. According to the same authors, other workers found no change in the amino acid content of other proteins treated with H2O2. The effect of H2O2 on the amino acid content and protein digestibility of saithe was investigated by Raksakulthai et al. (1983), who found that treatment of samples with 3.2 g/L H2O2 for a period of 18 h oxidised methionine. Oxidation mainly occurred during the first five hours of treatment. Methionine was not entirely oxidised even after the addition of a further 3.2 g/L H2O2. The authors also stated that other amino-acids seemed to be essentially unaffected by the oxidant, while digestibility was slightly increased by the treatment. Also, many workers have reported the disinfecting action of H2O2 on various foods including fish (Stout and Carter, 1983; Wheaton and Lawson, 1985; Shibamoto et al., 1993). In spite of the reported difficulties using H2O2, Himonides et al. (1999) considered that this had a commercial and technical potential for whitening the mince. They observed that the discolouration of flaps is mainly a superficial phenomenon and consequently treated whole flaps with H2O2 prior to separation for the production of fish mince. This differs from previous works in which the whitening treatment occurred after the flesh was separated and minced. The application of the bleaching agent prior to mincing should minimise protein insolubilisation and also the loss of water-soluble proteins, with minimal effect on the nutritional value or texture compared to the direct application of H2O2 on fish mince. The effect of concentrations of peroxide and time of soaking is shown in Table 5.2. Samples were removed from the H2O2 solution after the time shown, soaked in water for 10 min and then assessed visually for colour improvement, and textural damage. Immediately after treatment, flaps (particularly those of small size) may appear to have a loose flesh texture, with areas of skin that appear to be swollen owing to the oxygen trapped under the surface of the skin. This textural effect, however, is minimised after soaking flaps in water, or even after leaving the flaps out of solution, for a short period of time (30 min), during which the oxygen bubbles dissipate. For industrial applications, concentrations of 5±8 g H2O2/kg were recommended, with a soaking time of 90 min. Visual sensory evaluation of the mince derived from standard and treated flaps (soaking for 1.5 h in 8 g/L H2O2) showed noticeable improvement in the elimination of the red colouration. The degree of improvement depends on the initial condition of flaps and presumably on the depth of blood discolouration in the flesh. Colourimetric measurements confirmed a highly significant reduction of the red pigment (a values) and also a reduction of the yellow pigment towards the grey area (b values). No significant difference was detected between the taste

Increased processed flesh yield by recovery from marine by-products 101 Table 5.2 Whitening effect of a range of H2O2 concentrations on flaps, for different time intervals H2O2 (g/L)

5 min

10 min

30 min

40 min

60 min

120 min

0.5 1 2.5 5 8 10

0 0 s.i. n.i. n.i. 3 p.t.c.

0 0 s.i. n.i. n.i. 3 p.t.c.

0 0 s.i. n.i. n.i. 3 p.t.c.

0 s.i. s.i. 3 3 3 t.c.

0 s.i. s.i. 3 3 3 t.c.

0 s.i. s.i. 3 3 3 t.c.

0: no improvement, s.i.: small improvement, n.i.: noticeable improvement, 3: good whitening effect, p.t.c.: possible textural change, t.c.: textural change.

and odour of treated and standard cooked mince, by the sensory evaluation carried out before and after 8 months cold storage (±28ëC) of the samples. Microbiological analysis showed that treatment with H2O2 significantly reduces the total viable count of cod flaps. No residual H2O2 was detected in flaps immediately after treatment, using oxygen electrode technique (the limit of detection was 3 g/g) (Himonides et al., 1999). Immediately after treatment and washing of flaps, it is possible that there may be residual H2O2 trapped in the flesh. It has been demonstrated, however, that hyperoxidase (catalase), which exists in the flesh cells, converts residual H2O2 into O2 and H2O, particularly during mincing, owing to the release of the enzyme from the cells. The existence of the enzyme was confirmed in cod and haddock mince and was found to be present in the fish mince even after the eight months of frozen storage period. Since H2O2 cannot be detected after treatment and the products (O2 and H2O) are non-toxic, H2O2 can be characterised as a processing aid rather than a food additive. As a result, and according to the food legislation, the declaration of the use of H2O2 by the processor is not necessary. The procedure developed for and used by industry is shown in Fig. 5.4. 5.4.5 Whitening the mince with titanium dioxide (TiO2) An alternative to removing the discolouration, or bleaching the mince, is to mask the colour using whitening chemicals, such as TiO2 (Grantham, 1981; Connell and Hardy, 1982), vegetable fat based agents (Ravichander and Keay, 1976), hydrophilic colloids such as milk, gum hydrocolloids, mixtures of sugars, surfactants, and fats. Polyphosphates can have a similar whitening effect via the dispersion of myosin sol. Common problems with colour masking include the generation of a nonuniform colour, (given the highly variable colour of fish mince), the possible artificial appearance of the resultant colour, and the necessity to produce an emulsion, or suspension, which is capable of surviving the cooking/freezing process, in order to maintain the uniform colour. Consequently, Meacock et al.

102

Maximising the value of marine by-products

Fig. 5.4

Improved whitening of cod and haddock flaps using H2O2.

(1997) investigated the use of TiO2 coupled with a variety of dispersing agents with the aim of identifying a system to overcome these problems and which also would not affect the taste, odour, or texture of the material. To determine which suspension agents might be most suitable, a series of dispersed suspensions using TiO2 were prepared, using mixtures which were chosen on their existing use as suspension agents in a variety of products. Stable dispersions were achieved with several agents but only xanthan gum gave a stable and non-odorous dispersion. However, a `musty' taste and odour developed after a few weeks storage at room temperature of the dispersed TiO2 suspension dispersed with xanthan gum. Thus, such dispersed suspensions

Fig. 5.5

Titanium dioxide based whitener.

Increased processed flesh yield by recovery from marine by-products 103 should be used within 3 days of production. Fish mince produced using TiO2 in water produce an uneven dispersion, with a toothpaste-type appearance to fish mince products after cooking. The TiO2/xanthan gum dispersion is easily incorporated into the mince, with excellent distribution. It gives an even increase in whiteness which is stable after frying the product or microwaving. Although at concentrations of up to 250 ppm of TiO2, the whitening effect is negligible, the whiteness increases with further increasing concentrations of TiO2. A level of 1 g of TiO2/kg mince gives a most acceptable level of whiteness compared with the colour of cooked cod fillet. At this level, no differences were detected by expert panels, triangle tests, or the texture analyser regarding the resulting texture of whitened fish mince. Figure 5.5 summarises the whitening procedure. 5.4.6 Masking colour As a major problem with full acceptance of minced flesh from white fish is the colour, a possible alternative approach is to develop products which the consumer would accept as being of a definite acceptable colour, other than white. Products such as curried fish cakes and tomato flavoured fish cakes have been developed (Meacock, 1994), but not yet produced on a commercial scale. Initial consumer trials indicated that masking of the colour of the mince is an acceptable method of upgrading, but suitable retail outlets have to be identified for products such as curried cod fish burgers.

5.5

Future trends

Fish mince production is a viable process and is currently carried out using both white fish and salmon filleting wastes. However, in the case of white fish waste the use and hence the market for the mince is limited due to the colour of the resultant mince. The value of white fish mince is approximately £800/tonne, but does depend on quality, which is measured primarily by whiteness. The recovery of 10% of the weight of the fish as fish mince is potentially financially viable, and is practised by several firms. The additional costs include purchase of a flesh-bone separator (which could be from £14,000 for a second-hand one to £40,000), labour, electricity, and packaging/freezing. There are possible techniques for improvement of the colour and the alternative approach to develop products which the consumer would accept as being of a definite colour. Technological developments are likely to assist in the improved and economic recovery of edible flesh from the by-products of filleting and other primary processes. For example, an EU project funded to develop a process for automated tuna head meat recovery reported that optimisation of the prototype machine will make the production of automatically recovered meat economically feasible (Stefansson, 2001). The incentive for improved utilisation

104

Maximising the value of marine by-products

is threefold as it 1) reduces waste, and the problems of disposal are likely to increase, 2) increases the utilisation of harvested fish for food, and 3) could increase the profitability of processing firms.

5.6

Sources of further information and advice

In addition to the standard scientific literature, the following organisations are a source of advice concerning the recovery of flesh from fishery by-products. Sea Fish Industry Authority St Andrews Dock Hull, HU3 4QE United Kingdom Tel: +44 (0)1482 327 837 Fax: +44 (0)1482 223 310 Web-site: www.seafish.org Baader UK Ltd Nautilus House, Prospect Point 35 Waterloo Quay Aberdeen, AB11 5BS United Kingdom Tel: +44 (0) 1224 597320 Fax: +44 (0) 1224 597321 E-mail: [email protected] Web-site: www.baader.com

5.7

References

(2001), `Fish waste production in the United Kingdom ± The quantities produced and opportunities for better utilisation', Hull, UK, Seafish Report No. SR537, The Seafish Industry Authority. BABBITT J K, CRAWFORD D L, LAW D K (1974), `Quality and utilisation of minced fish muscle', 2nd Technical Seminar on Mechanical Recovery and Utilisation of Fish Flesh, Boston, MA. BLIGH E G, REGIER L W (1976), `The potential and limitations of minced fish', in Keay, J N, Proceedings of the Conference on the Production and Utilisation of Mechanically Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 73± 77. CONNELL J J, HARDY R (1982), Trends in fish utilisation, Surrey, UK, Fishing News Books Ltd. DREWS J (1976), `Development of fish boning machines', in Keay, J N, Proceedings of the Conference on the Production and Utilisation of Mechanically Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 25±27. ARCHER M, WATSON R, DENTON J W

Increased processed flesh yield by recovery from marine by-products 105 (1981), Minced fish technology: a review, Rome, Italy, FAO Fisheries Technical Paper No. 216. HALL G M, AHMAD N H (1997), `Surimi and fish-mince products', in Hall, G M, Fish Processing Technology, 2nd edn, London, Blackie Academic & Professional, 74±92. HIMONIDES A T (2001), `The improved utilisation of fish waste with particular reference to the enzymic hydrolysis of fish frames for the production of fish protein hydrolysates', PhD Thesis, University of Lincoln, Lincoln, UK. HIMONIDES A T, TAYLOR K D A, KNOWLES M J (1999), `The improved whitening of cod and haddock flaps using hydrogen peroxide', J Sci Food Agric, 79, 845±850. HOOVER D G, METRICK C, PAPINNEAU A M, FARKAS D F, KNORR D (1989), `Biological effects of high hydrostatic pressure on food micro-organisms', Food Technol, 43, 99±107. JAMES A L, MCCRUDDEN J E (1976), `Whitening of fish with hydrogen peroxide', in Keay, J N, Proceedings of the Conference on the Production and Utilisation of Mechanically Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 54±55. KING F J (1976), `Past, present and future uses of minced fish in the USA', in Keay, J N, Proceedings of the Conference on the Production and Utilisation of Mechanically Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 78± 81. MEACOCK G (1994), `Unpublished data', University of Lincoln, Lincoln, UK. MEACOCK G, TAYLOR K D A, KNOWLES M J, HIMONIDES A T (1997), `The improved whitening of minced cod flesh using dispersed titanium dioxide', J Sci Food Agric, 73, 221± 225. RAKSAKULTHAI N, AKSNES A, NJAA L R (1983), `Effects of hydrogen peroxide and of sulphite and humidity on the amino acid composition and digestibility of fish protein', J Sci Food Agric, 34, 619±626. RAVICHANDER N, KEAY J N (1976), `The production and properties of minced fish from several commercially important species', in Keay J N, Proceedings of the Conference on the Production and Utilisation of Mechanically Recovered Fish Flesh (Minced Fish), Aberdeen, UK, Torry Research Station, 18±24. REGENSTEIN J M (1980), `The Cornell experience with minced fish', in Connell J J, Advances in Fish Science and Technology, Surrey, UK, Fishing New Books Ltd., 192±199. REGENSTEIN J M (2004), `Total utilization of fish', Food Technol, 58, 28±30. SHIBAMOTO T, LEONARD B, TAYLOR S (1993), Introduction to food toxicology, San Diego, CA, Academic Press. STEFANSSON G (2001), `Developing a process for automated tuna head meat recovery', Final project report to the EU 35-01. FAIR CT-98-9079. STEINBERG M A, SPINELLI J, MIYAUCHI D (1977), `Minced fish as an ingredient in food combinations', in Proceedings of the Conference on the Handling, Processing and Marketing of Tropical Fish, London, Tropical Products Institute and Ministry of Overseas Development, 245±248. STOUT V, CARTER G (1983), `Ames test for mutagenicity on Pacific whiting treated with hydrogen peroxide', J Food Sci, 48, 492±495. SUZUKI T (1981), Fish and krill protein: processing technology, Essex, Applied Science Publishers Ltd. TAYLOR T, ALASALVAR C (2002), `Improved utilisation of fish and shellfish waste', in Alasalvar, C, Taylor T, Seafoods ± Quality, Technology and Nutraceutical Applications, Berlin, Germany, Springer, 123±136. GRANTHAM G J

106

Maximising the value of marine by-products

(1985), Processing aquatic food products, New York, John Wiley & Sons Inc. WINDSOR M, BARLOW S (1981), Introduction to fishery by-products, Surrey, UK, Fishing News Books Ltd. YOUNG K W, NEUMANN S L, MCGILL A S, HARDY R (1980), `The use of dilute solutions of hydrogen peroxide to whiten fish flesh', in Connell J J, Advances in Fish Science and Technology, Surrey, UK, Fishing New Books Ltd., 242±250. WHEATON F W, LAWSON T B

6 Enzymatic methods for marine by-products recovery F. Guerard, University of Western Brittany, France

6.1

Introduction

About 30% of total fishery landings is composed of by-catch (unconventional, unexploited), but fish processing wastes can also be considered as underutilized (Venugopal and Shahidi, 1995). In the past, part of this wasted material has often been dumped or used without treatment for animal feed or as fertilizer. However, due to the worldwide decline of fish stocks, a better use of by-catch and byproducts is deemed necessary. Today, particularly with the introduction of refined enzyme technologies, the potential for taking advantage of value added molecules already present in fish and invertebrate by-products is very high. Employing enzymatic methods for protein or lipid recovery in fish or shellfish processing as an alternative to mechanical or chemical treatments, which often damage the products and reduce product recovery, renders it possible to produce a large and diversified range of products for different applications. Among products of interest are enzymes, lipids, chitin and chitosan, calcium, nucleic acid, pigments, and biologically active peptides from fish protein hydrolysates. Enzyme technology has evolved to become an integral part of the food industry. This is due to the highly specific nature of enzymes. Enzymes are active at very low concentrations and under mild conditions of pH and temperature, which results in fewer unwanted side-effects in the production process (Shahidi and Kamil, 2001). The enzymatic processes vary according to the nature of the treated by-products. Usually, the type of enzyme which is employed for extracting or solubilizing by-products may be directly associated with the nature of the molecules to be extracted. For example, proteases are utilized for the solubilization of the protein part of by-products but in recent works, proteases were also used to enhance lipid extraction from fish tissues.

108

Maximising the value of marine by-products

This chapter surveys the recent literature on the possibilities of processes involving enzymatic biotransformation of fisheries and aquaculture by-products. The interest of the extracted molecules from a nutritional or a medical point of view will also be described.

6.2

Overview of by-products extracted by enzymatic methods

6.2.1 Definition of wastes and by-products According to Rustad (2003), there is not any single definition of what is a `byproduct', but usually, by-product indicates something that is not regarded as an ordinary saleable product but can be used after treatment. Thus `waste' refers to products that cannot be used for feed or food but have to be composted, or destroyed (e.g., by burning). Recovery of edible portions in seafood processing is traditionally low, ranging only from 20 to 50% and the resulting non-edible part consists of head, viscera (entirely or partly: heart, spleen, stomach, intestines, piloric caeca, liver, gall bladder, gonads, milt and roe), shell, skin and flesh remaining on the bone (An and Visessanguan, 2000; Aspmo et al., 2005a; GueÂrard et al., 2004; Taylor and Alasalvar, 2002). In addition, when harvesting fish and crustaceans, many species that are not used for human food are also caught. These `trash' fish can consequently be processed into useful products. The other by-product sources are fresh fish, crustaceans, and molluscs, of high or good edible quality but not having found any buyer. These could then either be frozen for subsequent selling, or destroyed according to the regulations governing the fish markets. Therefore, a large part of such material could alternatively be used as high quality raw material. All these examples indicate clearly that there is no established and permanent distinction between by-products and wastes. 6.2.2 Estimate of the available quantities of by-products In the literature, there are only a few papers dealing with the yield of by-product collected starting from the entire animal in terms of weight or percentage of head, frame, skin, viscera (Fig. 6.1). For example, in cod fillet production, as much as 60% of the whole fish is by-products, the backbone yielding about 15% of the fish weight (Gildberg et al., 2002). In a typically automated salmon filleting line, the fillets account for approximately 59±63% of the total weight for a salmon with body weight of 5±6 kg. Other products from the filleting line are salmon frame (9±15%), head (10±12%) and trimmings (1±2%) (Liaset et al., 2003). Shell waste from shrimp Crangon crangon processing is a good source of chitin and proteins with 17.8% and 40.6% in amount, respectively (Synowiecki and Al-Khateeb, 2000). The lobster heads correspond to two-thirds of the total weight with about 20% meat (Viera et al., 1995). In 2002, more than 133 million tonnes (MT) of fish and shellfish were landed (capture fisheries) or produced (aquaculture) (FAO report, 2004), of which about

Enzymatic methods for marine by-products recovery

Fig. 6.1

109

Fish by-products and their possible use (compiled from Andrieux, 2004; Gildberg et al., 2002; GueÂrard et al., 2004; Liaset et al., 2003).

100 MT were used for direct human consumption. The remainder (32 MT) was used for the manufacture of fishmeal including feed for aquaculture, fish oil and other non-food uses. The amount of processed fish (frozen, cured and canned) for human consumption was estimated at 39 MT. This could be translated into about 20 MT of fish by-products potentially available as a source of raw material. These data reveal that a large potential source of raw material exists globally from the fisheries industry. The basic recoverable components of fish frames and guts are mainly proteins and oils, while crustacean shell wastes provide calcium carbonates and chitin.

6.3

Enzymatic extraction methods

Enzymatic methods have become an important and indispensable part of the process used by the modern food and feed industry in order to produce a large and diversified range of products for human and animal consumption (Shahidi and Kamil, 2001). 6.3.1 Extraction of lipids Health effects and PUFA sources The beneficial effects of marine oils on human health were scientifically recognized three decades ago and have been well supported by many scientific

110

Maximising the value of marine by-products

studies and reviews over the past decade (Berge and Barnathan, 2005; Hunter et al., 2000; Kroes et al., 2003; Shahidi and Wanasundara, 1998; Vanschoonbeek et al., 2003 ; Ward and Singh, 2005). They have been attributed to the long chain n-3 polyunsaturated fatty acids (PUFAs) characteristic of marine oils, notably cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19docosahexaenoic acid (DHA). The n-3 PUFAs inhibit tissue eicosanoid biosynthesis, reduce inflammation, and lower serum triacylglycerol and cholesterol levels (Osborn and Akok, 2002). EPA has a beneficial effect on the cardiovascular system and inhibits very low-density lipoprotein (VLDL) formation, cell proliferation, and the inflammatory and allergic response (Uauy and Valenzuela, 2000). DHA is a major structural component of the grey matter of the brain and the eye retina and an important component of heart tissue, consequently DHA plays an important role in brain development and retinal function of the foetus and infants (Ward and Singh, 2005). The long chain n-3 PUFA may be obtained from marine mammals' oils (e.g., seal and whale blubber), and fish oils (e.g., menhaden, salmon, mackerel, cod, herring, sardine, capelin and tuna), as well as marine algae or derived from linoleic acid by a series of chain elongations and desaturations (Ward and Singh, 2005). In 2003±2004, the global production of fats and oils was expected at 12 805 million tonnes from which 82% were of vegetable origin (Berge and Barnathan, 2005). Fish oils are still the least expensive natural source of preformed long-chain PUFA and the n-3 PUFA content and EPA/DHA ratios in marine oils tend to vary with season, both in quality and quantity (Wanasundara et al., 2002). Aidos et al. (2002a) studied the oil composition changes of herring fillets and by-products over the year. They demonstrated that even if the herring by-products were throughout the year an adequate raw material for fish oil production, herring by-products were richer in PUFAs during the summer. On the contrary, farmed salmon (Salmo salar) slaughterhouses generate highquality offal at a relatively constant rate. In Norway, by-products of farmed salmon species were estimated at 133 000 tonnes in 2002, of which 8000 tonnes were used directly for salmon oil production (Skara et al., 2004). Efficient transportation or close proximity to a processing plant facilitates transformation of very fresh raw materials, in which initial post-mortem oxidation processes are limited. Today, the main sources of fish oils are pelagic species caught in large quantities and often not used for other purposes than fishmeal and oil production, but fish oil can also be produced from offal from the processing industry (Aidos et al., 2001). In fatty fish species, lipids are localized mainly under the skin, around the intestines or in the white muscle and the oil content varies but it can reach up to 21% in herring and 18% in sardine (Berge and Barnathan, 2005). The type of fat differs in various parts of the fish and organs (Aidos et al., 2002b). Pre-cooked tuna heads are known to be rich in DHA (Chantachum et al., 2000). In the cod viscera, the lipid content varies within the range of 1±10%, while the liver contains 42±78% lipids (Dauksas et al., 2005). The lipid concentrations of the farmed Atlantic salmon (Salmo salar L.) viscera

Enzymatic methods for marine by-products recovery

111

and fillet were 24.1 and 5.5%, respectively. Except for C18:0, C18:2, C20:1(n9), C20:4(n-6), C22:5(n-3), there were no significant differences in fatty acid concentration between the viscera and the fillet total lipid extracts (Sun et al., 2002). On the contrary, herring head by-products and their oil presented the highest amount of saturated fatty acids and the lowest -tocopherol and PUFA contents compared to mixed and headless herring by-products (Aidos et al., 2002b). Concentration of n-3 PUFA Typical processes for extraction/concentration of marine oils commonly involve cooking the raw material by steam under pressure with or without the presence of water. Partial cooking sterilizes the oil, denatures protein and facilitates oil release. Following this stage, the cooked material is pressed and centrifuged and/ or filtered to recover the oil from the micelle. Then PUFA concentrates may be obtained by several methods including chromatographic methods, distillation method, low temperature crystallization, supercritical fluid extraction, urea complexation and enzymatic methods including lipase-catalyzed hydrolysis and lipase-catalyzed esterification (Aidos et al., 2003; Shahidi and Wanasundara, 1998; Shimada et al., 2001). Experimental designs have been used to optimize the process parameters from the fish oil pilot plan in terms of oil quality (oxidative status) and yield (Aidos et al., 2003; Linder et al., 2005b) and to investigate the storage stability (lipid oxidation) of different processed oils (Skara et al., 2004). Each technique has its own advantages and drawbacks, as reviewed by Wanasundara et al. (2002). Enzyme-catalyzed process for lipid extraction The main enzymatic processes applied to fish oil recovery from by-products and whole fish can fall into two categories. The first one is the protease-catalyzed hydrolysis in order to extract the polyunsaturated fatty acids from the starting material (whole fish or selected by-products). This is a recent approach for lipid extraction by using a pre-hydrolysis step with a large spectrum of proteases in order to disrupt tissues and cell membranes. The second category concerns the lipase-catalyzed hydrolysis and esterification in order to enrich PUFAs in the oils and/or to produce different forms and compositions of PUFAs and/or to produce structured lipids. These enzymatic transformations occur once the oil is extracted from the starting material by traditional processes or by the use of proteases. Lipases may offer a degree of selectivity for particular fatty acids, not observed with acid or base catalyst (see Chapter 17). The examples presented below describe some applied enzymatic procedures using proteases for their ability to release oil content from the marine by-products. Use of proteases on lipid extraction from fish by-products Several commercial enzymes from plant origin such as papain and animal origin such as chymotrypsin or microbial sources such as ProtamexTM, FlavourzymeÕ,

112

Maximising the value of marine by-products

NeutraseÕ 0.5L, AlcalaseÕ 2.4L can be used for this purpose (Dumay et al., 2004; Linder et al., 2005a; Dauksas et al., 2005; Slizyte et al., 2005a). Linder et al. (2005a) carried out solvent-free extraction of oil from by-products of Atlantic salmon at moderate temperature for a short duration (50±60ëC; 30±120 min) by selective enzymatic hydrolysis using three commercial proteases (NeutraseÕ 0.5L, AlcalaseÕ 2.4L and FlavourzymeÕ). Reaction kinetics were monitored by measuring the degree of hydrolysis using the pH-stat method, in order to preserve the functional and nutritional values of hydrolysates. The amount of oil (17%) obtained after 2 hours was close to that obtained using the classical method with solvent. In a second step, the lipolysis of the oil was carried out with Novozym SP398 and followed by a filtration using a hydrophobic membrane (Fig. 6.2). DHA increased from 9.9% to 11.6% and EPA changed from 3.6% to 5.6%. A re-esterification of the free fatty acids in the permeate was achieved using glycerol and Lypozyme IM to increase the amount of long chain acylglycerols. Dumay et al. (2004) also compared yield extraction for total lipids, phospholipids, EPA and DHA, using four proteases (papain, chymotrypsin, ProtamexTM,

Fig. 6.2

Flowsheet for the production of hydrolysate and crude oil from salmon byproducts (adapted from Linder et al., 2005a).

Enzymatic methods for marine by-products recovery

113

FlavourzymeÕ) to those obtained by organic extraction. The authors observed that, despite using non-optimizing hydrolysis conditions, the use of proteolytic enzymes for partially disrupting lean fish tissue had, most of the time, enhanced the fat extraction. However, this increase was sometimes higher for total lipids than for those complex lipids such as phospholipids, DHA and EPA which, in fact, led to a reduction in the proportion of phospholipids, DHA and EPA among the total lipid compounds extracted this way. Liaset et al. (2003) isolated approximately 77% of total lipids present in salmon frames using ProtamexTM at 55ëC, pH 6.5 for 60 min. The resulting salmon oil was high in both EPA and DHA. Slizyte et al. (2005a) and Dauksas et al. (2005) performed hydrolysis of by-products from cod Gadus morhua (viscera, backbone, digestive track) using respectively FlavourzymeÕ, NeutraseÕ or no enzyme. Four lipid containing fractions were generated: oil, emulsion, FPH (fish protein hydrolysate) and sludge. The authors demonstrated that the most important factor influencing the yield of the different fractions was `added water' rather than the type of enzyme used, the highest yield of oil being obtained was when no water was added. The fatty acids were distributed unequally among the four fractions. The sludge contained up to 50% lipids with phospholipid content of up to 60% and the highest amount of PUFA (EPA/DHA). In the oil fraction, the major lipid class was triacylglycerols and the amount of triacylglycerols was similar in all samples (95±98%), the second major compound in the oil fraction was cholesterol (1.0 to 3.4% of total lipids) while the phospholipids were more difficult to extract from the mixtures. The monounsaturated fatty acids (MUFA) were the main compounds of the fatty acid methyl esters (FAMEs) in the oil fraction and made up more than 50% of all FAMEs. The oil produced from viscera tended to develop high levels of free fatty acids (FFA). FFAs are more susceptible to oxidation than esterified fatty acids and a low level of FFA is an important quality criterion in many food-related applications. After the preliminary step of oil extraction by conventional methods or by using proteases as described above, the enzymatic processes applied to fish oils are lipase-catalyzed hydrolysis, esterification, or exchange of fatty acids in esters. This provides an opportunity for the fat and oil industry to produce new types of triacylglycerols, esters and fatty acids, and to improve the quality of the existing products produced by employing conventional technologies (Shahidi and Kamil, 2001; Osborn and Akok, 2002). Lipase-catalyzed hydrolysis and esterification Lipases (E.C.3.1.1.3) are enzymes that are primarily responsible for the hydrolysis of acylglycerols in aqueous conditions. In a typical lipase-catalyzed hydrolysis run, the first step consists of obtaining freshly prepared, refined, bleached and deodorized marine oil. Then the lipase is dissolved in a selected buffer and the oil is added to it and stirred at a predetermined temperature. Samples are withdrawn at fixed time intervals until a desired hydrolysis percentage is reached. Alcohol is added quickly to deactivate the enzyme, and the sample is titrated in order to determine its free fatty acid content and con-

114

Maximising the value of marine by-products

Table 6.1 The two main categories in which lipase-catalyzed reactions may be classified (Gandhi, 1997) RCOOR0 + H2O , RCOOH + R0 OH

Hydrolysis Synthesis

Esterification Interesterification Alcoholysis Acidolysis

RCOOH + R0 OH , RCOOR0 + H2O RCOOR0 + R00 COOR* , RCOOR* + R00 COOR0 RCOOR0 + R00 OH , RCOOR00 + R0 OH RCOOR0 + R00 COOH , R00 COOR' + RCOOH

sequently, the percentage of enzymatic hydrolysis. Then separation of the enriched fraction is carried out (Shahidi and Kamil, 2001). However, since lipase reactions are reversible, the enzymes can be used to promote synthesis reactions such as esterification, interesterification, alcoholysis, acidolysis reactions, the last three reactions often being grouped together into a single term, i.e. transesterification (Table 6.1). These reactions are favoured when the amount of water in the reaction mixture is restricted (Gandhi, 1997). Thus, it is possible to attempt enzymatic purification of PUFA by selective esterification or alcoholysis. In theory, by choosing the right lipase judiciously, with respect to lipase triacylglycerol positional specificity, ester specificity and fatty acid chain length specificity, and by varying substrate, water content and use of hydrophobic solvents and other conditions, the reaction can be tailored to produce different product forms (Shimada et al., 2001; Ward and Singh, 2005). In addition, lipases have been frequently used to discriminate between EPA and DHA in concentrates containing both of these fatty acids, thus providing the possibility of producing n-3 PUFA concentrates with dominance of either EPA or DHA (Wanasundara et al., 2002).There is an extensive literature concerning different approaches to the preparation of n-3 fatty acid concentrates from natural sources using lipases (Linder et al., 2002; Shahidi and Wanasundara, 1998; Shimada et al., 1998; Skara et al., 2004; Ward and Singh, 2005). Sun et al. (2002) estimated the yield of EPA and DHA concentration in Atlantic salmon viscera using lipase C. rugosa. One kilogram viscera produced 0.17 kg viscera oil. One gram of viscera oil with 27.1% of EPA + DHA can be obtained from 4 g of viscera oil with 11.7% EPA + DHA after incubation with lipase from C. rugosa at 35ëC. The recovery of EPA + DHA from salmon viscera oil was about 58%. Therefore, from 1 kg salmon viscera, 11.4 g EPA + DHA can be produced. In some cases, experimental design was used to optimize the production of PUFA from marine oils (Wanasundara and Shahidi, 1998b; Linder et al., 2005b). Some examples of modification of lipids from marine oil involving lipase-catalyze process are given in Tables 6.2 and 6.3. In conclusion of this section, applications of enzymes in bioprocessing are especially advantageous because they act under mild conditions: mild temperature and pH conditions, ambient pressure, less solvent, give cleaner productsattributes of `green chemistry', and can exert region- or stereospecific control over reactions (Scrimgeour, 2005). Long chain PUFAs are highly labile and reaction methods, which exploit extremes of pH and temperature can destroy the

Enzymatic methods for marine by-products recovery

115

Table 6.2 Summary of general PUFA enrichment process using lipase (adapted from Ward and Singh, 2005) Method

Procedure

Enzyme splitting

· Promote lipase selective esterification of more saturated fatty acids · Separate concentration of PUFAs in FFA fraction from esterified saturated fatty acid · Repeat esterification for further PUFA enrichment · Esterify PUFA free fatty acids to produce esters (ethyl-, glyceryl-, sugar-, other) · Interesterification to enrich non-HUFA oils with PUFAs

PUFA transformations

PUFA: Polyunsaturated fatty acid HUFA: Highly unsaturated fatty acid

all cis nature of n-3 fatty acids, such as EPA or DHA, by oxidation, cis-trans isomerization or migration of double bond. A number of recent reviews and books cover topics developed in this part (Ward and Singh, 2005; Osborn and Akok, 2002; Shahidi and Wanasundara, 1998). Enzymatic oil extraction using food-grade proteases could provide an interesting alternative as it presents two major interests: the first one is the mild condition in which the enzymatic process is performed, coupled or not with membrane technology. The valuable components in the fish oil are not destroyed and oils with higher quality will obtain higher prices. The second one is its versatility: optimal processing conditions must be found to obtain tailor-made products according to the final objective such as the purification of phospholipids, a high oil yield and/or a high quality fish protein hydrolysate. Subsequent modification of the extracted lipids by using lipases having high stereoselectivity has found applicability in the site-specific modification of triacylglycerol products enriched with EPA or DHA. Lipases offer many advantages over traditional methods of concentration (chromatographic separation, molecular distillation, etc.) because such procedures involve extremes of pH and high temperature, which may partially destroy the natural all cis n-3 PUFA by oxidation and by cis-trans isomerization or double bond migration. Therefore, the lipase-catalyzed process provides a good alternative that could also save energy and increase product selectivity. In addition, the enzymatic hydrolysis method produces n-3 fatty acids in the acylglycerol form, which is considered to be nutritionally favourable (Wanasundara and Shahidi, 1998a; Wanasundara et al., 2002). The lipase catalytic efficiency is high, therefore a relatively low amount of enzyme is required, especially when immobilized, which enables their reuse and enhances their productivity. The susceptibility to oxidation of fish oil during storage is a major problem since the formation of undesirable odours and flavours caused by oxidation often limit the shelf life (Skara et al., 2004). Most fish oils are produced under inert gas or in closed containers to reduce oxidation by atmospheric oxygen (Ward

Table 6.3

Examples of lipase-transformations of PUFAs

Enzyme process

1. Lipaseassisted hydrolysis

PUFA substrate

Specific PUFA concentration

Reaction conditions

Transformation %

References

before optimization

Seal blubber oil (SBO)

EPA 6.4% DPA 4.7% DHA 7.6%

4g Oil, 6 mL phosphate buffer, lipase CC* 200 U/g oil, 40 h

EPA 9.75% DPA 8.6% DHA 24%

Wanasundara and Shahidi (1998a)

after optimization using CCRD**

Seal blubber oil (SBO)

EPA 6.4% DHA 7.6%

lipase CC* 297 U/g oil, 26 h, 36ëC

EPA 16.5%

Wanasundara and Shahidi (1998b)

lipase CC* 342 U/g oil, 51 h, 39ëC

DHA 28.1%

Salmon viscera oil

EPA 67 mg/g DHA 74 mg/g

4 g Oil, 6 mL phosphate buffer, lipase C. rugosa (800 U/g oil), 35ëC

EPA 106 mg/g DHA 184 mg/g

Sun et al. (2002)

Menhaden oil (MHO)

EPA 13.2% DPA 2.4% DHA 10.1%

4 g Oil, 6 mL phosphate buffer, lipase CC* 200 U/g oil, 40 h

EPA 18.5% DPA 3.6% DHA 17.3%

Wanasundara and Shahidi (1998a)

2. Lipaseassisted hydrolysis 3. Lipaseassisted hydrolysis

before optimization

after optimization using CCRD**

lipase CC* 370 U/g oil, 31 h, 37ëC

EPA 21.1%

lipase CC* 314 U/g oil, 34 h, 36ëC

DHA 25.9%

Wanasundara and Shahidi (1998b)

4. Lipaseassisted hydrolysis

Sardine oil

EPA 14.5% DPA 1.3% DHA 12.5%

12 g Oil, 18 mL phosphate buffer, lipase*** (0.25, 0.5, 0.75% w/w oil basis) pH7

EPA 46.2% DPA 2.16% DHA 40.32%

Gamez-Meza et al. (2003)

5. Acidolysis (incorporation of capric acid)

Seal blubber oil (SBO)

5.4% 20:5n-3, 7.9% 22:6n-3

Oil/fatty acid mole ratio of 1:3, 45ëC, hexane, 24 h, 1% water, 10% Lipozyme-IM

SL containing 3.2% 20:5n-3, 7.5% 22:6n-3, 27.1% 10:0

Senanayake and Shahidi (2002)

*CC: Candida cylindracea from Amano Pharmaceutical; **CCRD: central composite rotatable design; ***lipase: Pseudomonas cepacia lipase immobilized on chemically modified ceramic (CMC).

118

Maximising the value of marine by-products

and Singh, 2005) but efforts to keep these products from oxidative rancidity during processing, cooking, and storage are necessary (Uauy and Valenzuela, 2000). In the case of direct consumption of PUFA-containing oils, this is overcome through use of capsules while microencapsulation techniques can address this problem for incorporation of oils into dried foods (Ward and Singh, 2005). In addition to the oxidative instability of PUFA-containing oils is the presence of co-extracted contaminants. Nevertheless, the crucial problem of fish oils is their sustainability due to the worldwide decline of fish stocks. A better use of raw material by-catch and by-products from fisheries as well, may be one solution (Berge and Barnathan, 2005). 6.3.2 Proteins Enzymatic proteolysis as applied to proteinaceous fish by-products has been the objective of numerous studies (reviewed by Kristinsson and Rasco, 2000; Mackie, 1982). Fish protein hydrolysates (FPH) and other hydrolysates have a range of potential applications, as ingredients in animal feed or food, as peptone ingredient in microbial growth media, as fertilizer or as a new source of bioactive peptides (Aspmo et al., 2005b; Byun and Kim, 2001; Dufosse et al., 2001, 2003; GueÂrard et al., 2001a, 2003, 2005a; Je et al., 2005; Liaset et al., 2000; Rousseau et al., 2001). Proteases are among the best characterized enzymes and their use is well established in the food industry. Proteases are classified in four major classes according to the specificity of their peptide bond cleavage: serine proteinases (E.C.3.4.21), cysteine proteinases (E.C.3.4.22), aspartic proteinases (E.C.3.4.23) and metalloproteinases (E.C.3.4.24). Proteases are further characterized by their hydrolyzing mechanism into endoproteinases or exoproteinases. The breakdown of proteins into peptides is brought about primarily by endoproteinases which cleave the peptide bond within protein molecules, usually at specific residues to produce relatively large peptides (Kristinsson and Rasco, 2000). The exopeptidases, including carboxypeptidases, aminopeptidases, and di- and tripeptidase, systematically reduce the peptides into amino acids. Endoproteinases may be combined with exopeptidases in order to achieve a more complete degradation. Autolysis versus enzymatic hydrolysis Biochemical processes for converting fish processing wastes and by-products into fish protein hydrolysates may be carried out by employing an autolytic process or by using added proteolytic enzymes. Two important examples of utilization of autolytic digestion in product manufacturing are fish sauce and fish silage production, in which hydrolytic enzymes from the fish itself play a key role in the solubilization and degradation of the tissue proteins (Gildberg et al., 2000). The biochemistry of the process is not well understood in detail (Martin and Patel, 1991).

Enzymatic methods for marine by-products recovery

119

Enzymatic hydrolysis with added enzymes On the other hand, the enzymatic hydrolysis using added proteases presents a lot of advantages compared to autolytic process and chemical hydrolysis (Table 6.4). Addition of commercial exogenous enzymes to the fish tissue reduces the time needed to obtain a similar degree of hydrolysis (DH) and allows a good control of the hydrolysis, and subsequently of the size of the obtained peptide. In addition, hydrolytic degradation products via racemization reaction observed with both acid and alkaline hydrolysis, are not produced. The choice of the hydrolysis process will depend on the targeted applications. For dietary use, or in order to obtain a hydrolysate with a high nutritional and therapeutic value, the protein hydrolysates should be rich in low molecular weight peptides, with as few free amino acids as possible whereas large molecular weight peptides (more than 20 amino acid residues) are presumed to be associated with the improvement in the functionality of hydrolysates. Research during the past 20 years has greatly enhanced understanding about better processing of fish or shellfish by-products. A list of the most frequent underutilized species or processing wastes investigated for the production of hydrolysates, is presented in Table 6.5. Generally, underutilized fish, fish frames or crustacean wastes are suspended in water and enzyme is added to the slurry. In some cases, the meat is first heated in order to denature the endogenous proteases (GueÂrard et al., 2001b). The reaction is allowed to proceed for less than one hour to several hours, depending on the activity of the enzyme employed, process temperature and other factors. After separation of solids, pH is adjusted and the aqueous layer is clarified, and then dehydrated. Figure 6.3 outlines the main steps of the process in the enzymatic solubilization of proteinaceous fish or invertebrate by-products. The need to inactivate added enzymes by pH or heat treatment at the end of the batch reaction adds to the processing cost and may be improved by coupling the enzymatic hydrolysis with membrane technology in order to perform continuous process. Continuous processes are used by many companies for large-scale production of hydrolysates. Most of the commercial proteases have been used successfully to solubilize proteins from underutilized species or processing waste. Industrial proteinases are mostly derived from GRAS microorganisms (AlcalaseÕ 2.4L, NeutraseÕ, FlavourzymeÕ, UmamizymeÕ, ProtamexTM), and to a lesser extent from plant (papain, bromelain, ficin) and animal sources (pepsin, trypsin). According to Simpson (2000), there is only very limited use of marine proteinases by the industry. The reasons for the rather limited use of marine digestive proteinases include the relative paucity of basic information on these enzymes, the cyclical nature of the source material (which precludes supply in a steady manner), and the stereotypical attitude of the general public toward the source material: fish offal. Some examples of proteolytic enzymes used to hydrolyze marine byproducts are presented in Table 6.5. By selecting both the enzyme and the conditions of digestion, various degrees of hydrolysis or breakdown of the proteins can be achieved in order to obtain products with a range of functional and/or biological properties. The introduction of new proteases capable of

120

Maximising the value of marine by-products

Table 6.4 Comparison between autolysis, chemical and enzymatic hydrolysis (adapted from Diniz and Martin, 1997b; Kristinsson and Rasco, 2000; GueÂrard et al., 2005a) Process

Specificity

Autolysis process Action of (fish sauce and the digestive silage) enzymes of the fish itself

Advantages

Disadvantages

Low cost Simple operation

Slow reaction Molecular weight out of control Subsequent deactivation of the enzyme Large amount of salt (in fish sauce) Enzymes with different activity requirements High variations in the presence of enzymes Final product with bad functionality

Mild reaction conditions No destruction of amino acids High nutritional value No enzyme addition Improvement of organoleptic characteristics

Acid/alkaline hydrolysis

Random process

Fast reaction Complete hydrolysis Low cost High solubility

Enzymatic hydrolysis

Unique specificity of action of the enzyme(s)

Control of the molecular weight Mild reaction conditions Attractive functional product characteristics (solubility, dispersibility, foaming, capacity and foam stability) Control of the properties of the resulting products Few side reactions No destruction of amino acids High nutritional value

High temperatures Molecular weight out of control Large amount of salt Undesirable side reactions (destruction of tryptophan, racemization, etc.) Cost of enzyme(s) Subsequent deactivation of the enzyme(s) Complex process

Table 6.5

Some examples of proteolytic enzymes used to hydrolyze marine by-products including `trash fish' or underutilized species

Enzymes

Suppliers

Substrates

Papain (EC 3.4.22.2)

Solvay Enzymes, Inc. Sigma Sigma Biochem Europe (7 ¨/kg)a

Herring (Clupea harengus) Lobster cephalothorax (Palinurus sp) Capelin (Mallotus villosus) Atlantic cod (Gadus morhua L.) viscera

Applications 1 1 1,2 3

Evaluation of hydrolysis

References

TCA soluble N, TN, colour, sensory, MWDP TL, FP, NSI pH-stat, FP, PER, ED DM, -amino groups**, MWDP, TN, MS, SDS-PAGE

Hoyle and Merrit (1994) Vieira et al. (1995) Shahidi et al. (1995) Aspmo et al. (2005a,b)

Actinidin (30 ¨/kg)a

Biochem Europe

Atlantic cod (Gadus morhua L.) viscera

3

DM, -amino groups**, Aspmo et al. MWDP, TN, MS, SDS-PAGE (2005a,b)

Bromelain (EC 3.4.22.4)

Pineapple juice

Yellowfin tuna (Thunnus albacares) canning waste

np

NR

Biochem Europe (20 ¨/kg)a

Atlantic cod (Gadus morhua L.) viscera

3

DM, -amino groups**, Aspmo et al. MWDP, NT, MS, SDS-PAGE (2005a,b)

Trypsin (EC 3.4.21.4)

Merck

Sardine (Sardina pilchardus) viscera and heads

2

MWDP

Cancre et al. (1999)

Pepsin (EC 3.4.23.1)

Sigma

Lobster cephalothorax (Palinurus sp)

1

TL, FP, NSI

Vieira et al. (1995)

Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) frames

1

pH-stat, NP, MWDP

Liaset et al. (2000)

Raghunath (1993)

Table 6.5

Continued

Enzymes

Suppliers

Substrates

Crude proteinases from tuna (Thunnus thynnus) pyloric caeca

Cod (Gadus macrocephalus) frame

Crude proteinase from mackerel intestine

Hoki frames (Johnius belengerii)

FlavourzymeÕ

Novozymes

Gold carp (Carassius auratus) filleting by-products Fish soluble concentrate (a byproduct from canning industry) Atlantic cod (Gadus morhua L.) viscera

Applications 1,2

Evaluation of hydrolysis

References

10% TCA soluble nitrogen content, LPC, MWDP, FP

Kim et al. (1997); Jeon et al. (1999)

2

1

Kim et al. (2003)

% -amino acids released*, NR, ED DH%=AN/TN, ED, AAC, SE -amino groups/TN; PER, free AA; MWDP; FP

Sumaya-Martinez et al. (2005) Nilsang et al. (2005) Slizyte et al. (2005a,b)

DH%=AN/TN,

Nilsang et al. (2005)

KojizymeTM

Novozymes

Fish soluble concentrate (byproduct from canned fish industry)

Fungal protease type II from A. oryzae

Sigma

Lobster cephalothorax (Palinurus sp)

1

TL, FP, NSI

Vieira et al. (1995)

Newlase A from Rhizopus niveus

Amano

Atlantic Cod (Gadus morhua) frames

4

Viscosity, MWPD

Ferreira and Hultin (1994)

ED, SE

AlcalaseÕ 2.4 L (25 ¨/kg in 2004)a

Novozymes

Capelin (Mallotus villosus) 1,2 Shrimp (Crangon crangon) processing 1 discards Atlantic cod (Gadus morhua L.) viscera 3

Herring (Clupea harengus)

1

Herring (Clupea harengus)

1

Pacific whiting (Merluccius productus) solid wastes Harp seal (Phoca groenlandica)

1

Shrimp (Pandalus borealis) waste Cod head (Gadus morhua) Shrimp (Pandalus borealis) waste

2 2 1

Threadfin bream (Nemipterus japonicus)

1

Shark muscle (Squalus acanthias)

1

Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) frames Atlantic salmon (Salmo salar) heads

1

Shahidi et al. (1995) Synowiecky and AlKhateeb (2000) DM,-amino groups**, Aspmo et al. MWDP, TN, MS, SDS-PAGE (2005a,b) pH-stat, MWDP GueÂrard et al. (2001a,b) TCA soluble N, TN, colour, Hoyle and Merrit SE, MWDP (1994) DH-TNBS method; Liceaga-Gesualdo and SDS-PAGE, FP Li-Chan (1999) DH-TNBS method, NR, Benjakul and colour, SDS-PAGE Morrissey (1997) pH-stat, NSI, FP Shahidi et al. (1994); Shahidi and Synowiecki (1997) MWDP Cancre et al. (1999) MWDP Cancre et al. (1999) TN Gildberg and Stenberg (2001) pH-stat, SE, MWDP, Normah et al. (2004) SDS-PAGE pH-stat, NR, ED, PER Diniz and Martin (1996, 1997a, 1998) pH-stat, NP, NSI, FP, MWDP Liaset et al. (2000)

1

pH-stat, ED, MWDP

Tuna stomach (Tunus albacora)

1,2,3

1

pH-stat, FP, PER, ED pH-stat, PER, SE, EEA

Gbogouri et al. (2004); Linder et al. (2005a,b)

Table 6.5

Continued

Enzymes

Suppliers

Substrates

AlcalaseÕ 0.6 L

Novozymes

Capelin (Mallotus villosus)

NeutraseÕ 0.5 L

Novozymes

Pacific whiting (Merluccius productus) solid wastes Harp seal (Phoca groenlandica) Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) frames Atlantic cod (Gadus morhua L.) viscera

1

Atlantic cod (Gadus morhua L.) viscera

3

NeutraseÕ 0.8 L (15 ¨/kg)a

Novozymes

Applications 1,2

1 1 1

Evaluation of hydrolysis

References

pH-stat, FP, PER, ED

Shahidi et al. (1995)

DH-TNBS method, NR, color, Benjakul and SDS-PAGE Morrissey (1997) pH-stat, NSI, FP Shahidi et al. (1994) pH-stat, NP, MWDP Liaset et al. (2000) -amino groups/TN; PER; free AA; MWDP; FP DM, -amino groups**, MWDP, TN, MS, SDS-PAGE

Slizyte et al. (2005a,b) Aspmo et al. (2005a,b) GueÂrard et al. (2003)

UmamizymeÕ

Novozymes A/S

Tuna stomach (Tunus albacora)

1

pH-stat, MWDP, NR

ProtamexTM (42 ¨/kg)a

Novozymes

Atlantic cod (Gadus morhua L.) viscera

3

DM,-amino groups**, Aspmo et al. MWDP, TN, MS, SDS-PAGE (2005a,b)

Atlantic Salmon (Salmo salar L) frames

1

NR, Lipid recovery

a

Liaset et al. (2003)

: approximative price as specified by suppliers. Prices may vary depending on purchase order quantity (Aspmo et al., 2005a). 1: Dietary protein source; 2: biological activities; 3: peptone; 4: fertilizer; np: not precise. AAC: amino acid composition; -amino groups; **: concentration of -amino groups evaluated using OPA; DM: dry matter, EEA: essential amino acids; ED: experimental design; FD: factorial design; FP: functional properties; FVFA: free volatile fatty acids; LPC: length of the peptide chain in hydrolysate using the TNBS method; MS: mass spectrometry; MWDP: molecular weight distribution of peptides; N: nitrogen content; NP: nutritional properties; NR: nitrogen released; NSI: nitrogen solubility index; PER: protein efficiency ratio; pH-stat: pH-stat method; SC: soluble content; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SE: sensory evaluation; TL: tyrosine level; TCA: TCA soluble nitrogen; TN: total nitrogen.

Enzymatic methods for marine by-products recovery

125

Fig. 6.3 Flowsheet for the enzymatic solubilization of proteinaceous fish or invertebrate by-products (adapted from Kristinsson and Rasco, 2000; GueÂrard et al., 2005a).

degrading bitter peptides (such as Flavourzyme from Novozymes) has contributed to eliminating the problem of bitter hydrolysates. The screening for a suitable enzyme in any process or experiment is very important if the product is to have predetermined properties (Kristinsson and Rasco, 2000). The screening process can be conducted in a variety of ways, and there is no standard methodology for this selection. Recently, the relative activities of six commercial enzymes (AlcalaseÕ 2.4L, NeutraseÕ 0.8L, ProtamexTM, papain, bromelain, actinidin and a mix of plant proteases) were measured for the hydrolysis of cod viscera at the natural pH of the substrate without pH-control, in order to avoid adding more salt to the hydrolysate and to limit the cost of the process (Aspmo et al., 2005a). However, in this experiment, viscera endogenous enzymes worked together with the added proteases and maximized the process efficiency, thus making it difficult for a comparative study of the proteases. The results showed that there is a great variation among the performances of commercial enzymes. Highest yields in solubilized dry matter were obtained with AlcalaseÕ 2.4L and papain, AlcalaseÕ 2.4L clearly performing the best and leading to solubilization of close to 95% of the dry matter. Endogenous enzymes made an important contribution to viscera solubilization. The fish viscera hydrolysates gave good performances as peptones for microbial growth (Aspmo et al., 2005b). Shahidi et al. (1995)

126

Maximising the value of marine by-products

used AlcalaseÕ 2.4L to optimize the preparation of capelin protein hydrolysates. Alcalase-treated hydrolysates exhibited superior protein recovery (70.6%) compared with NeutraseÕ (51.6%) and papain (57.1%). AlcalaseÕ 2.4L and NeutraseÕ were studied by Benjakul and Morrissey (1997) on the solubilization of Pacific whiting solid wastes (solid wastes resulting from surimi production). AlcalaseÕ 2.4L had a higher proteolytic activity than NeutraseÕ. The final hydrolysate had high protein content (about 80%) and an amino acid composition comparable to fish muscle. Other enzyme preparations from microbial origin have shown good potential for hydrolyzing proteinaceous by-products to make highly functional hydrolysates, including FlavourzymeÕ, ProtamexTM, and UmamizymeÕ (Table 6.5). UmamizymeÕ performed as effectively as AlcalaseÕ 2.4L for the tuna waste solubilization, however, UmamizymeÕ stability during the hydrolysis process was lower than that of AlcalaseÕ 2.4L (GueÂrard et al., 2002). Although proteases operating at alkaline pH have often been used for hydrolysis of fishery by-products, Ferreira and Hultin (1994) reported the use of a fungal enzyme (the Newlase A from Rhizopus niveus, Amano) in order to liquefy cod fish frames under acidic conditions. The main advantage of working at low pH is the stabilizing effect towards microbial spoilage. The authors concluded that Newlase A was a useful catalyst in any situation where a pH between 3 and 4 was to be used and where it was desired to reduce the viscosity of fish frames without excessive hydrolysis. In some cases, experimental designs were employed to optimize hydrolysis conditions. Shahidi et al. (1995) used response surface regression (RSREG) procedure of the statistical analysis system to fit a quadratic polynomial equation to the experimental data. The three-dimensional response surface indicated that both the AlcalaseÕ 2.4L concentration (13.9±83.9 AU/kg crude protein) and the treatment temperature (45±65ëC) affected the degree of hydrolysis and thus the protein recovery. Response surface methodology was also used by several authors in order to study the effects of pH, temperature, enzyme/substrate ratio and substrate concentration on the degree of hydrolysis of crayfish by-products (Baek and Cadwaller, 1995), canned tuna processing (Nilsang et al., 2005), dogfish muscle (Diniz and Martin, 1996, 1997a), and gold carp by-products (Sumaya-Martinez et al., 2005). The resulting equations were adequate for predicting the DH under any combination of values of the variables. For example, Sumaya-Martinez et al. (2005) used a Box-Behnken factorial design with four independent variables (Temperature T, pH, substrate/ buffer ratio S:B, and enzyme concentration E). All regression coefficients were significant and the model obtained showed a good fit with the experimental data. A maximum DH% was obtained at the following critical values for FlavourzymeÕ: pH ˆ 5.9, T ˆ 53ëC, S:B ˆ 14.7%, and E ˆ 80 LAPU (leucine aminopeptidase units) gÿ1. Thus, among numerous proteases tested, AlcalaseÕ 2.4L, an alkaline enzyme produced from Bacillus licheniformis and developed by Novozymes for the detergent industry, has been repeatedly proven by many researchers to be one of

Enzymatic methods for marine by-products recovery

127

the most effective enzymes to solubilize proteins (Diniz and Martin, 1997b; Aspmo et al., 2005a). Unfortunately, these comparative studies were not undertaken using standardized relative enzyme activity and most researchers compare enzyme activity on (i) a weight basis of enzymes used in the reaction mixture, or (ii) when using a system based on Anson Units, did not use the enzymes at the same AU, or (iii) did not inactivate the endogenous proteases before adding exogenous enzymes. Kristinsson and Rasco (2000) suggested assaying enzymes using a synthetic substrate such as azocoll or casein in order to obtain a uniform level of proteolytic activity for all the enzymes used. Quantification of the proteolysis extent The hydrolysis reaction must be carefully controlled in order to maintain a uniform quality of the end products. The degree of hydrolysis (DH), which is defined as the percentage of cleaved peptide bonds, is most commonly used to describe hydrolysis of food protein and serves as the controlling parameter for the hydrolysis reaction. The pH-stat technique consists of adding base for titration of the released -amino groups, thus maintaining the pH constant (at pH values above 6.5). Equation (6.1), which relates the DH to alkali consumption, is as follows (Adler-Nissen, 1982): 1 1 1  100 …6:1†  %DH ˆ B  Nb    MP htot where B is the volume (mL) of base added, Nb is the normality of the base,  is the average degree of dissociation of the -NH groups, MP is the gram of protein in the reaction mixture, htot is the total number of peptide bonds. The principle of the pH-stat method has been used by many workers for kinetic studies and the DH gives a measure of the enzyme hydrolytic efficiency (Table 6.5). In addition to the pH-stat method, the extent of proteolysis may also be quantified by the depression of the freezing point, which is indicative of the increasing osmolarity (osmometry), or by the increase in solubility in trichloracetic acid (Kristinsson and Rasco, 2000). DH values determined by different methods are often not directly comparable. Base consumption and osmometry methods are easy to perform, allowing continuous monitoring of the hydrolysis process, whereas the estimation of soluble nitrogen content using the Kjeldahl method is time-consuming and cannot be used as an on-line process control tool (Panyam and Kilara, 1996). The trinitrobenzenesulfonic acid (TNBS) method developed in 1979 by Adler-Nissen is always used to determine the degree of hydrolysis of food protein hydrolysates. However, the o-phtaldehyde (OPA) method used for analysing the protein hydrolysate DH, has been found to be more accurate, easier and faster to carry out than the TNBS method (Nielsen et al., 2001). This method has a broader application range and is environmentally safer. Silvestre (1997) provided a review of various methods used for the analysis of protein hydrolysates and discussed the potential and limitations of the different techniques. Finally, hydrolysates can be characterized according to the peptide

128

Maximising the value of marine by-products

size in order to check that hydrolysates can be produced in a reproducible manner. This is a very important point when the objective is the production of hydrolysates with biological activities. In this case, size exclusion chromatography is a simple and quick method for the evaluation of peptide molecular weight. New size exclusion chromatography supports in FPLC mode such as the SuperdexÕ Peptide HR 10-30 (Pharmacia Biotech, Sweden), with fractionation range from 100 to 7,000 Daltons allowed accurate separations of enzymatic hydrolysates. However, the fractionation range values can only serve as guidelines, especially because the elution behaviour of peptides in non-dissociating media is influenced by adsorption and aggregation and because of the underestimation of small peptides and free amino acids (GueÂrard et al., 2001b). Some properties of enzymatically hydrolyzed by-product proteins Functional properties The functional properties of proteins can be defined as those properties that affect the processing, storage stability and organoleptic quality of the formulated food in which they are present (Vojdani and Whitaker, 1994). It has been reported that FPH possess desirable functional properties such as high solubility over a wide range of pH and excellent wettability (Shahidi et al., 1995; Vieira et al., 1995). These properties are the consequence of enzymatic degradation of the original protein to smaller peptides (Diniz and Martin, 1997b). As observed by Kristinsson and Rasco (2000), the emulsifying capacity (EC) of hydrolysates decreased with increasing DH. Hydrolysates with low DH, have high EC while extensive hydrolysis resulted in a drastic loss of emulsifying properties. Shahidi and Synowiecki (1997) reported the use of a seal protein hydrolysate (bland taste) as phosphate alternative for enhancing the water-binding capacity and improving the functional properties of thermally processed meat products. Functional properties of fish protein hydrolysates are extensively discussed by Kristinsson and Rasco (2000). Biological activities In an extensive paper, FPH were recently described as a new source of biologically active substances (GueÂrard et al., 2005a). A wide range of biological activities including antioxidant activities (GueÂrard et al., 2005b; Jun et al., 2004; Je et al., 2005), neuroactive peptides (Bernet et al., 2000; Bordenave et al., 2002), hypotensive (Byun and Kim, 2001) and immunoactive peptides (Bogwald et al., 1996; Gildberg et al., 1996), hormonal and hormonal regulating peptides such as calcitonin gene related peptide (CGRP), cholecystokinins and gastrins (Fouchereau-PeÂron et al., 1999; Rousseau et al., 2001; Ravallec-Ple et al., 2001) have been associated with FPH or with some purified sequences derived from FPH. In addition, the results of several studies have shown that fish peptones may be an excellent nitrogen source for microorganisms including bacteria requiring complex growth media (Aspmo et al., 2005b; Dufosse et al., 2001, 2003; Gildberg et al., 1989; Ghorbel et al., 2005; Martone et al., 2005; Vasquez et al., 2004).

Enzymatic methods for marine by-products recovery

129

In conclusion to this section, enzymatic extraction and/or solubilization of proteinaceous by-products (e.g., heads, frames) have been developed for many years. The resulting hydrolysates have great potential to be produced and sold as functional food or feed ingredients. The occurrence of many biologically active peptides in marine by-products is now well established. In all cases, the control of the enzymatic reaction is very important. Uncontrolled or prolonged hydrolysis of proteins may result in the formation of highly soluble peptides, completely devoid of the functional properties of native proteins and may promote the formation of bitter peptides. 6.3.3 Skin, bones, fin, scales, cartilage By-products from the fish processing industry create large amounts of skin, bones and scales in which structural proteins such as keratin, collagen and, to a lesser extent, elastin have been found (Jongjareonrak et al., 2005a,b). Extraction of collagen and collagen-derived products According to Gomez-Guillen et al. (2002), about 30% of fish wastes consists of skin and bone with a high collagen content. Heat denaturation of collagen produces gelatine, a high digestible protein characterized by its rheological properties varying according to the starting raw material. Until now, the main sources of collagen were limited to those of land-based animals, such as bovine and porcine skin and bone. However, the outbreak of bovine spongiform encephalopathy (BSE) has resulted in anxiety among users of collagen and collagen-derived products of land origin. Due to religious objections, the collagen obtained from porcine sources cannot be used as a component of some foods. As a consequence, increasing attention has been paid to alternative collagen sources, especially fish skin and bone from seafood processing wastes (Jongjareonrak et al., 2005a; Kittiphattanabawon et al., 2005). Collagen type I is found in all connective tissue, including bones and skin. It is a heteropolymer of two 1 chains and one 2 chain. It consists of one third glycine, contains no tryptophan or cysteine and is very low in tyrosine and histidine (Muyonga et al., 2004). Isolation of acid- and pepsin-solubilized collagens In general, the preparation of collagen from fish skin and bone is performed according to the procedure described by Nagai and Suzuki (2000) with or without slight modifications. Typically, the collagen extraction combine treatments by NaOH to remove non-collagenous proteins and pigments then by butyl alcohol to remove fat followed by an acid extraction using acetic acid. The product obtained is referred to as acid-solubilized collagen (ASC) and, in some cases, is subjected to limited hydrolysis with pepsin (E.C. 3.4.23.1). The product is referred to as pepsin-solubilized collagen (PSC). With the limited pepsin digestion, the cross-linkages at the telopeptide region are cleaved without damaging the integrity of the triple helix (Hickman et al., 2000). As a

130

Maximising the value of marine by-products

consequence, the triple helix structure is still predominant in both ASC and PSC, resulting in similar thermal characteristics for both fractions. Jongjareonrak et al. (2005a) used pepsin in order to solubilize collagens from the skin of Browstripe red snapper (Lutjanus vitta). A yield of 4.7% on the basis of wet weight was obtained. The pepsin-solubilized collagen (PSC) consisted of two different  chains (1 and 2), and was characterized to be type 1 with no disulphide bond. The PSC peptide pattern was totally different from those of calf skin collagen type I, suggesting differences in amino acid sequences and collagen conformation. The thermal stability of PSC was much lower than that of calf skin collagen, due to its lower hydroxyproline content (86 residues versus 94 residues per 1000). Morimura et al. (2002) developed a procedure for the extraction of protein and production of peptides by enzymatic hydrolysis from the spine of yellowtail fish wastes containing collagen by evaluating the effectiveness of 16 commercial enzymes for degradation of pretreated fish bone. One protease from Bacillus species appeared to be superior to those originating from fungi for the degradation of collagen with an optimal 83% degradation efficiency using 5 g.lÿ1 of substrate and 0.25 g.lÿ1 enzyme. The resulting hydrolysate showed a mean degree of polymerization of about three, and appeared to be a composite of oligopeptides. The hydrolysates had a high antiradical activity (IPOX50, 0.18 and 0.45 mg/mL) and a high potential for decreasing blood pressure (IC50, 0.16 and 0.41 mg/mL), suggesting the hydrolysate had suitable properties for use as additive in food materials. A three-step membrane reactor was designed to prepare the enzymatic hydrolysis of gelatine extracted from Alaska pollack skin. Successive digestions with AlcalaseÕ 2.4L, PronaseÕE, and collagenase were performed in order to produce angiotensin converting enzyme (ACE) inhibitory effect peptides and antioxidant peptides (Byun and Kim, 2001; Kim et al., 2001). The isolated antiACE peptides were composed of Gly-Pro-Leu and Gly-Pro-Met and showed IC50 values of 2.6 and 17.13 M, respectively. Two antioxidant peptides composed of 13 and 16 amino acid residues were also purified. Ogawa et al. (2003) isolated acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) from the skins of black drum (Pogonias cromis) and sheepshead seabream (Archosargus probatocephalus). The yields of ASCs on dry basis from black drum and sheepshead seabream, were estimated at 2.3 and 2.6%, and the yields of PSC were 15.8 and 29.3%, respectively. Analyses of molecular weight profile, amino acid composition, and secondary structure showed that the skin collagens from both species were typical type-I collagen. The amino acid composition of ASC and PSC for both species was closer to calf skin ASC than to cod skin ASC. Jung et al. (2005) reported the enzymatic solubilization of the skeletons discarded from industrial processing of hoki (Johnius belengerii). The efficiency of an enzymatic preparation extracted from the intestine of bluefin tuna (Thunnus thynnus) containing tryptic and collagenic enzymes was compared to other commercial enzymes such as AlcalaseÕ 2.4L, pepsin, collagenase and papain. The tuna intestine crude enzyme (TICE) efficiently degraded the hoki

Enzymatic methods for marine by-products recovery

131

bone matrices composed of collagen, non-collagenous proteins, carbohydrates and minerals, in comparison with other enzymes tested. Total bone hydrolysates liberated by TICE were 32.1% of total bone, from which an oligophosphopeptide with 23.6% phosphorus, a molecular mass of 3.5 kDa and calcium binding activity was purified. The fish bone oligophosphopeptide prepared by enzymatic digestion of the bone could be utilized as a nutraceutical with potential calcium-binding activity. Gildberg et al. (2002) used a combination of gentle enzymatic hydrolysis and chemical extraction to recover both gelatine and a calcium-rich residual bone fraction with favourable nutritional properties. The functional properties of gelatine extracted from shark cartilage were lower than that of porcine skin gelatine but their fat-binding capacity was higher (Cho et al., 2004). Some characteristics of fish collagens are presented in Table 6.6. The quality and, consequently, the specific applications of both collagen and gelatine are highly related to their purity and functional properties. The denaturation temperatures (Td) of collagen from cold-water fish are lower than those of warm-water fish and seemed to be correlated with a lower content of imino acids (proline and hydroxyproline) compared to those of temperate and warm-water fish (Jongjareonrak et al., 2005a; Kittiphattanabawon et al., 2005). The presence of pigments and fish odours may restrict the potential use of fish collagen and gelatine. However, although fish gelatine does not form particularly strong gels, it is well-suited for certain industrial applications, e.g. microencapsulation, lightsensitive coating and low-set-time glues (Rustad, 2003). Chondroitin sulphate from shark cartilage Shark cartilage and purified components of cartilage have traditionally been credited with a number of medical benefits, including anticancer effects (Hassan et al., 2005), fibrinolytic activities (Ratel et al., 2005), and beneficial effects on osteoarthritis, progressive systemic sclerosis and neurovascular glaucoma, as well as other diseases. The immunomodulating activity of shark cartilage was associated with large molecular masses and the active compounds are extracted from shark cartilage pulverized in a complex medium without any enzymatic process (Kravolec et al., 2003). Chondroitin sulphate, a bioactive compound of shark cartilage, is known to have the same therapeutic functions as described above. Chondroitin sulphate is a typical mucopolysaccharide sulphate. There are three isomers differing in the position of the sulphuric acid groups. The promotion of crude shark cartilage extracts as a cure for cancer is highly criticized by Ostrander et al. (2004) for at least two significant negative outcomes: a dramatic decline in shark populations and a diversion of patients from effective cancer treatments. Lignot et al. (2003) described a low cost process producing chondroitin sulphate (CS) in non-denaturing conditions. The first step consisted of an enzymatic extraction using papain at 65ëC, pH 6.5 for 3 hours, followed by a tangential filtration to concentrate and purify CS up to a volume concentrate ratio (VCR) of 4. The performances of UF and MF membranes were compared

Table 6.6

Imino acid content and thermal transition temperature of acid soluble collagen (ASC) and pepsin soluble collagen (PSC) (type I)

Source of collagen

Imino acids (Pro+Hyp) content per 1000 residues

Tm (ëC)

Td (ëC)

Yield (%)

Black drum (Pogonias cromis) skin ASC Sheepshead skin ASC Black drum (Pogonias cromis) skin PSC Sheepshead skin PSC Cod (Gadus morhua) skin ASC Brownstripe red snapper (Lutjanus vitta) ASC Brownstripe red snapper (Lutjanus vitta) PSC Hake (Merluccius merluccius) skin collagen Trout (Salmo irideus) skin collagen Fish (Pagrus major) scale collagen PSC Bigeye snapper (Priacanthus macracanthus) skin ASC

199.8 205.1 197.1 198.1 130.3 212.0 221.0 191.0 180.0 180.0 211.0

34.2 34.0 35.8 34.3 ND np np np np 28.85 np

2.3dwb 2.6dwb 15.8dwb 29.3dwb np 9 4.7wwb np np np 6.4wwb

Jongjareonrak et al. (2005a) Jongjareonrak et al. (2005a) Montero et al. (1990) Montero et al. (1990) Ikoma et al. (2003) Jongjareonrak et al. (2005b)

Bigeye snapper (Priacanthus macracanthus) skin PSC

187.0

34.8 33.5 35.1 33.6 13.0 31.5 31.02 np np np 30.37* 28.85** 30.87* 29.38** np np 25.6 25.0 np np

1.1wwb

Jongjareonrak et al. (2005b)

Ocellate puffer fish (Takifugu rubripes) skin PSC Adult Nile perch (Lates niloticus) ASC Chub mackerel skin (Scomber japonicus) PSC Bullhead shark skin (Heterodontus japonicus) PSC Porcine dermis PSC Porcine skin ASC

170.0 193.0±200.0 np np 220.0 205.0

dwb: dry weight basis; np: not precise; Tm: thermal transition; wwb: wet weight basis *in deionized water; ** in 0.05 mol.kgÿ1 acetic acid.

28.0 36.0 np np 40.69 37.0

44.7dwb 58.7±63.1 49.8dwb 50.1dwb np np

References

Ogawa Ogawa Ogawa Ogawa

et et et et

al. al. al. al.

(2003) (2003) (2003) (2003)

Nagai et al. (2002) Muyonga et al. (2004) Nagai and Suzuki (2000) Nagai and Suzuki (2000) Ikoma et al. (2003) Morimura et al. (2002)

Enzymatic methods for marine by-products recovery

133

in terms of flux and selectivity. The 0.1 m-pore size membrane appeared to be most efficient to separate CS from other compounds. 6.3.4 By-products from the shellfish industry By-products from shellfish processing (crabs, shrimps, lobsters, etc.) are a good source of chitin, chitosan and proteins, which consist of approximately 33% protein and 33% chitin (Martin and Patel, 1991). Chitin is the second most abundant natural biopolymer polymer after cellulose. Chitosan is the deacetylated (to varying degrees) form of chitin, which, unlike chitin, is soluble in acidic solutions. Chitin and chitosan are interesting polysaccharides with unique properties that offer a wide range of industrial applications. They may be employed to solve numerous problems in environmental and biomedical engineering (Ravi Kumar, 2000). The food applications of chitin and chitosan are numerous: these include preservation of foods from microbial deterioration due to their antimicrobial activity, formation of biodegradable films, clarification and deacidification of fruit juices, etc. (Shahidi et al., 1999). Chitosan may find applications as a preservative coating for herring and Atlantic cod in reducing or preventing moisture loss, lipid oxidation, and microbial growth (Jeon et al., 2002). The processing of crustacean shells mainly involves the removal of proteins and the dissolution of calcium carbonate, which is present in crab shells in high concentration (Ravi Kumar, 2000). Usually, a simple base extraction is employed. The alkali removes the protein and deacetylates chitin simultaneously. However, this process produces waste liquid containing base, proteins and protein degradation products, and results in commercial products of inconsistent physico-chemical characteristics. Chitinolytic enzymes would be a prime tool for converting chitin into oligomeric units without the use of the chemical depolymerization such as concentrated hydrochloric acid. There are many reports of purification and identification of chitinolytic activities in marine species, the origin of the gastrointestinal chitinases (endogenous origin or from microflora) being disputed (see the review of Shahidi and Kamil, 2001). However, the main enzymatic processes use proteolytic enzymes for the shell digestion in order to recover chitin and nutritionally valuable protein hydrolysates. For example, Synowiecky and Al-Khateeb (2000) reported the digestion of Crangon crangon processing discards preliminary demineralized using AlcalaseÕ 2.4L at 55ëC and pH 8.5. Recovered protein hydrolysate contained, on a dry basis, 64.3% of protein (N  6.25), 6.24% lipids and 23.4% of sodium chloride. The authors concluded that the enzymatic deproteinization of the shrimp shells using AlcalaseÕ 2.4L was suitable for isolation of the chitin containing only about 4% of protein impurities and also for production of protein hydrolysate with good essential amino acid index (125.4) and protein efficiency ratio (2.99). Simpson and Haard (1985) performed extraction of carotenoproteins from shrimp processing discards using trypsin and a chelating agent. The product recovered contained about 80% of the protein and carotenoid pigments present

134

Maximising the value of marine by-products

in shrimp offal. A new process for advanced utilization of shrimp wastes including enzymatic hydrolysis was recently described by Gildberg and Stenberg (2001). The authors demonstrated the recovery of amino acids, nitrogen and astaxanthin by AlcalaseÕ 2.4L pre-treatment of shrimp (Pandalus borealis) processing wastes before further processing in chitosan. The AlcalaseÕ 2.4L treatment did not influence negatively either the recovery or the quality of chitosan produced from the shrimp heads and scales. The nitrogen recovery was about 70% as compared to only 15% by conventional method. The yield and quality of chitosan was not affected by the enzymatic treatment. In addition, a concentrate of astaxanthin was recovered and could constitute a valuable supplement in salmon feed, improving both the growth and the disease resistance of the fish. According to Shahidi et al. (1999), most physiological activities and functional properties of chitin and chitosan oligomers clearly depend upon their molecular weights and a chain length of at least five residues is required. These oligomers may be more advantageous than chitin and chitosan as polymers in the field of food additives and nutraceuticals in human health, because chitin and chitosan could not be degraded in the human intestine due to the absence of enzymes such as chitinase and chitosanase. In this context, chitin and chitosan may behave as dietary fibres which are excreted without any degradation in the intestine.

6.4

Traceability of by-products

All commodities recovered from by-products may be prepared from a large number of species and from starting materials that exhibit much wider compositional variations. The following section will examine the question of accurate determination of fish species at the various steps of processing, when the by-products are presented in the form of fragments, the origin of which it is impossible to determine. Another aspect of the traceability of the products, in terms of contaminant and toxin contents will not be discussed in this section. A method of genetic identification applicable to fresh fish samples as well as to derived products, has recently been described (GueÂrard et al., 2005a). The first step of the protocol consisted of extraction, purification and amplification of a DNA fragment from muscle, skin, liver, bone or cartilaginous material using PCR technology. The DNA can also be directly sequenced using either one or the other external primer that was used for the amplification process or with an internal primer. This protocol was illustrated with data obtained for two processed (boiled, washed and dried) samples of `dry shark fin' collected from a fish market in Asia, for which it was impossible to obtain any indication of origin. After checking for possible misinterpretations of the results of electrophoresis, the sequences obtained in an easily exploitable forms from the two `dry fin' samples were aligned and compared using the program (Blast) and a sequence library established for phylogenetic purposes. From a comparison of

Enzymatic methods for marine by-products recovery

135

shark sequences, using only one (partial) marker sequence, the authors demonstrated the processed sample was composed of two different species of shark from the genus Sphyrna. Thus, the use of genetic markers for studying genetic diversity and population structure of marine resources, for stock identification and fishery management, is a powerful, reliable and easy technique on fresh, ethanol-preserved, dry, boiled or even processed samples. However, there is a need to complete this approach, assessing methodologies for acquiring data on a large panel of commercial fish and shellfish species and related by-products. It is also necessary to draw up consensual protocols and to adapt the choice of the markers to the level of the need for identification. In addition, it is of primary importance that the sequences used as references were given with all the desired precision on specimens identified without any ambiguity.

6.5

Conclusions and future trends

Bioconversion of fishery processing by-products is receiving increasing attention with the realization that the by-products contain valuable components which can be utilized for conversion into useful and high-value products. Physical and chemical processes applied to fishery by-products have demonstrated their limits in terms of functionality and quality of the final products, while enzymatic processes appear to be prominent among those which will need to be developed for the upgrading of fishery by-products, because they offer the possibility of obtaining tailored products. With regard to future trends, emphasis should be laid on the following aspects: · The production of fish oils is a mature technology, with more than one decade of experience and refinement. Due to (i) environmental pressures demanding cleaner processes, (ii) existence of a market for new products based on fish oils, and (iii) highly labile nature of PUFAs, the application of enzymes in bioprocessing is especially advantageous due to environmentally friendly processes. Increased efforts should be focused on research for more specific lipases and proteases in order to control production of tailor-made products. · Fish protein hydrolysates prepared using enzymatic processes under controlled conditions will probably find new uses and markets. Additional research is needed for optimization of the enzymatic processes (e.g., choice of more specific enzymes, development of models for prediction of hydrolysis degree and biological activities) in order to develop hydrolysates enriched in tailored peptides suitable for the production of specific food with active compounds (antihypertensive, antioxidant, etc.). · Further research work is needed on scaling up of laboratory-tested processes to commercial applications based on improved understanding of the mechanisms of enzymatic hydrolysis in heterogeneous media and on the reproducibility of the enzymatic processes.

136

Maximising the value of marine by-products

· The potential for genetic traceability of the raw material needs to be developed with the use of genetic markers for accurate determination of fish species and by-products at various steps of processing. · Much greater efforts are required to recover by-products of higher quality (for example, low microbial load, low content in heavy metals and toxins) and for the training of people working on the recovery of fishery by-products. In addition, efforts should be focused on the recovery and primary stabilization of by-products such as storage at low temperature or freezing. · The potential for upgrading of by-products from fish processing industries needs to be improved. Efficient transportation or close proximity to a processing plant will facilitate processing of very fresh raw material, in which initial post-mortem oxidation processes are limited. · Last but not least, so that the by-products have a durable future and may be transformed in new products with high added value, they will have to be treated with the same care as the products intended for human consumption.

6.6

Acknowledgements

This work was performed within the Integrated Research Project SEAFOODPlus, Contract N FOOD-CT-2004-506359. The partial financing of this work by the European Union is gratefully acknowledged. In addition, we thank Mr. Jean-Jacques Le Yeuc'h for reviewing the English language of this document.

6.7

References and further reading

(1979), `Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid', J Agric Food Chem, 27(2), 1256± 1262. ADLER-NISSEN J (1982), `Limited enzymic degradation of proteins : a new approach in the industrial application of hydrolases', Chem Tech Biotechnol, 32, 138±156. AIDOS I, VAN DER PADT A, BOOM R M, LUTEN J B (2001), `Upgrading of maatjes herring byproducts: production of crude fish oil', J Agric Food Chem, 49, 3697±3704. AIDOS I, VAN DER PADT A, LUTEN J B, BOOM R M (2002a), `Seasonal changes in crude and lipid composition of herring fillets, byproducts, and respective produced oils `, J Agric Food Chem, 50, 4589±4599. AIDOS I, MASBERNAT-MARTINEZ S, LUTEN J B, BOOM R M, VAN DER PADT A (2002b), `Composition and stability of herring oil recovered from sorted by-products as compared to oil from mixed by-products `. J Agric Food Chem, 50, 2818±2824. AIDOS I, KREB N, BOONMAN M, LUTEN J B, BOOM R M, VAN DER PADT A (2003), `Influence of production process parameters on fish oil quality in a pilot plant', J Food Sci, 68, 581±587. ALASALVAR C, SHAHIDI F, QUANTIK P (2002), `Food and health applications of marine nutraceuticals : a review' in Alasalvar C and Taylor T, Seafoods ± Quality, Technology and Nutraceutical Applications, Berlin, Springer, pp. 174±204. ADLER-NISSEN J

Enzymatic methods for marine by-products recovery

137

(2000), `Recovery of enzymes from seafood processing wastes', in Haard NF and Simpson E, Seafood Enzymes ± Utilization and influence on postharvest seafood quality, New York, Marcel Decker, pp. 641±664. ANDRIEUX G (2004), `La filieÁre francËaise des co-produits de la peÃche et de l'aquaculture: eÂtat des lieux et analyse'. Office national interprofessionnel des produits de la mer et de l'aquaculture (OFIMER), Paris, France. ASPMO SI, HORN SJ, EIJSINK VGH (2005a), `Enzymatic hydrolysis of Atlantic cod (Gadus morhua L.) viscera', Process Biochem, 40, 1957±1966. ASPMO SI, HORN SJ, EIJSINK VGH (2005b), `Hydrolysates from Atlantic cod (Gadus morhua L.) viscera as components of microbial growth media', Process Biochem, 40, 3714±3722. BAEK HH, CADWALLER KR (1995), `Enzymatic hydrolysis of crayfish processing byproducts', J Food Sci, 60, 929±934. BENJAKUL S, MORRISSEY M T (1997), `Protein hydrolysates from Pacific whiting solid wastes', J Agric Food Chem, 45, 3423±3430. BERGEÂ J P, BARNATHAN G (2005), `Fatty acids from lipids of marine organisms: molecular biodiversity, roles as biomarkers, biologically active compounds, and economical aspects', in Le Gal Y and Ulber R, Marine Biotechnology I, Series: Advances in Biochemical Engineering/Biotechnology, Vol. 96, Springer-Verlag Berlin Heidelberg, pp. 49±126. BERNET F, MONTEL V, NOEL B, DUPOUY JP (2000), `Diazepam-like effects of a fish protein hydrolysate (Gabolysat PC60) on stress responsiveness of the rat pituitary-adrenal system and sympathoadrenal activity', Psychopharmacology, 149, 34±40. BOGWALD J, DALMO R, MCQUEEN LEIFSON R, STENBERG E, GILDBERG A (1996), `The stimulatory effect of a muscle protein hydrolysate from atlantic cod, Gadus morhua L., on atlantic salmon, Salmo salar L., head kidney leucocytes', Fish Shellfish Immunol, 6, 3±16. BORDENAVE S, FRUITIER I, BALLANDIER I, SANNIER F, GILDBERG A, BATISTA I, PIOT JM (2002), `HPLC preparation of fish waste hydrolysate fractions. Effect on guinea pig ileum and ACE activity', Prep Biochem Biotechnol, 32, 65±77. BRENNAN M, GORMLEY R (2002), `Eating quality of deep-water fish species and their products', in Alasalvar C and Taylor T, Seafoods ± Quality, Technology and Nutraceutical Applications, Berlin, Springer, pp. 33±42. BYUN HG, KIM SK (2001), `Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin', Process Biochem, 36, 1155±1162. CANCRE I, RAVALLEC R, VANVORMHOUDT A, STENBERG E, GILDBERG A, LE GAL Y (1999), `Secretagogues and growth factors in fish and crustacean protein hydrolysates', Mar Biotechnol, 1, 489±494. CHANTACHUM S, BENJAKUL S, SRIWIRAT N (2000), `Separation and quality of fish oil from precooked and non-precooked tuna heads', Food Chem, 69, 289±294. CHO S M, KWAK K S, PARK D C, GU Y S, JI C I, JANG D H, LEE Y B, KIM S B (2004), `Processing optimization and functional properties of gelatin from shark (Isurus oxyrinchus) cartilage', Food Hydrocolloids, 18, 573±579. DAUKSAS E, FALCH E, SLIZYTE R, RUSTAD T (2005), `Composition of fatty acids and lipid classes in bulk products generated during enzymatic hydrolysis of cod (Gadus morhua) by-products', Process Biochem, 40, 2659±2670. DINIZ FM, MARTIN AM (1996), `Use of response surface methodology to describe the combined effects of pH, temperature and E/S ratio on the hydrolysis of dogfish AN H, VISESSANGUAN W

138

Maximising the value of marine by-products

(Squalus acanthias) muscle', Int J Food Sci Technol, 31, 419±426. (1997a), `Optimization of nitrogen recovery in the enzymatic hydrolysis of dogfish (Squalus acanthias) protein. Composition of the hydrolysates', Int J Food Sci Technol, 48, 191±200. DINIZ FM, MARTIN AM (1997b), `Fish protein hydrolysates by enzymatic processing', Agro Food Ind Hi-Tec, May/June, pp. 9±13. DINIZ FM, MARTIN AM (1998), `Influence of process variables on the hydrolysis of shark muscle protein', Food Sci Technol Int, 4, 91±98. DUFOSSE L, DE LA BROISE D, GUEÂRARD F (2001), `Evaluation of nitrogenous substrates such as peptones from fish: a new method based on Gompertz modeling of microbial growth', Curr Microbiol, 42, 32±38. DUFOSSE L, RICHERT L, GUEÂRARD F (2003), `Microbial nitrogenous substrate from protein hydrolysate of non tradable oysters, a marine equivalent to yeast extract?' in Colliec-Joualt S, Marine biotechnology: an overview of leading fields, Editions IFREMER, Plouzane (France), pp. 169±175. DUMAY J, BARTHOMEUF C, BERGE J P (2004), `How enzymes may be helpful for upgrading fish by-products: Enhancement of fat extraction', J Aquatic Food Prod Technol, 13(2), 69±84. FAO REPORT (2004), `The state of world fisheries and aquaculture', FAO Fisheries department, Food and Agriculture Organization of the United Nations, Rome. FERREIRA NG, HULTIN HO (1994), `Liquifying cod fish frames under acidic conditions with a fungal enzyme', J Food Process Preserv, 18, 87±101. FOUCHEREAU-PEÂRON M, DUVAIL L, MICHEL C, GILDBERG A, BATISTA I, LE GAL Y (1999), `Isolation of an acid fraction from a fish protein hydrolysate with a calcitoningene-related-peptide-like biological activity', Biotechnol Appl Biochem, 29, 87± 92. DINIZ FM, MARTIN AM

 MEZ-MEZA N, NORIEGA-RODRIGUEZ JA, MEDINA-JUAÂREZ LA, ORTEGA-GARCIÂA J, MONROYGA  LO-GUERRERO O (2003), RIVERA JA, TORO-VAZQUEZ JF, GARCI A HS, ANGU

`Concentration of Eicosapentaenoic Acid and Docosahexaenoic Acid from Fish Oil by Hydrolysis and Urea-Complexation', Food Res Int, 36, 721±727. GANDHI N (1997), `Applications of lipase', J Am Oil Chem Soc, 74, 621±634. GBOGOURI GA, LINDER M, FANNI J, PARMENTIER M (2004), `Influence of hydrolysis degree on the functional properties of salmon byproducts hydrolysates', J Food Sci, 69, 615±622. GHORBEL S, SOUISSI N, TRIKI-ELLOUZ R, DUFOSSEÂ L, GUEÂRARD F, NASRI M (2005), `Preparation and testing of Sardinella protein hydrolysate as nitrogen source for extracellular lipase production by Rhizopus oryzae', World J Microbiol Biotechnol, 21, 33±38. GILDBERG A, STENBERG S (2001), `A new process for advanced utilisation of shrimp waste', Process Biochem, 36, 809±812. GILBERG A, BATISTA I, STROM E (1989), `Preparation and characterization of peptones obtained by a two-step enzymatic hydrolysis of whole fish', Biotechnol Appl Biochem, 11, 413±423. GILDBERG A, BéGWALD J, JOHANSEN A, STENBERG E (1996), `Isolation of acid peptide fractions from a fish protein hydrolysate with strong stimulatory effect on Atlantic salmon (Salmo salar) head kidney leucocytes', Comp Biochem Physiol B-Biochem Mol Biol, 114B, 97±101. GILDBERG A, SIMPSON B, HAARD N (2000), `Uses of enzymes from marine organisms', in Haard NF and Simpson BK, Seafood enzymes, New York, Marcel Dekker, pp. 619±639.

Enzymatic methods for marine by-products recovery

139

(2002), `Utilisation of cod backbone by biochemical fractionation', Process Biochem, 38, 475±480.

GILDBERG A, ARNESEN JA, CARLEHOG M

GOMEZ-GUILLEN M C, TURNAY, J, FERNANDEZ-DIAZ M D, ULMO N, LIZARBE M A, MONTERO P

(2002), `Structural and physical properties of gelatine extracted from different marine species: a comparative study', Food Hydrocolloids, 16, 25±34. GUEÂRARD F, RAVALLEC-PLE R, DE LA BROISE D, BINET A, DUFOSSE L (2001a), `Enzymic solubilisation of proteins from tropical tuna using Alcalase and some biological properties of the hydrolysates', in Thonart P and Hofman M, Engineering and Manufacturing for Biotechnology, Focus on Biotechnology, Vol. IV, New York, Kluwer Academic Publishers, pp. 39±53. GUEÂRARD F, DUFOSSE L, DE LA BROISE D, BINET A (2001b), `Enzymatic hydrolysis of proteins from yellowfin tuna (Thunnus albacares) wastes using Alcalase', J Mol Catal B-Enzym, 11, 1051±1059. GUEÂRARD F, GUIMAS L, BINET A (2002), `Production of tuna waste hydrolysates by a commercial neutral protease preparation', J Mol Catal B-Enzym, 19-20, 489±498. GUEÂRARD F, SUMAYA-MARTINEZ MT, BINET A (2003), `An example of shrimp waste upgrading: the production of hydrolysates with antioxidative ± free radical scavenging properties', in Colliec-Joualt S, Marine biotechnology: an overview of leading fields, Editions IFREMER, Plouzane (France), pp. 155±162. GUEÂRARD F, BATISTA I, PIRES C, LE GAL Y (2004), `Report on sources and selection criteria for raw material', in Integrated Project SEAFOODplus 2004-2008, Pilar 4 ± PROPEPHEALTH, workpackage 4.1.1. GUEÂRARD F, SELLOS D, LE GAL Y (2005a), `Fish and Shellfish Upgrading, Traceability' in Le Gal Y and Ulber R, Marine Biotechnology I, Series: Advances in Biochemical Engineering/Biotechnology, Vol. 96, Springer-Verlag, Berlin, pp. 127±164. GUEÂRARD F, SUMAYA-MARTINEZ MT, LINARD B, DUFOSSE L (2005b), `Marine protein hydrolysates with antioxidant properties', Agro Food Ind Hi-Tec, 16, 16±18. HALLDORSSON A, KRISTINSSON B, GLYNN C, HARALDSSON GG (2003), `Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol', J Am Oil Chem Soc, 80, 915±921. HASSAN Z M, FEYZI R, SHEIKHIAN A, BARGAHI A, MOSTAFAIE A, MANSOURI K, SHAHROKHI S, GHAZANFARI T, SHAHABI S (2005), `Low molecular weight fraction of shark cartilage can modulate immune responses and abolish angiogenesis', Int Immunopharmacol, 5, 961±970. HICKMAN D, SIMS TJ, MILES CA, BAILEY AJ, DE MARI M, KOOPMANS M (2000), `Isinglass/ collagen: denaturation and functionality', J Biotechnol, 79, 245±257. HOYLE NT, MERRIT JH (1994), `Quality of fish protein hydrolysates from Herring (Clupea harengus)', J Food Sci, 59, 76±79. HUNTER BJ, ED B, ROBERTS DCK (2000), `Potential impact of the fat composition of farmed fish on human health', Nutr Res, 20, 1047±1058. IKOMA T, KOBAYASHI H, TANAKA J, WALSH D, MANN S (2003), `Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas', Int J Biol Macromol, 32, 199±204. JE JY, PARK PJ, KIM SK (2005), `Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate', Food Res Int, 38, 45±50. JEON YJ, BYUN HG, KIM SK (1999), `Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes', Process Biochem, 35, 471± 478.

140

Maximising the value of marine by-products

(2002), `Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod', J Agric Food Chem, 50, 5167±5178. JONGJAREONRAK A, BENKAJUL S, VISESSANGUAN W, NAGAI T, TANAKA M (2005a) `Isolation and characterization of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper (Lutjanus vitta)', Food Chem, 93, 475±484. JONGJAREONRAK A, BENKAJUL S, VISESSANGUAN W, TANAKA M (2005b) `Isolation and characterization of collagen from bigeye snapper (Priacanthus macracanthus) skin', J Sci Food Agric, 85, 1203±1210. JUN SY, PARK PJ, JUNG WK, KIM SK (2004), `Purification and characterization of an antioxidative peptide from enzymatic hydrolysate of yellowfin sole (Limanda aspera) frame protein', Eur Food Res Technol, 219, 20±26. JUNG W-K, PARK P-J, BYUN H-G, MOON S-H, KIM S-K (2005), `Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase', Food Chem, 91, 333±340. KIM SK, JEON YJ, BYEUN HG, KIM YT, LEE CK (1997), `Enzymatic recovery of cod frame proteins with crude proteinase from tuna pyloric caeca', Fish Sci, 63, 421±427. KIM SK, KIM YT, BUYN HG, NAM KS, JOO DS, SHAHIDI F (2001), `Isolation and characterization of antioxidative peptides from gelatine hydrolysate of Alaska pollack skin', J Agric Food Chem, 49, 1984±1989. KIM S-K, PARK P-J, BYUN H-G, JE JY, MOON S-H (2003), `Recovery of fish bone from hoki (Johnius belengerii) frame using a proteolytic enzyme isolated from mackerel intestine', J Food Biochem, 27, 255±266. KITTIPHATTANABAWON P, BENJAKUL S, VISESSANGUAN W, NAGAI T, TANAKA M (2005), `Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus)', Food Chem, 89, 363±372. KRALOVEC JA, GUAN Y, METERA K, CARR RI (2003), `Immunomodulating principles from shark cartilage. Part 1. Isolation and biological assessment in vitro', Int Immunopharmacol, 3, 657±669. KRISTINSSON HG, RASCO BA (2000), `Fish protein hydrolysates: production, biochemical and functional properties', Crit Rev Food Sci Nutr, 40, 43±81. KROES R, SCHAEFER EJ, SQUIRE RA, WILLIAMS GM (2003), `A review of the safety of DHA 45-oil', Food Chem Toxicol, 41, 1433±1446. LIASET B, LIED E, ESPE M (2000), `Enzymatic hydrolysis of by-products from the fishfilleting industry; chemical characterisation and nutritional evaluation', J Sci Food Agric, 80, 581±589. LIASET B, JULSHAMN K, ESPE M (2003), `Chemical composition and theoretical nutritional evaluation of the produced fractions from enzymatic hydrolysis of salmon frames with Protamex', Process Biochem, 38, 1747±1759. LICEAGA-GESUALDO AM, LI-CHAN ECY (1999), `Functional properties of fish protein hydrolysate from Herring (Clupea harengus)', J Food Sci, 64, 1000±1004. LIGNOT B, LAHOGUE V, BOURSEAU P (2003), `Enzymatic extraction of chondroitin sulphate from skate cartilage and concentration-desalting by ultrafiltration', J Biotechnol, 103, 281±284. LINDER M, MATOUBA E, FANNI J, PARMENTIER M (2002), `Enrichment of salmon oil with n-3 PUFA by lipolysis, filtration and enzyme re-esterification', Eur J Lipid Sci Technol, 104, 455±462. LINDER M, FANNI J, PARMENTIER M (2005a), `Proteolytic extraction of salmon oil and PUFA concentration by lipases', Mar Biotechnol, 15, 70±76. LINDER M, KOCHANOWSKI N, FANNI J, PARMENTIER M (2005b), `Response surface JEON Y-J, KAMIL J Y V A, SHAHIDI F

Enzymatic methods for marine by-products recovery

141

optimisation of lipase-catalysed esterification of glycerol and n-3 polyunsaturated fatty acids from salmon oil', Process Biochem, 40, 273±279. MACKIE IM (1982), `General review of fish protein hydrolysate', Anim Feed Sci Technol, 7, 113±124. MARTIN AM, PATEL TR (1991), `Bioconversion of wastes from marine organisms', In Martin AM, Bioconversion of Waste Materials to Industrial Products, Elsevier Applied Science, London, pp. 417±440. MARTONE CB, BORLA OP, SANCHEZ JJ (2005), `Fishery by-product as a nutrient source for bacteria and archea growth media', Bioresour Technol, 96, 383±387. MONTERO P, BORDERIAS J, TURNAY J, LEYZARBE MA (1990), `Characterization of hake (Merluccius merluccius L.) and Trout (Salmo irideus Gibb) collagen', J Agric Food Chem, 38, 604±609. MORIMURA S, NAGATA H, UEMURA Y, FAHMI A, SHIGEMATSU T, KIDA K (2002), `Development of an effective process for utilization of collagen from livestock and fish waste', Process Biochem, 37, 1403±1412. MUYONGA JH, COLE CGB, DUODU KG (2004), `Characterisation of acid soluble collagen from skins of young and adult Nile perch (Lates niloticus)', Food Chem, 85, 81±89. NAGAI T, SUZUKI N (2000), `Isolation of collagen from fish waste material ± skin, bone and fins', Food Chem, 68, 277±281. NAGAI T, ARAKI Y, SUZUKI N (2002), `Collagen of the skin of ocellate puffer fish (Takifugu rubripes)', Food Chem, 78, 173±177. NIELSEN PM, PETERSEN D, DAMBMANN C (2001), `Improved method for determining food protein degree of hydrolysis', J Food Sci, 66, 642±646. NILSANG S, LERTSIRI S, SUPHANTHARIKA M, ASSAVANIG A (2005), `Optimization of enzymatic hydrolysis of fish soluble concentrate by commercial proteases', J Food Eng, 70, 571±578. NORMAH I, JAMILAH B, SAARI N, CHE MAN YB (2004), `Chemical and taste characteristics of threadfin bream (Nemipterus japonicus) hydrolysate', J Sci Food Agric, 84, 1290± 1298. OGAWA M, MOODY MW, PORTIER RJ, BELL J, SCHEXNAYDER MA, LOSSO JN (2003), `Biochemical properties of black drum and sheepshead seabream skin collagen', J Agric Food Chem, 51, 8088±8092. OSBORN HT, AKOK CC (2002), `Structured lipids-novel fats with medical, nutraceutical, and food applications', Compr Rev Food Sci Food Safety, 1, 93±103. OSTRANDER GK, CHENG KC, WOLF JC, WOLFE M (2004), `Shark cartilage, cancer and the growing threat of pseudoscience', Cancer Res, 64, 8485±8491. PANYAM D, KILARA A (1996), `Enhancing the functionality of food proteins by enzymatic modification', Trends Food Sci Technol, 7, 120±125. RAGHUNATH MR (1993), `Enzymatic protein hydrolysate from tuna canning wastes ± Standardisation of hydrolysis parameters', Fish Technol, 30, 40±45. RATEL D, GLAZIER G, PROVENCAL M, BOIVIN D, BEAULIEU E, GINGRAS D, BELIVEAU R (2005), `Direct-acting fibrinolytic enzymes in shark cartilage extract. Potential therapeutic role in vascular disorders', Thromb Res, 115, 143±152. RAVALLEC-PLEÂ R, CHARLOT C, PIRES C, BRAGA V, BATISTA I, VAN WORMHOUDT A, LE GAL Y, FOUCHEREAU-PEÂRON M (2001), `The presence of bioactive peptides in hydrolysates

prepared from processing waste of sardine (Sardina pilchardus)', J Sci Food Agric, 81, 1120±1125. RAVI KUMAR M N V (2000), `A review of chitin and chitosan applications', React Funct Polym, 46, 1±27.

142

Maximising the value of marine by-products

(2001), `Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates', Electronic J Biotechnol, 4, 1±8. RUSTAD T (2003), `Utilisation of marine by-products', Electron J Envir Agric Food Chem, 2, 458±463. SCRIMGEOUR C (2005), `Chemistry of fatty acids' in Shahidi F, Bailey's Industrial Oil and Fat Products, Vol 1, Edible Oil and Fat Products: Chemistry, Properties, and Health Effects, 6th edn, Wiley, pp. 1±43. SENANAYAKE SPJN, SHAHIDI F (2002), `Enzyme-catalyzed synthesis of structured lipids via acidolysis of seal (Phoca groenlandica) blubber oil with capric acid', Food Res Int, 35, 745±752. SHAHIDI F, KAMIL JYVA (2001), `Enzymes from fish and aquatic invertebrates and their application in the food industry', Trends Food Sci Technol, 12, 435±464. SHAHIDI F, SYNOWIECKI J (1997), `Protein hydrolyzates from seal meat as phosphate alternatives in food processing applications', Food Chem, 60, 29±32. SHAHIDI F., WANASUNDARA UN (1998), `Omega-3 fatty acid concentrates: nutritional aspects and production technologies', Trends Food Sci Technol, 9, 230±240. SHAHIDI F, SYNOWIECKI J, BALEJKO J (1994), `Proteolytic hydrolysis of muscle proteins of harp seal (Phoca groenlandica)', J Agric Food Chem, 42, 2634±2638. SHAHIDI F, HAN X Q, SYNOWIECKI J (1995), `Production and characteristics of protein hydrolysates from capelin (Mallotus villosus)', Food Chem, 53, 285±293. SHAHIDI F, VIDANA ARACHCHI JK, JEON YJ (1999), `Food applications of chitin and chitosans', Trends Food Sci Technol, 10, 37±51. ROUSSEAU M, BATISTA I, LE GAL Y, FOUCHEREAU-PEÂRON M

SHIMADA Y, MARUYAMA K, SUGIHARA A, BABA T, KOMEMUSHI S, MORIYAMA S, TOMINAGA Y

(1998), `Purification of ethyl docosahexaenoate by selective alcoholysis of fatty acid ethyl esters with immobilized Rhizomucor miehei lipase', J Am Oil Chem Soc, 75, 1565±1571. SHIMADA Y, SUGIHARA A, TOMINAGA Y (2001), `Enzymatic purification of polyunsaturated fatty acids', J Biosci Bioeng, 91, 529±538. SILVESTRE MPC (1997), `Review of methods for the analysis of protein hydrolysates', Food Chem, 60, 263±271. SIMPSON BK (2000), `Digestive proteinases from marine animals', in Haard NF and Simpson B, Seafood enzymes ± Utilization and influence on postharvest seafood quality, Marcel Dekker, New York, pp. 191±213. SIMPSON BK, HAARD NF (1985), `The use of proteolytic enzymes to extract carotenoproteins from shrimp wastes', J Appl Biochem, 7, 212±222. SKARA T, SIVERTSVIK M, BIRKELAND S (2004), `Production of Salmon oil from filleting byproducts ± Effects of storage conditions on lipid oxidation and content of !-3 polyunsaturated fatty acids', J Food Sci, 69, 417±421. SLIZYTE R, DAUKSAS E, FALCH E, STORRé I, RUSTAD T (2005a), `Yield and composition of different fractions obtained after enzymatic hydrolysis of cod (Gadus morhua) byproducts', Process Biochem, 40, 1415±1424. SLIZYTE R, DAUKSAS E, FALCH E, STORRé I, RUSTAD T (2005b), `Characteristics of protein fractions generated from hydrolysed cod (Gadus morhua) by-products', Process Biochem, 40, 2021±2033. SUMAYA-MARTINEZ T, CASTILLO-MORALES A, FAVELA-TORRES E, HUERTA-OCHOA S, PRADO-

(2005), `Fish protein hydrolysates from gold carp (Carrassius auratus): 1. A study of hydrolysis parameters using response surface methodology', J Sci Food Agric, 85, 98±104.

BARRAGAN LA

Enzymatic methods for marine by-products recovery

143

(2002), `Lipase-assisted concentration of n-3 polyunsaturated fatty acids from viscera of farmed Atlantic salmon (Salmo salar L.)', J Food Sci, 67, 130±136. SYNOWIECKY J, AL-KHATEEB NAAQ (2000), `The recovery of protein hydrolysate during enzymatic isolation of chitin from shrimp Crangon crangon processing discards', Food Chem, 68, 147±152. TAYLOR T, ALASALVAR C (2002), `Improved utilisation of fish and shellfish waste. in Alasalvar C and Taylor T, Seafoods ± Quality, Technology and Nutraceutical Applications, Berlin, Springer, 123±136. UAUY R AND VALENZUELA A (2000), `Marine oils: the health benefits of n-3 fatty acids', Nutrition, 16, 680±684. VANSCHOONBEEK K, DE MAAT M P M, HEEMSKERK J W M (2003), `Fish oil consumption and reduction of arterial disease', J Nutr, 133, 657±660. VASQUEZ JA, GONZALEZ MP, MURANO MA (2004), `Peptones from autohydrolysed fish viscera for nisin and pediocin production', J Biotechnol, 112, 299±311. VENUGOPAL V, SHAHIDI F (1995), `Value-added products from underutilized fish species', Crit Rev Food Sci Nutr, 35, 431±453. VIEIRA GHF, MARTIN AM, SAKER-SAMPAIAO S, OMAR S, GONCALVES RCF (1995), `Studies on the enzymatic hydrolysis of Brazilian lobster (Panulirus spp) processing wastes', J Sci Food Agric, 69, 61±65. VOJDANI F, WHITAKER JR (1994), `Chemical and enzymatic modification of proteins for improved functionality', in Hettiarachy NS and Ziegler GR, Protein functionality in food system, Marcel Dekker, New York, pp. 261±309. WANASUNDARA UN, SHAHIDI F (1998a), `Lipase-assisted concentration of n-3 polyunsaturated fatty acids in acylglycerols from Marine Oils', J Am Oil Chem Soc, 75, 945±951. WANASUNDARA UN, SHAHIDI F (1998b), `Concentration of !-3 polyunsaturated fatty acids of marine oils using Candida cylindracea lipase: optimization of reaction conditions', J Am Oil Chem Soc, 75, 1767±1774. WANASUNDARA UN, WANASUNDARA J, SHAHIDI F (2002), `Omega-3 fatty acid concentrates: a review of production technologies' in Alasalvar C and Taylor T, Seafoods ± Quality, Technology and Nutraceutical Applications, Berlin, Springer, 157±174. WARD O P, SINGH A (2005), `Omega-3/6 fatty acids: Alternative sources of production', Process Biochem, 40, 3627±3652. XU X (2000), `Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a review', Eur J Lipid Sci Technol, 102, 287±303. SUN T, PIGOTT GM, HERWIG RP

7 Chemical processing methods for protein recovery from marine by-products and underutilized fish species H. G. Kristinsson, A. E. Theodore and B. Ingadottir, University of Florida, USA

7.1

Introduction

Fish has an amino acid composition which makes it an excellent source of nutritive and easily digestible protein. Fish proteins also possess properties that make them good agents of water holding, gelation, fat binding, emulsification and foaming (Xiong, 1997; Kristinsson and Rasco, 2000). For these reasons they are an attractive food source and ingredient for various food applications. The great demand for quality fish protein in the world is growing at a faster pace than can be met with traditional resources, and has in many places led to significant over fishing, often requiring governmental intervention (Kristinsson and Rasco, 2002). Despite the current bleak situation and the economic disruption this has caused, there are still abundant sources of fish that are underutilized in the sense that they are not utilized as human food, most notably the fatty pelagic fish species and processing by-products. The underutilized species are normally small dark muscle pelagic fish species, and make up to 40±50% of the world fish catch (FAO, 2000). Only about 40% of the small pelagic species caught are utilized for human consumption (Unido, 1990). The potential exists to develop high-value functional protein products from these species. Furthermore, many fisheries operations lead to substantial amounts of by-catch consisting of a complex array of different fish species. For example, it has been reported that over four pounds of by-catch is caught by shrimp boats in the Gulf of Mexico for every pound of shrimp using conventional fishing gear (Cushman, 1998). This material is typically discarded back to the sea with little attempt at recovery, and represents an enormous amount of high quality protein which can be utilized for

Chemical processing methods for protein recovery

145

human consumption and furthermore could create a substantial dividend for the local fishing industry. Even with the best preventative gear available to reduce by-catch it will inevitably always be a sizable portion of the catch or about 2±3 pounds fish per pound of shrimp (Gulf and South Atlantic Fisheries Foundation, Inc, personal communications). Discarding the product makes little economic and environmental sense. The same holds true for fish processing by-products which are typically composed of fish frames generated from filleting, including visceral material, and are usually discarded or utilized in animal feed or fertilizer. Using conventional technologies to process fish and creating value added fish products generally leads to limited utilization of the animal and large amounts of protein-rich by-product materials are lost and not recovered. For example, even the most efficient filleting operations will always yield great amounts of proteinrich by-products, up to 60±70% of the fish depending on species (Mackie, 1982; Kristinsson and Rasco, 2000). This material is high in quality protein and lipids, and other valuable compounds which could be utilized for human consumption. The global aquaculture industry is also growing at a rapid rate and should not be overlooked as it will lead to more processing by-products in the coming years, which could provide a sizable source of quality food protein and lipids. The advantages of a process aimed at isolating high-quality food protein from underutilized fish species and by-products are obvious. To upgrade products made from pelagic species and by-products would not only add economic value and assist the seafood industry, it would also be a more responsible use of these sources. Major efforts have been undertaken in both academia and industry in the past century to reach the goal of economic recovery and utilization of proteins from underutilized species of fish and byproducts (Kristinsson and Rasco, 2000, 2002). Most of these efforts have been met with limited success. Some of the key hurdles in the successful and economic recovery of fish proteins include: (a) the processes have to be able to process the material with as little pre-processing as possible (ideally whole fish), (b) the processes have to be able to utilize low-value sources of fish such as fatty pelagic fish species, trimmings and frames, and (c) the processes have to be able to yield a consistent, functional, palatable and stable product (Kristinsson and Rasco, 2000, 2002). Pelagic species, by-catch and by-products present the fish processor with numerous difficulties with respect to their utilization. These raw materials are very complex, including bones, skins, connective tissue, abundance of oxidatively unstable lipids, large amounts of pro-oxidants (blood and heme proteins), unstable muscle proteins of low functionality and in some cases high levels of active proteases (Okada, 1980; Hultin, 1994; Hultin and Kelleher, 2000). The above factors hamper their direct consumption and greatly limit the possibilities to economically recover functional proteins from them using conventional techniques. Various attempts have been taken towards this goal in the past but with limited success. Both chemical and enzymatic processes have been developed with the goal to recover functional ingredients from these materials, most notably proteins and lipids. One of the oldest methods to recover proteins

146

Maximising the value of marine by-products

from fish muscle is surimi processing which includes washing ground fish muscle with water to leech out undesirable water soluble proteins and lipids. This approach, however, has limited utility on very complex raw materials such as whole fish or by-products. Early attempts on complex raw materials included harsh chemical extraction processes where both proteins and lipids could be effectively extracted, but in many cases functionality was lost and palatability was low. This led to a growing interest in the production of fish protein hydrolysates, which are extracted with enzymes under milder conditions, and are discussed in detail in Chapter 10. Using proteases to extract fish proteins does, however, lead to modifications in their functionality, and often a reduction in certain key functional properties. Therefore, there was still a need to develop a mild chemical extraction process, which could be used on challenging raw materials but not compromise protein functionality. This has recently been achieved with a novel acid and alkaline solubilization/precipitation process developed by Hultin and coworkers (Hultin and Kelleher, 1999; Hultin et al., 2004). This chapter discusses some of the principal chemical processes developed primarily with the goal of extracting functional fish proteins.

7.2

Chemical extraction: fish protein concentrate

One of the earliest attempts to recover protein from by-products and underutilized species for use as a human food was the production of fish protein concentrates (FPC). Fish protein concentrates are produced by using chemical solvents and sometimes high temperatures to extract and separate proteins from other components of the raw material (e.g., fat). The National Marine Fisheries Service (NMFS) in the US (then Bureau of Commercial Fisheries) initiated a large research program in this area in the early 1960s with the goal of finding ways to produce FPC on a large scale to stimulate the US seafood industry and also fight the global protein malnutrition problem (Snyder, 1967). The process of making FPC is relatively straightforward. Solvent extracted FPC (type-A FPC) is produced by extraction with isopropanol or azeotropic extraction with ethylene dichloride. Ethanol has been successfully used as well. Figure 7.1 shows one example of FPC processing (Sikorski and Naczk, 1981). The raw material is ground and then extracted with isopropanol at 20±30ëC for 50 minutes. The supernatant is then collected and extracted two times, first at 75ëC for 90 minutes with isopropanol and then at 75ëC for 70 minutes with azeotropic isopropanol. This gives a final supernatant fraction which is then dried, milled and screened to separate out bone pieces. The final product should be largely colorless and odorless and primarily consist of protein (<1% lipids) with high biological value. This relatively harsh process does, however, take its toll on the functionality of the proteins. Type-A FPC is poorly soluble or dispersible in foods, which greatly limits its applicability (Cheftel et al., 1971; Mackie, 1974; Venugopal et al., 1996). Relatively poor emulsification properties have also been reported (Cheftel et al., 1971; Mackie, 1974; Venugopal et al., 1996).

Chemical processing methods for protein recovery

147

Fig. 7.1 An example of fish protein concentrate production using isopropanol as the extracting solvent (adapted from Sikorski and Naczk, 1981).

Temperature during extraction has an impact on the functionality of the protein. For example, it has been reported that FPC produced at 50ëC had significantly lower emulsifying properties compared to FPC produced at 20ëC (Dubrow et al., 1973). Both had very low solubility. Some studies have, however, reported that FPC has good foaming properties over a wide pH range (pH 2±11), although this functional property may be of limited interest for a fish protein ingredient (Sheustone, 1953; Hermansson et al., 1971; Kinsella, 1976). Despite major problems with protein functionality, solvent extraction has been the method of choice for fatty pelagic fish species (e.g., sardine, herring and capelin) since the protein is effectively separated from the oil, thereby improving oxidative stability. It has been reported that isopropanol is a slightly more efficient solvent than ethanol for fatty fish species since it removes more oil (Moorjani et al., 1968). However absolute ethanol was able to produce FPC of lighter color and a more neutral flavor (Moorjani et al., 1968). Very few recent studies have been reported on the production and use of FPC, since more successful protein extraction techniques are now available (e.g., fish protein hydrolysates (Chapter 10) and fish protein isolates made with pH-shift processing, see below). There are, however, a handful of papers from the last 10±15 years which demonstrate that solvent extracted FPC may find good uses if

148

Maximising the value of marine by-products

it is produced properly. For example Vareltzis and coworkers (1990) used ethanol extraction to make FPC from sardines and added it to hamburger patties. These authors reported that that the overall functional properties of the hamburger, i.e. water binding and cooking yield, increased with addition of FPC. Penetration depth and shear force value of the FPC added hamburger also indicated a better hamburger patty. However, on the downside, the hamburgers were found to have a slightly unfavorable fishy flavor. Although FPC on its own may possess poor properties in many cases, several studies have shown that FPC may be a good substrate for enzymatic hydrolysis to make fish protein hydrolysates (FPH) (Cheftel et al., 1971; Hale, 1972; Spinelli et al., 1972; Quaglia and Orban, 1987a,b; Hoyle and Merritt, 1994). This is because it provides a largely oil-free substrate and has partially denatured proteins which are highly susceptible to enzymatic hydrolysis (Kristinsson and Rasco, 2002). Enzymatically hydrolyzed FPC have generally greatly improved solubility and dispersibility compared to the parent FPC, while some other functional properties such as foaming would be reduced (Hermansson et al., 1971; Kristinsson and Rasco, 2002). Taste and odor problems are generally minimized for FPH when FPC is the starting material (Hale, 1972). For example, Hoyle and Merritt (1994) found that FPC made from herring with ethanol extraction and then hydrolyzed with either Alcalase or papain produced a FPH with a reduced bitterness and less fishy odor compared to FPH made directly from the herring. However, general poor functionality, off-flavors and colors, high cost of production and possible traces of solvent in the final product have made solvent extracted FPC commercially unsuccessful regardless of intensive efforts (Mackie, 1982).

7.3

Chemical hydrolysis

Proteins can be chemically hydrolyzed with either acid or base with the help of high temperatures. Chemical hydrolysis is a relatively inexpensive and simple method to extract fish proteins from by-products and several processes have been proposed for the acid or alkaline hydrolysis of fish (Hale, 1972). Chemical hydrolysis is, however, a difficult process to control and because of that leads to end products with variable composition and functionality (Blenford, 1994; Skanderby, 1994). Furthermore, hydrolyzing proteins at very low or high pH, sometimes in the presence of chemical solvents at very high extreme temperatures, generally yields products with reduced nutritional qualities, poor functionality and restricts their use to products such as seafood flavorings (Webster et al., 1982; Loffler, 1986). Acid hydrolysis is a more commonly used method to hydrolyze fish proteins than alkaline hydrolysis. Acid hydrolysis of fish protein normally involves adding strong hydrochloric acid or sulfuric acid to the fish raw material and then extensively hydrolyzing the proteins at a high temperature, sometimes under high pressure. Total hydrolysis can be achieved in 18 hours at 118ëC in 6N

Chemical processing methods for protein recovery

149

hydrochloric acid (Thomas and Loffler, 1994), although those conditions would rarely be used. The resulting fraction containing the hydrolyzed proteins is then neutralized to pH 6.0±7.0 and dried or concentrated (Thakar et al., 1991). The extensive hydrolysis leads to a product of very high solubility and dispersibility, while other functional properties are largely destroyed (Kristinsson and Rasco, 2002). Due to the pH neutralization, the hydrolysate can contain a large amount of salt (NaCl) which can reduce the palatability of the product. Another downside of the acid hydrolysis process is the destruction of tryptophan, which is an essential amino acid. This limits its use as a protein ingredient in food or animal feed. A handful of publications have shown that acid hydrolysis may find a use as a protein recovery process. Orlova and coworkers (1979) reported a relatively promising process where they used acid hydrolysis on whole fish, followed by steam distillation to remove aromatic substances, then filtering and concentrating the extracted and hydrolyzed protein. The concentrate was successfully used in dehydrated soup cubes and as a microbial media (Orlova et al., 1979). Acid hydrolysis (sometimes with the aid of acidic proteases) is also commonly used to convert underutilized species and processing by-products into fertilizer, due to the simple operation, low production cost and extensive hydrolysis which makes the peptides/amino acids easily utilized by plants (Kristinsson and Rasco, 2002). The use of alkali (mainly sodium hydroxide) to hydrolyze protein can result in poor functionality and adversely affect the nutritive value of the final product. Despite these drawbacks, limited alkali treatment is used in the food industry to recover and solubilize a broad range of proteins (Kristinsson and Rasco, 2002). Very few studies have been published on the alkaline hydrolysis of fish proteins. One of the reported key benefits of alkaline hydrolysis is to help modify and improve functional properties of otherwise highly insoluble FPC (Sikorski and Naczk, 1981). For example, Tannenbaum and coworkers (1970a,b) developed a small-scale batch process that utilizes very high pH (12.5) and 95ëC for 20 min. The final product consisted of large peptides, some which were relatively insoluble at the isoelectric point of myofibrillar proteins, but demonstrated an overall improvement in functionality compared to the original FPC. These authors reported that the alkaline-treated FPC could be used as a milk substitute, giving a product of far superior properties to that obtained with the original FPC, which had very low solubility and dispersibility.

7.4

Surimi processing

Surimi originated in Japan where it has been a traditional food source for centuries. Currently, Japan consumes about 70% of the surimi produced worldwide. In Japan, the popular surimi-based products include: satsuma-age, chikuwa, kamaboko, flavored kamaboko, hanpen/naruto, and crab sticks. Imitation crab chunks, flakes, and sticks are the most popular form of surimi consumed in the

150

Maximising the value of marine by-products

United States and Europe where it's gaining much popularity, and consumption is growing at a rapid rate every year and the product receives a premium price (Anonymous, 2001). China, Russia, and South America have also recently discovered surimi-based crab sticks and are becoming major users (Park, 2000). Surimi once referred to the ground fish paste formed during the manufacturing of the surimi-based product kamaboko. Surimi now describes mechanically deboned then washed fish muscle which is used as an ingredient for a range of imitation seafood products, primarily crustacean and shellfish substitutes. It is important not to confuse fish mince with surimi. Fish mince is a starting material of surimi, not surimi itself. Briefly, in conventional surimi processing the raw material is minced, mechanically deboned, washed with water, strained, dewatered, cryoprotectants added and the product packaged and finally frozen in blocks until used. The remaining myofibrillar protein concentrate demonstrates enhanced functional properties, such as gel-forming ability, water holding capacity, and fat-binding (Okada, 1992). Surimi is often modified for long-term storage or further processed into other seafood products, such as imitation crab meat, by incorporating additional components such as flavoring agents, sugars, and salts. The primary fish species used to make surimi in Japan and in the United States is Alaskan pollock. However, other species such as menhaden (Brevooritia tyrannus), red hake (Urophycis chuss), Pacific whiting (Merluccius productus), and spiny dogfish (Squalus acanthias) are being used in the surimi industry (Gwenn, 1992). For many years the industry was dependent on supply and availability of fresh fish. However, the discovery of adding cryoprotectants to surimi in order to prevent protein denaturation during freezing revolutionized the industry (Park and Lanier, 2000) which was no longer dependent on fluctuations in supply of fresh fish. Surimi manufacture is a multi-step process, as shown in Fig. 7.2. Fish heads are removed, guts are cleaned, and bones are removed with large amounts of water to separate the waste material from the muscle tissue. The muscle is then minced by passing the material through a perforated screen and collecting the mince. During the mincing process, tough cartilage, skin, and bones do not pass through the mesh screen, thus removing further undesirable material from the muscle. By removing blood, skin, membranes, and other materials, the muscle becomes more stable and yields a higher quality product (Park and Morrissey, 2000). The next phase in surimi processing is the washing step. The number of washing cycles and water volume depend on many factors, such as fish species, facility type and capacity, initial fish quality, and desired final surimi quality. Generally, the fish to water ratio is between 1:5 and 1:10, although more modern operations are able to use ratios as low as 1:2. Washing with water removes components that can have negative effects on gelation (e.g., sarcoplasmic proteins, although this is debatable) and compounds that can cause flavor, odor, stability and color problems. The product after washing primarily consists of myofibrillar protein, with a significant decrease in amount of blood, soluble proteins, connective tissue and fat (which are mostly removed during washing) as compared with the starting

Chemical processing methods for protein recovery

Fig. 7.2

151

Processing steps in conventional surimi processing.

material. By removing or at least decreasing the amount of undesirable compounds in the fish, the surimi texture, color, flavor, and storage quality is increased. During the entire process the temperature should be maintained low enough to prevent protein denaturation which varies according to species (Ohshima et al., 1993; Park, 2000). The washed muscle is then refined, which removes any remaining bone pieces, skin, scales and connective tissue. The material is then dewatered two or three times by centrifugation, screening or pressing. Dewatering is necessary because during the process water is absorbed (approximately 100% increase) due to repulsion of negatively charged proteins in the washed mince (which is at pH 6.4 to 7.0). The water reduces the repulsion by separating the proteins. Addition of salt (0.1 to 0.3% NaCl or a combination of NaCl and CaCl2) to the wash water further reduces the repulsive forces by shielding negative charges which allows the proteins to be in closer contact with each other, thus expelling water and reducing the tendency of the tissue to absorb water. Since surimi is generally frozen after dewatering, it is important to protect the functional properties of the product during storage. By adding cryoprotectants to the washed refined material prior to freezing, protein denaturation and aggregation are reduced, which would otherwise result in reduced gelation ability of the proteins (Park and Lanier, 2000). The most common cryoprotectants used in the surimi industry are sorbitol and sucrose at ~8±9%, along with a 1:1 mixture of sodium tripolyphosphate, and tetrasodium pyrophosphate at ~0.2±0.3%. These

152

Maximising the value of marine by-products

compounds are uniformly distributed throughout the surimi by using a silent cutter (Park and Morrissey, 2000). Prior to freezing, proteolytic enzyme inhibitors are sometimes added along with cryoprotectants to prevent proteolytic degradation of proteins during heating. For example in Pacific whiting surimi manufacturing enzyme inhibitors have to be added as well as the application of a very rapid heating rate to minimize proteolytic degradation of muscle proteins (Klesk et al., 2000; Park and Lanier, 2000). Surimi is then frozen in blocks or in chips or chunks (Park and Morrissey, 2000). High quality surimi has generally only been produced from lean white fleshed fish such as Alaska pollock. However, much effort has been put into how to make good surimi from dark fleshed underutilized species as well as by-products. Most of these attempts have led to products with poor gelation properties, in part due to the low pH of the muscle of these species and different protein isomers in dark vs. white muscle. Considerable color and lipid stability problems are also encountered with surimi from dark muscle species and by-products due to the high amount of lipids, pro-oxidants and pigments (Okada, 1980; Hultin and Kelleher, 2000). Studies have shown that oxidative problems can be reduced with proper processing techniques. For example, having the wash-water alkaline or mincing fish tissue underwater (preferably in an alkaline solution) may reduce rancidity and improve gel strength (Hultin and Kelleher, 2000). Also applying antioxidants early on during processing may significantly increase gel strength and oxidative stability (Kelleher et al., 1994). Many researchers have also proposed ways to remove the dark muscle of these species and process only the light muscle (e.g., Langmyhr et al., 1988; Shimizu et al., 1992; Spencer and Tung, 1994). In some cases this has led to high quality surimi, comparable to that from Alaska pollock, which is the industry standard (Ishikawa et al., 1977; Suzuki and Watabe, 1987). However, this practice becomes very expensive as it requires filleting and then deep skinning the fillets to remove the dark muscle, which is highly unfeasible for small pelagic fish species due to prohibitive cost of labor. Also, the more dark muscle is removed so is some light muscle and thus yield drops. In addition, utilizing only the light muscle of fish will lead to substantial loss of protein. Even just obtaining the fillets from these species can be too cost prohibitive. Also, even if fish can be headed and gutted, some tissues such as the black skin layer on their belly flaps and the kidney tissue along the backbone make processing whole fish far more difficult than just the muscle (Hultin and Kelleher, 2000). The requirement to use very fresh, preferably lean white fleshed fish fillets and the high labor needed for conventional surimi processing, therefore makes it a relatively expensive process. The large quantities of water used also increase the cost of this process.

7.5

Fish protein isolates: pH-shift processing

Two recent processes, developed by Dr Herbert O. Hultin and coworkers (Hultin and Kelleher, 1999; Hultin et al., 2004, 2005), involving acid and alkaline

Chemical processing methods for protein recovery

153

solubilization and isoelectric precipitation of muscle protein are specifically designed to recover highly functional, stable proteins from low value underutilized fish and by-products. These new processes have shown great promise for both cold and warm water fish species and are currently being commercialized for several species. Fish proteins can be solubilized without the addition of salt at very low and high pH values. The high and low pH values give the muscle proteins a large net charge, causing them to solubilize. At the same time as the proteins solubilize at extremes of pH, the cellular lipid membrane encasing the myofibrillar proteins is disrupted causing a dramatic drop in solution viscosity (Kristinsson, 2002; Kelleher et al., 2004). This allows new approaches to be taken to economically recover fish muscle proteins to produce functional protein isolates from fish sources of low value (Hultin and Kelleher, 1999; Hultin et al., 2004). The process (Fig. 7.3) involves subjecting a diluted slurry (5±10 fold dilution) of finely homogenized muscle tissue to either a very low pH (~2.5±3) or very high pH (~10.8±11.2) at low temperatures. The solubilization of the muscle proteins, cellular membrane disruption and dramatic

Fig. 7.3 Schematic representation of the acid and alkaline processes used in the production of functional fish protein isolates. The process involves solubilizing muscle proteins at low or high pH, separating them from undesirable muscle components via centrifugation and recovery of the proteins of interest by isoelectric precipitation. The final protein isolate can then be used directly, or stabilized with cryoprotectants and frozen until used. (Adapted from Kelleher and Hultin, 2000.)

154

Maximising the value of marine by-products

Fig. 7.4 The three phases that develop after the first centrifugation step at low or high pH. The soluble protein is collected and sedimented by isoelectric precipitation aided by centrifugation to prepare the protein isolate.

drop in viscosity enable the cellular lipid membranes to be separated from the soluble proteins by centrifugation (Kelleher and Hultin, 2000), at the same time removing solids such as bones, scales and neutral fat, which are not desired in the final product (Fig. 7.4). The soluble proteins are then collected and recovered by adjusting the pH to ca. 5.2±5.5, the isoelectric pH of most muscle proteins (primarily myofibrillar proteins), causing them to aggregate and precipitate to give a protein pellet, i.e. the protein isolate. There are several significant benefits of these processes compared to harsh chemical extraction and hydrolysis processes and surimi processing. Whole fish with skin and bones, and fatty fish can potentially be utilized in the acid- and alkali-aided processes because proteins are selectively separated and recovered from undesirable muscle components. This is not feasible using typical surimi processing without greatly negatively affecting the recoveries and quality (Hultin, 2002). In the acid- and alkali-aided processes protein recoveries are normally significantly higher compared to many other recovery processes. Significant amounts of proteins are often lost during the production of FPC as well as hydrolysis. Conventional surimi processing leads to the loss of almost all sarcoplasmic protein during the washing steps, upwards of 35% of the total protein. Multiple washings furthermore lead to myofibrillar protein solubilization and consequently some loss of these proteins as well (Lin and Park, 1996). The loss of these proteins during conventional surimi processing is responsible for the significant decrease in yield. The sarcoplasmic proteins are, however, largely recovered in the acid- and alkali-aided processes, thus substantially increasing yield. As a testament to this, using Pacific whiting fillets as the starting material, a conventional three-washing cycle surimi processing yielded only 40% recovery, compared with 60% recovery using acid-aided processing (Choi and Park, 2002). Kristinsson and coworkers (2005) published data on catfish muscle, and found that the acid- and alkali-aided processes had

Chemical processing methods for protein recovery

155

Table 7.1 Comparison of protein recovery and lipid reduction for the acid-aided, alkaliaided and surimi processes (adapted from Kristinsson et al., 2005)

Alkali-aided process Acid-aided process Surimi processing

Protein recovery

Lipid reduction (neutral and polar)

70.3  2.9%b 71.5  4.5%b 62.3  3.1%a

88.6  2.8%c 85.4  2.0%b 58.3  7.8%a

Means within one column having different superscript letters are significantly different (p < 0:05).

significantly higher protein recovery than a lab-scale surimi process (Table 7.1). Similar recoveries were recorded for herring light muscle; 74% for acid-aided process and 68% for alkali-aided process (Undeland et al., 2002). The protein recovery can be further increased when the first centrifugation step is omitted (Kristinsson et al., 2005). This is possible for certain raw materials which are stable towards lipid oxidation, and can bring the protein recovery close to 90%. The bottom sediment from the first centrifugation can also be reprocessed to increase the level of protein extracted (Kristinsson and Demir, 2003). The isoelectric precipitation step in the acid- and alkali-aided process also aids in the higher protein yields as compared to conventional surimi processing. This is due to the protein having a zero net charge at their pI, thus leading to aggregation and precipitation of the proteins. Surimi processing, on the other hand, does not involve reducing the native pH, resulting in a moderate negative charge on the protein molecules which gives them more solubility and thus more proteins are leached out during washing. Generally, the acid-aided process has resulted in slightly higher protein yields compared to the alkali-aided process with a few exceptions (e.g., tilapia). Studies by Kristinsson and coworkers (2005) have demonstrated that more of the sarcoplasmic proteins are recovered for the acid-aided process compared to the alkali-aided process, due to more aggregation at pH 5.5 as a consequence of a more improperly refolded protein structure. Many of the sarcoplasmic proteins are not greatly affected by the high pH in the alkaline process, and are thus partly or fully native when readjusted to pH 5.5. That pH is away from the isoelectric point of many sarcoplasmic proteins, and thus they don't ready aggregate and co-precipitate with the aggregated myofibrillar proteins. The higher recovery of sarcoplasmic proteins for the acid process, however, means that more heme proteins are recovered with the isolate, which can have negative consequences on color, odor and eventually flavor. More retention on sarcoplasmic proteins in the isolate does, on the other hand, translate to fewer pollution problems since the processing water has a low biological oxygen demand due to its relatively low protein content. This is not the case for surimi processing. A major advantage of the acid and alkali isolation processes is that undesirable compounds, like skin, bones, microorganisms, cholesterol, membrane lipids, and other contaminating materials are removed during the first centrifug-

156

Maximising the value of marine by-products

ation step (Hultin and Kelleher, 2000). Work on catfish has demonstrated that both processes lead to a significant reduction in aerobic bacteria and also a longer bacterial shelf life compared to surimi from the same starting raw material (Kristinsson, 2004). This work demonstrated that bacteria are both killed/injured by the high and low pH and are also removed during the first centrifugation step in the bottom sediment. The processes also have the potential to remove lipid soluble toxins such as mercury and PCBs, and along with the reduction in bacteria give a safer fish protein product. Removal of most lipid components in the acid and isolation procedures can lead to greater oxidation stability and decreased off-odor development as compared with conventional surimi where membrane lipids mostly remain (Hultin and Kelleher, 2000). The great reduction in lipids is a key step in this process, since many materials of interest are rich in triacylglycerols (neutral storage lipids) and in particular membrane phospholipids due to high amounts of mitochondria in dark muscle (Hultin, 1994). The substantial absence of membranes and neutral lipids in the protein isolate clearly distinguishes the acid and alkali processes from presently available processes. Cholesterol is also reduced in the process due to the removal of membranes (Mireles Dewitt et al., 2002). Due to the higher unsaturation of phospholipids vs. neutral lipids and their greater surface area exposed to the cell aqueous phase, they are known to be the main substrate for oxidative reactions in muscle foods (Shewfelt, 1981; Gandemer, 1999). Their removal is therefore expected to greatly enhance the oxidative stability of the final protein isolate. This is not achieved in conventional processes unless organic solvents are used, which destroys protein functional properties. Kristinsson and Demir (2003) studied four different species and reported total lipid reduction of 58.3, 72.1, 10.4 and 16.7% for surimi processing (catfish, Spanish mackerel, mullet and croaker, respectively), 85.4, 76.9, 58, 38.1% for the acid-aided process, and 88.6, 79.1, 81.4 and 68.4% for the alkali-aided process. Undeland and coworkers (2002) reported ~70% reduction in neutral lipids and ~50% reduction in membrane phospholipids for herring white muscle using both acid and alkali-aided processes. Undeland and coworkers (2005) later reported that including centrifugation in the solubilization step to remove membranes led to about 50% less development of secondary oxidation products (as measured by TBARS analysis). Kristinsson (2004) also demonstrated that catfish protein isolates have lower TBARS values on storage when centrifugation is included in the solubilization step, compared to skipping the centrifugation. Kristinsson and Demir (2003) showed that the alkali-aided process gives a more oxidatively stable protein isolate at pH 5.5 than the acid-aided process, and in many cases is more stable than surimi (Fig. 7.5). Substantial lipid oxidation was seen during the acid-aided process. Petty and Kristinsson (2004) investigated oxidation at low and high pH in detail for Spanish mackerel muscle homogenates and showed extensive oxidation development at low pH but almost no development at high pH. When the homogenate was adjusted from low and high pH to pH 5.5 or 7, the isolates from the acid-aided process oxidized significantly more than those form the alkali-aided process. The same was seen

Chemical processing methods for protein recovery

157

Fig. 7.5 Levels of oxidation for surimi and acid and alkali isolates at day 3 of refrigerated storage (4ëC) as assessed by TBARS (secondary oxidation products). Acid isolates were made using pH 2.5 as the solubilization pH, while alkali isolates were made using pH 11. Isolates were recovered at pH 5.5 and stored at 4ëC. Surimi was made by by washing ground muscle in 3 volumes of water 3 times, with the last wash containing 0.3% NaCl to aid in dewatering. The pH of the surimi was not adjusted and was from pH 6.5± 6.6 for all species. Surimi was also stored at 4ëC.

in a model system with trout hemoglobin and washed cod muscle, and it appears that the hemoglobin (one of the main catalysts/mediators of oxidation in fish muscle) was effectively stabilized at high pH but became highly pro-oxidative at low pH (Kristinsson and Hultin, 2004). Undeland and coworkers (2005) also reported extensive oxidation for herring isolates made with the acid-aided process. This oxidation could be effectively reduced and delayed by employing proper antioxidant treatments, such as erythorbate, EDTA and sodium tripolyphoshpate (Undeland et al., 2005). Undeland and coworkers (2005) demonstrated that if antioxidative treatments (metal reducing agents and metal chelators) are incorporated early on in the extraction process, e.g. during homogenization, oxidative stability of the isolate can be improved. Speed of processing at the extreme pH appears to be important. Lipid oxidation can be somewhat reduced if the system is at low pH for a very brief time before being adjusted to pH 5.5 (Hultin, 2004; Petty and Kristinsson, 2004). Heme proteins are more effectively removed with the alkali-aided process compared to surimi processing, resulting in a product that is whiter and more stable to lipid oxidation. Heme proteins are also protected from denaturation and autoxidation during high pH treatment at low temperature (Kristinsson, 2002). The acid aided process, however, leads to the denaturation of heme proteins and thus co-precipitation with muscle proteins when they are adjusted to pH 5.5 (Kristinsson and Hultin, 2004; Kristinsson et al., 2005). This leads to an isolate with darker color and more oxidative problems (Kristinsson and Demir, 2003). Choi and Park (2002) reported that whiteness was lower in acid-treated Pacific whiting isolates as compared with conventional surimi. This lower whiteness of

158

Maximising the value of marine by-products

Table 7.2 Color of protein isolates and surimi according to Hunter L*, a* and b* values (adapted from Kristinsson and Demir, 2003) Sample

L*

a*

b*

Catfish alkali PI Catfish acid PI Catfish surimi

75.0  0.7 73.8  0.4 70.4  1.1

ÿ3.0  0.2 ÿ3.6  0.2 ÿ0.9  0.2

0.2  0.4 5.7  0.3 0.7  0.4

Croaker alkali PI Croaker acid PI Croaker surimi

67.6  0.4 69.8  0.7 64.7  2.4

ÿ2.1  0.2 ÿ2.0  0.3 0.2  0.1

4.2  0.4 7.8  0.4 6.4  0.9

the acid isolates was attributed to higher b* values, indicating a more yellow appearance. Kristinsson and Demir (2003) demonstrated that color was generally good for isolates made from several warm water species using the acid- and alkali-aided processes as compared to surimi from the same species. Table 7.2 shows the color for catfish and croaker isolates and surimi. Isolates had higher L* values and whiteness than surimi, however the acid isolates had higher yellowness, possibly since more denatured heme proteins are found in these isolates (Kristinsson and Demir, 2003). It has been found that functional properties are retained, decreased (in few cases for the acid process) or often significantly improved (most notably for the alkali process) using the pH-shift processes to recover fish proteins. The main functional property of extracted fish proteins is their ability to form strong and elastic gels with high water-holding capacity. Research shows that the ability of isolates to form gels varies, depending on species and conditions used to make the isolate. Hultin and Kelleher (2000) reported that acid-aided isolates made from Atlantic cod and mackerel produces good gels. Later it was found that Pacific whiting surimi from a 3-cycle washing method made stronger gels than gels from the acid-aided process (Choi and Park, 2002). Work by Kristinsson (2004) has demonstrated that the acid-aided process in some cases does form better gels than surimi but in some cases worse. All studies by this group, however, clearly show that the alkali-aided process produces superior gels over both the acid-aided process and the surimi process. For example, Ingadottir and Kristinsson (2004, 2005) reported significantly higher gel strength and elasticity for tilapia isolates made with the alkali-aided process compared to the acidaided process and surimi (Fig. 7.6). Davenport and Kristinsson (2004) did also report using oscillatory rheology and torsion testing that catfish protein isolates from the acid-aided process have significantly lower gel-forming ability than isolates from the alkali-aided process, and hypothesize that this could be due to some very different effects on protein structure at acid vs. alkali pH. Another study by Yongsawatdigul and Park (2001), demonstrated that rockfish protein isolates produced from the alkali-aided process had better gel-forming ability as compared to the acid-aided and conventional surimi processes. The lower performance of the isolates from the acid-aided process could be due to

Fig. 7.6 Shear stress (kPa) and strain values of gels produced from tilapia acid protein isolate, alkali protein isolate and surimi. The acid isolates were made by using pH 2.9 as the solubilization pH, while the alkali isolates were made using pH 11. Isolates were recovered at pH 5.5. The surimi was made by washing ground muscle in 3 volumes of water 3 times, with the last wash containing 0.3% NaCl to aid in dewatering. A protein paste was made with or without the addition of 2% NaCl, and adjusted to pH 7.2. No cryoprotectants were added. Pastes were cooked in steel tubes at 80ëC for 30 min to form a gel. The gels were stored in a cold room at 4ëC for 48 hours prior to testing with a Torsion Gelometer. (Adapted from Ingadottir and Kristinsson, 2005.)

160

Maximising the value of marine by-products

proteolysis which has been seen during the low pH solubilization step (Choi and Park, 2002; Undeland et al., 2002; Ingadottir and Kristinsson, 2004). Proteolysis is a major problem for any muscle protein extraction process as it will lead to adverse effects on protein functionality, particularly gelation and water-binding. Both Ingadottir and Kristinsson (2005) and Undeland et al. (2002) found proteolysis of myosin at low pH. Kristinsson and Demir (2003) reported the same findings for Spanish mackerel. For some species, proteolysis can also occur at the recovery pH, i.e. pH 5.5. Choi and Park (2002) showed that cathepsin B and L activity was higher in an acid-treated Pacific whiting as compared with a 3-cycle washed surimi, leading to poorer gel-forming ability for the isolate. The differences between the cathepsin activity levels were due to cathepsin B and H removal during repeat washing and pH 5.5 being the optimum pH for cathepsin L activity (An et al. 1994; Choi and Park, 2002). However, cathepsin H was removed from the surimi and inactivated in the acid isolate. Therefore, this enzyme did not contribute to decreased gel-forming ability in either sample. Some species have, however, not demonstrated any proteolysis at low pH, e.g. cod (Hultin and Kelleher, 2000) and catfish (Kristinsson et al., 2005). Cod isolate made with the acid-aided process makes a good gel, while the catfish isolate made with the acid-aided process makes a poorer gel than isolates from the alkali-aided process. The difference has been linked to how the muscle proteins respond differently to changes in pH. Davenport and Kristinsson (2003) reported that catfish myosin adjusted to a low pH (2.5) and then readjusted to pH 7 had significantly less gel-forming ability compared to myosin adjusted to high pH (11) and then readjusted to pH 7. The difference was not attributed to proteolysis, but rather some changes within the protein, which are yet to be fully understood. Previous work with cod myosin demonstrated that acid and alkali treated myosin performed about the same as untreated myosin (Kristinsson and Hultin, 2003). This demonstrates that proteins from different species do respond differently to these processes. The theory has long been that the sarcoplasmic proteins interfere with gel formation, possibly by binding to the myofibrillar proteins on heating (Okada, 1980; Shimizu et al., 1992; Park et al., 1997). This is one of the arguments why these proteins are removed in surimi. However, some recent studies have challenged this belief and have shown that gel strength is either equal or, in fact, enhanced by the presence of the sarcoplasmic proteins (Morioka and Shimizu, 1990; Ko and Hwang, 1995). The presence of the sarcoplasmic proteins in the isolates from the acid and alkali process does not appear to negatively impact the gel strength of the final product (Hultin and Kelleher, 1999) but gel mechanism may be different as these proteins have been acid or alkali denatured. This may, however, be species dependent. The acid-aided process does recover more of the sarcoplasmic proteins, and as has been discussed above, some species produce acid isolates of poor functionality, which could be linked to the higher levels of denatured sarcoplasmic proteins. Kristinsson and Crynen (2003) studied the gelforming ability of myofibrillar and sarcoplasmic proteins from catfish, indivi-

Chemical processing methods for protein recovery

161

dually and in combination. The results indicated that sarcoplasmic proteins subjected to a low and high pH and added to myofibrillar proteins subjected to the same treatment improve the gel-forming ability of the overall system compared to myofibrillar proteins alone. The results suggested that adverse changes in the myofibrillar proteins at low pH (pH 2.5) and positive change in the myofibrillar proteins at high pH (pH 11) may explain the difference between the performance of the acid and alkali isolates, rather than changes in the sarcoplasmic proteins. This is being investigated in more detail. It is a commonly held view that denaturing fish muscle proteins has a detrimental impact on their functional properties (Konno et al., 1997; Visessanguan and An, 2000). As the muscle proteins experience very low or high pH in the acid and alkaline processes, one might assume that they lose their functionality since they are partly denatured. Preliminary work by Kristinsson (unpublished data) on the cellular organization of the muscle cell using phase contrast microscopy showed that as pH is either lowered or increased, the contractile element within the cell is being distorted as its protein constituents are being progressively more charged, repelling each other and eventually they become solubilized and any remnants of the muscle cell are lost. Upon pH readjustment to pH 5.5 the cell structure is clearly not recovered and an aggregate of partially denatured muscle proteins is observed. These findings are supported by recent electron microscope data by Wright and Lanier (2005). Studies on the molecular level with myosin have demonstrated that the protein subunit assembly and tertiary structure are greatly affected by the low and high pH, and are not reversibly fully refolded or reassembled on pH readjustment to neutrality (Kristinsson and Hultin, 2003). Furthermore, the ATPase activity of myosin is almost completely lost on acid or alkali treatment (Kristinsson and Hultin, 2003). Other workers have also shown that isolates made with the process have little or no ATPase activity, yet they have good gel-forming ability. Studies frequently report on the positive relationship between functional ATPase activity and functionality of muscle proteins (Katoh et al., 1979; Ooizumi et al., 1981; Konno et al., 1997). Interestingly, even when all ATPase activity is lost in the isolate, some of them have improved gelling capability compared to proteins still with high ATPase activity. These findings suggest that a native structure is not required for good gel-forming ability, and a partly pH denatured protein may, in fact, be better suited to form quality gels, perhaps through different mechanisms than native proteins.

7.6

Other processes using low or high pH

Only a handful of other workers have described processes for fish proteins that utilize high or low pH. These processes, however, differ considerably in nature to the one described above and the end uses are quite different. Cuq and coworkers (1995) reported on the acid solubilization of fish muscle proteins at pH 3 using aqueous acetic acid for the purpose of producing edible packaging

162

Maximising the value of marine by-products

films. A process was reported by Shahidi and Venugopal (1993) where minced Atlantic mackerel, herring or capelin is homogenized in aqueous liquids, including acetic acid at pH 3.5. Venugopal and Shahidi (1994) later reported a process where Atlantic mackerel is suspended in water and acetic acid at a pH of 3.5. The use of acetic acid in these processes, however, in many cases increases viscosity of these fish protein suspensions and in some cases reduces it insufficiently so that cellular lipid membranes cannot be separated from the fish proteins. The volatile acetic acid also potentially leads to a strong odor to the final material which may limit their use as food products. Some of the above processes also involved washing steps which remove the water soluble sarcoplasmic muscle proteins, which are retained in the acid and alkaline processes described previously. Alkaline pH has also been used in the processing of fish muscle and muscle from other sources. One common use of high pH is to recover protein from deboned meat (McCurdy et al., 1986; Opiacha et al., 1994), not, however, involving separation of membrane lipids from the alkali solubilized protein. Alkaline conditions have been used previously in the manufacture of surimi from fish, where alkali or compounds with buffering capacity at high pH are added to the wash water to increase pH. The wash water pH in these processes is, however, considerably lower than the pH used in the alkaline process employed in this proposal. The increase of wash water pH reportedly yields a product with improved gelling abilities, brighter color and lower lipid content (Shimizu et al., 1992; Jiang et al., 1998; Hultin and Kelleher, 1999). On the other hand, yield drops considerably in these processes, since presumably increased protein solubility as pH is increased would increase the amount of proteins removed in the washing steps. For example, it has been reported that processing surimi from mackerel light muscle using alkaline wash water led to only 40% protein recovery (Hultin and Kelleher, 2000).

7.7

Future trends

To meet the increasing demand for quality fish proteins and products containing fish proteins, it is of great importance to utilize our raw materials more responsibly as well as to find new resources of fish to utilize. Processing methods which can employ inexpensive raw material and whole fish instead of fillets would create a significant economic advantage to the fish protein ingredient industry, as new sources of fish unsuitable for conventional processes could enter the market, and production could be increased to meet the world demand. This chapter has reviewed some of the chemical processes which can be employed to recover fish proteins, as well as lipids. From looking at research and industrial applications of these processes, the pH-shift processing appears to have the most promise on by-products and underutilized species. To increase the success of these processes and their products it is essential to put more research efforts into the commercial applications of these proteins as food ingredients or

Chemical processing methods for protein recovery

163

as food products. More research should be focused on the utilization of extracted fish proteins for human consumption rather than animal consumption, although the latter cannot be overlooked. One very promising food application being studied is the use of isolated fish proteins as water-binders in seafood products. It has been found that these proteins can effectively compete with and outperform phosphates as water-binders. This could have a significant meaning for the seafood processing industry. Future research efforts should also be directed towards ways to effectively stabilize the proteins against functional changes as well as finding efficient and economic ways to stabilize the isolated proteins against oxidative changes (e.g., lipid oxidation during processing and in the final isolate). The future for functional fish protein ingredients is promising and with the right mindset from industry, government and academia great progress can be made in the near future.

7.8

References and further reading

and MORRISSEY, M. T. (1994), Cathepsin degradation of Pacific whiting surimi proteins. J. Food Sci., 59, 1013±1033. ANONYMOUS (2001), Frozen surimi market booms in Europe, sparking new production to meet demand. Quick Frozen Foods Int., 6, 22. BLENFORD, D. E. (1994), Protein hydrolysates; functionalities and uses in nutritional products, Int. Food Ingr., 3, 45. CHEFTEL, C., AHERN, M., WANG, D. I. C. and TANNENBAUM, S. R. (1971), Enzymatic solubilization of fish protein concentrate: Batch studies applicable to continuous enzyme recycling processes, J. Agr. Food Chem., 19, 155. CHOI, Y. J. and PARK, J. W. (2002), Acid-aided protein recovery from enzyme-rich pacific whiting. J. Food Sci., 67, 2962±2967. CUQ, B., AYMARD, C., CUQ, J. L. and GUILBERT, S. (1995), Edible packaging films based on fish myofibrillar proteins: Formulation and functional properties. J. Food Sci., 60, 1369±1374. CUSHMAN JR., J. H. (1998), Cuts sought in wasteful fish kills. New York Times ± Science Times, January 13, 2. DAVENPORT, M. and KRISTINSSON, H. G. (2003), Low and high pH treatments induce a molten globular structure in myosin which improves its gelation properties. IFT Annual Meeting, Chicago, IL, Abstract 42-9. DAVENPORT, M. and KRISTINSSON, H. G. (2004), Effect of different acid and alkalitreatments on the molecular and functional properties of catfish muscle proteins. IFT Annual Meeting, July 12±16, Las Vegas, NV. Abstract 49G-14. DAVENPORT, M. and KRISTINSSON, H. G. (2005), Effect of cold storage and freezing of ground catfish muscle prior to acid or alkali-aided isolation of muscle proteins. IFT Annual Meeting, July 15±20, New Orleans, LA. Abstract 89B-33. DUBROW, D. L., KRAMER, A. and MCPHEE, A. D. (1973), Effects of temperature on lipid extraction and functional properties of fish protein concentrate (FPC). J. Food Sci., 38, 1012. FAO (2000), The State of World Fisheries and Aquaculture; FAO Fisheries Department, Food and Agricultural Organization of the United Nations: Rome, Italy. AN, H., WEERASINGHE, V., SEYMOUR, T. A.

164

Maximising the value of marine by-products

(1999), Lipids and meat quality: lipolysis, oxidation, Maillard reaction and flavour. Sci. Aliment. 19, 439±458. GWENN, S. E. (1992), Development of Surimi Technology in the United States. In T. C. Lanier and C. M. Lee (eds), Surimi Technology, New York, Marcel Dekker, Inc., 23±39. HALE, M. B. (1972), Making fish protein concentrate by enzymatic hydrolysis, NOAA Technical Report NMFS SSRF-675, US Department of Commerce, Seattle, WA, 1±31. HERMANSSON, A. M., SIVIK, B. and SKJOLDEBRAND, C. (1971), Factors affecting solubility, foaming and swelling of fish protein concentrate, Lebensm. Wiss. Technol., 4, 201. HOYLE, N. and MERRITT, J. H. (1994), Quality of fish protein hydrolysates from herring (Clupea harengus), J. Food Sci., 59, 76. HULTIN, H. O. (1994), Oxidation of lipids in seafoods. In F. Shahidi and J. R. Botta, Seafoods: Chemistry, Processing Technology and Quality, Glasgow, Blackie Academic, 49±74. HULTIN, H. O. (2002), Recent Advances in Surimi Technology. In M. Fingerman and R. Nagabhushanam, Recent Advances in Marine Biotechnology, Enfield, New Hampshire, Science Publisher, Inc., 241±251. HULTIN, H. O. (2004), Controlling lipid oxidation during processing at low and high pH. In symposium: Fish Protein Recovery Using pH Shifts. IFT Annual Meeting, Las Vegas, NV, July 12±16, 2004. HULTIN, H. O. and KELLEHER, S. D. (1999), Process for isolating a protein composition from a muscle source and protein composition. US Patent No. 6,005,073. HULTIN, H. O. and KELLEHER, S. D. (2000), Surimi processing from dark muscle fish. In J. W. Park Surimi and Surimi Seafood, New York, Marcel Dekker, 59±77. GANDEMER, G.

HULTIN, H. O., KELLEHER, S. D., FENG, Y., KRISTINSSON, H. G., RICHARDS, M. P., UNDELAND, I. A.

and KE, S. (2004), US Patent Application No. 10/363,612. `High Efficiency Alkaline Protein Extraction'. HULTIN, H. O., KRISTINSSON, H. G., LANIER, T. C. and PARK, J. W. (2005), Process for recovery of functional proteins by pH shifts. In J. W. Park, Surimi and Surimi Seafood, 2nd edn, New York, Marcel Dekker, 107±139. INGADOTTIR, B. and KRISTINSSON, H. G. (2004), Effects of low and high pH treatments on solubility and viscosity of tilapia light muscle homogenates. IFT Annual Meeting, Las Vegas, NV, July 12±16, 2004, Abstract 17E-20. INGADOTTIR, B. and KRISTINSSON, H. G. (2005), Recovery and gel forming ability of proteins recovered from tilapia muscle with acid and alkali aided solubilization and precipitation. IFT Annual Meeting, July 15±20, New Orleans, LA. Abstract 89B-31. ISHIKAWA, S., NAKAMURA, K. and FUJII, Y. (1977), Test program to manufacture sardinebased products and frozen surimi. I. Effects of freshness of material and fish dressing methods. Tokai Fisheries Research Agency Japan, 20, 59±66. JIANG, S-H., HO, M-L., JIANG, S-H., LO, L. and CHEN, H-C. (1998), Color and quality of mackerel surimi as affected by alkaline washing and ozonation. J. Food Sci., 63, 652±655. KATOH, N., NOZAKI, H., KOMATSU, K. and ARAI, K. (1979), A new method for evaluation of the quality of frozen surimi from Alaska pollack. Relationship between myofibrillar ATPase acitivity and Kamaboko forming ability of frozen surimi. Nippon Suisan Gakkaishi, 45, 1027±1032. KELLEHER, S. D. and HULTIN, H. O. (2000), Functional chicken muscle protein isolates prepared using low ionic strength, acid solubilization/precipitation. Rec. Meat. Conf. Proc., 53, 76±81.

Chemical processing methods for protein recovery

165

and WILHELM, K. A. (1994) Stability of mackerel surimi prepared under lipid-stabilizing processing conditions. J. Food Sci., 59, 269±271. KELLEHER, S. D., FENG, Y., KRISTINSSON, H., HULTIN, H. O. and MCCLEMENTS, D. J. (2004), Functional fish protein isolates prepared using low ionic strength, acid solubilization/precipitation. In: More Efficient Utilization of Fish and Fisheries Products. New York, Elsevier Science, 407±414. KINSELLA, J. E. (1976), Functional properties of proteins in foods: A survey, Crit. Rev. Food Sci. and Nutr., 8, 219. KLESK, K., YONGSAWATDIGUL, J., PARK, J. W., VIRATCHAKUL, S. and VIRULHAKUL, P. (2000), Gel forming ability of tropical tilapia surimi as compared with Alaska pollock and Pacific whiting surimi. J. Aquat. Food Prod. Technol., 9, 91±104. KO, W-C. and HWANG, M-S. (1995), Contribution of milkfish sarcoplasmic proteins to gel formation of fish meat. Nippon Suisan Gakkaishi, 61, 75±78. KONNO, K., YAMANODERA, K.-I. and KIUCHI, H. (1997), Solubilization of fish muscle myosin by sorbitol. J. Food Sci., 62, 980±984. KRISTINSSON, H. G. (2002), Conformational and functional changes of hemoglobin and myosin induced by pH: Functional role in fish quality. PhD dissertation. University of Massachusetts at Amherst. KRISTINSSON, H. G. (2004), Using high and low pH processing to produce functional protein ingredients from warm water fish species. In symposium: Fish Protein Recovery Using pH Shifts. IFT Annual Meeting, Las Vegas, NV, July 12±16, 2004. KRISTINSSON, H. G. and CRYNEN, S. (2003), The effect of acid and alkali treatments on the gelation properties of sarcoplasmic and myofibrillar proteins from channel catfish. IFT Annual Meeting, Chicago, IL, Abstract 87-3. KRISTINSSON, H. G. and DEMIR, N. (2003), Functional protein isolates from underutilized tropical and subtropical fish species and byproducts. In P. Bechtel, Advances in Seafood Byproducts, Alaska Sea Grant College Program, University of Alaska, Anchorage, 277±298. KRISTINSSON, H. G. and HULTIN, H. O. (2003), Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding. J. Agric. Food Chem., 51, 7187±7196. KRISTINSSON, H. G. and HULTIN, H. O. (2004), Changes in trout hemoglobin conformations and solubility after exposure to acid and alkali pH. J. Agric. Food Chem., 52, 3633±3643. KRISTINSSON, H. G. and RASCO, B. A. (2000), Fish protein hydrolysates: Production, biochemical and functional properties. CRC Crit. Rev. Food Sci. Hum. Nutr., 40, 43±81. KRISTINSSON, H. G. and RASCO, B. A. (2002), Fish protein hydrolysates and their potential use in the food industry. In M. Fingerman and R. Nagabhushanam, Recent Advances in Marine Biotechnology, Vol. 7, Enfield, NH, Science Publishers, Inc., 157±181. KRISTINSSON, H. G., THEODORE, A. E., DEMIR, N. and INGADOTTIR, B. (2005), The recovery of channel catfish muscle proteins using acid and alkali-aided processing vs. surimi processing. J. Food Sci., 70, C298±C306. LANGMYHR, E., OPSTVEDT, J., OFSTAD, R. and SORENSEN, N. K. (1988), Potential conversion of North Atlantic fatty species into surimi and surimi. In N. Davis, Fatty Fish Utilization: Upgrading from Feed to Food, UNC Sea Grant College Program Publication; Raleigh, NC, 1988. LIN, D. D. and PARK, J. W. (1996), Extraction of proteins from Pacific whiting mince at various washing conditions. J. Food Sci., 61, 432±438. KELLEHER, S. D., HULTIN, H. O.

166

Maximising the value of marine by-products

LOFFLER, A.

63.

(1986), Proteolytic enzymes: sources and applications, Food Technol., 40,

(1974), Proteolytic enzymes in recovery of proteins from fish waste, Proc. Biochem., 12, 12. MACKIE, I. M. (1982), Fish protein hydrolysates. Proc. Biochem., 17, 26±32. MCCURDY, S. M., JELEN, P., FEDEC, P. and WOOD, D. F. (1986), Laboratory and pilot plant scale recovery of protein from mechanically separated chicken residue. J. Food. Sci., 51, 742±747. MIRELES DEWITT, C. A., GOMEZ, G. and JAMES, J. M. (2002), Protein extraction from beef heart using acid solubilization. J. Food Sci., 67, 3335±3341. MOORJANI, M. N., NAIR, R. B. and LAHIRY, N. L. (1968), Quality of fish protein concentrate prepared by direct extraction of fish with various solvents, Food Technol., 22, 1557. MORIOKA, K. and SHIMIZU, Y. (1990), Contribution of sarcoplasmic proteins to gel formation of fish meat. Nippon Suisan Gakkaishi, 56, 929±933. OHSHIMA, T., USHIO, H. and KOIZUMI, C. (1993), High pressure processing of fish and fish products. Trends in Food Sci and Technol., 4, 370±375. OKADA, M. (1980), Utilization of small pelagic species for food. In R. E. Martin. Proceedings of the Third National Technical Seminar on Mechanical Recovery and Utilization of Fish Flesh, Washington, DC, National Fisheries Institute, 265±282. OKADA, S. (1992), History of Surimi Technology. In T. C. Lanier and C. M. Lee, Surimi Technology, New York, Marcel Dekker, Inc., 3±20. OOIZUMI, T., HASHIMOTO, A., OGURA, J. and ARAI, K. (1981), Quantitative aspect for protective effect of sugar and sugar alcohol against denaturation of fish myofibrils. Nippon Suisan Gakkaishi, 47, 901±908. OPIACHA, J. O., MAST, M. G. and MACNEIL, J. H. (1994), Composition of dehydrated protein extracts from poultry bone residue. J. Muscle Foods, 5, 343±353. ORLOVA, T. A., NELICHIK, N. N. and FLEIDER, K. A. (1979), Edible protein concentrate from fish raw material, Rybnoe Khozyaistvo, 10, 59. PARK, J. W. (2000), Surimi and Surimi Seafood, New York, Marcel Dekker Inc. PARK, J. W. and LANIER, T. C. (2000), Processing of Surimi and Surimi Seafoods. In R. E. Martin, Marine and Freshwater Products Handbook, Lancaster, PA, Technomic Publishing Company. PARK J. W. and MORRISSEY M. T. (2000), Manufacture of Surimi from Light Muscle Fish. In J. W. Park, Surimi and Surimi Seafood, New York, Marcel Dekker, Inc., 23±54. PARK, J. W., LIN, T. M. and YONGSAWATDIGUL, J. (1997), New developments in manufacturing of surimi and surimi seafood. Food Rev. Int., 13, 577±610. PETTY, H. T. and KRISTINSSON, H. G. (2004) Impact of antioxidants on lipid oxidation during acid and alkali processing of Spanish mackerel. IFT Annual Meeting, Las Vegas, NV, July 12±16, 2004, Abstract 49B-7. QUAGLIA, G. B. and ORBAN, E. (1987a), Enzymic solubilisation of proteins of sardine (Sardina pilchardus) by commercial proteases, J. Sci. Food Agric., 38, 263. QUAGLIA, G. B. and ORBAN, E. (1987b), Influence of the degree of hydrolysis on the solubility of the protein hydrolsyates from sardine (Sardina pilchardus), J. Sci. Food Agric., 38, 271. SHAHIDI, F. and VENUGOPAL, V. (1993), Notes and digest. Meat Focus Int. October, 443± 445. SHEUSTONE, F. S. (1953), Forming of fish protein concentrate. Food Preserv. Q., 13, 45. SHEWFELT, R. L. (1981), Fish muscle lipolysis ± a review. J. Food Biochem., 5, 79±100. MACKIE, I. M.

Chemical processing methods for protein recovery

167

and LANIER, T. C. (1992), Surimi production from fatty and darkfleshed fish species. In T. C. Lanier and C. M. Lee, Surimi Technology, New York, Marcel Dekker, 181±207. SIKORSKI, Z. E. and NACZK, M. (1981), Modification of technological properties of fish protein concentrates, Crit. Rev. Food Sci. Nutr., 4, 201. SKANDERBY, M. (1994), Protein hydrolysates: their functionality and applications, Food Technol. Int. Eur., 10, 141. SNYDER, D. G. (1967), Bureau of commercial fisheries program, Food Technol., 21(9), 70. SPENCER, K. E. and TUNG, M. A. (1994), Surimi processing from fatty fish. In F. Shahidi and J. R. Botta, Seafoods: Chemistry, Processing Technology and Quality, London, Blackie Academic and Professional. SPINELLI, J., KOURY, B. and MILLER, R. (1972), Approaches to the utilization of fish for the preparation of protein isolates; enzymic modifications of myofibrillar fish proteins, J. Food Sci., 37, 604. SUZUKI, T. and WATABE, S. (1987), New processing technology of small pelagic fish protein. Food Rev. Int., 2, 271±307. TANNENBAUM, S. R., AHERN, M. and BATES, R. P. (1970a), Solubilization of fish protein concentrate. 1. An alkaline process, Food Tecnol., 24, 604±606. TANNENBAUM, S. R., AHERN, M. and BATES, R. P. (1970b), Solubilization of fish protein concentrate. 2. Utilization of the alkaline-process product, Food Tecnol., 24, 607. THAKAR, P. N., PATEL, J. R. and JOSHI, N. S. (1991), Protein hydrolysates: A review. Indian J. Dairy Sci., 44, 557±588. THOMAS, D. and LOFFLER, F. (1994), Improved protein functionalities by enzymatic treatment, Food Marketing and Technology, 2. UNDELAND, I. A., KELLEHER, S. D. and HULTIN, H. O. (2002), Recovery of functional proteins from herring (Clupea harengus) light muscle by an acid or alkaline solubilization process. J. Agric. Food Chem., 50, 7371±7379. UNDELAND, I., HALL, G., WENDIN, K., GANGBY, I. and RUTGERSSON, A. (2005), Preventing lipid oxidation during recovery of functional proteins from herring (Clupea harengus) fillets by an acid solubilization process. J. Agric. Food Chem., 53, 5625±5634. UNIDO, SECTORAL STUDIES BRANCH (1990), Industrial development strategies for fishery systems in developing countries. Food Rev. Int., 6, 1±13. VARELTZIS, K., SOULTOS, N., ZETOU, F. and TSIARAS, I. (1990), Proximate composition and quality of a hamburger type product made from minced beef and fish protein concentrate, Lebensm. Wiss. u. Technol., 23, 112. VENUGOPAL, V. and SHAHIDI, F. (1994), Thermostable water dispersions of myofibrillar proteins from Atlantic mackerel (Scomber scombrus). J. Food Sci., 59, 265±276. VENUGOPAL, V., CHAWLA, S. P. and NAIR, P. M. (1996), Spray dried protein powder from threadfin beam: preparation, properties and comparison with FPC type-B, J. Muscle Foods, 7, 55. VISESSANGUAN, W. and AN, H. (2000), Effects of proteolysis and mechanism of gel weakening in heat-induced gelation of fish myosin. J. Agric. Food Chem., 48, 1024±1032. WEBSTER, J. D., LEDWARD, D. A. and LAWRIE, R. A. (1982), Protein hydrolysates from meat industry by-products, Meat Sci., 7, 147. WRIGHT, B. J. and LANIER, T. C. (2005), Microscopic evaluation of ultrastructural disintegration and dispersion of myofibrillar proteins as affecting gel formation. IFT Annual Meeting, July 15±20, New Orleans, LA. Abstract 50-8. XIONG, Y. L. (1997), Structure-function relationships of muscle proteins. In S. Damodaran SHIMIZU, Y., TOYOHARA, H.

168

Maximising the value of marine by-products

and A. Paraf, Food Proteins and Their Applications, New York, Marcel Dekker, Inc., 341±392. YONGSAWATDIGUL, J. and PARK, J. W. (2001), Gelation characteristics of alkaline and acid solubilization of fish muscle, IFT National Meeting, New Orleans, La. June 23±27, 2001, Institute of Food Technologists, Abstract number100-1.

Part II Food uses of marine by-products

8 By-catch, underutilized species and underutilized fish parts as food ingredients I. Batista, Fish and Sea Research Institute (IPIMAR), Portugal

8.1

Introduction: by-catch, discards and by-products

The evolution of the estimated world fish production from 1950 until around 1990 was basically characterized by an almost continuous growth, which was followed by a levelling off until the end of the 1990s. The increased global production further recorded was due to aquaculture catches, which in that period had an average annual growth rate of 5.3%, excluding China's production (FAO, 2002). In 2000, reported global capture fisheries attained 77 to 78 million tonnes (China not included) with relative percentage distribution shown in Fig. 8.1. In this FAO report it is also pointed out that 70.8% (63 million tonnes) of the estimated production was used for direct human consumption and the remaining was destined for non-food products, mostly for the manufacturing of fish meal and oil. The highest share of the total catch used for fish meal production was 38% in 1970. The global per capita food fish supply has increased from about 7 kg in 1950 to 15 kg in 2000 (Valdimarsson and James, 2001). However, some decrease occurred from 1987 to 2000 because the world's population increased more quickly than the total production of food fish (FAO, 2002). Nevertheless, these mean values mask important regional differences such as those between developed and developing countries where the per capita consumption is 29 and 12.5 kg, respectively (Valdimarsson and James, 2001). The previously mentioned production levels for the total world fisheries do not include the discards, which are potentially useful for food or feed. According to Alverson et al. (1994) such levels of catch include the portion of the catch

172

Maximising the value of marine by-products

Fig. 8.1 Distribution of the world fisheries production in 2000 (FAO, 2002).

returned to the sea as a result of economic, legal, or personal considerations. Another term frequently used is by-catch, which refers to the total catch of nontargeted animals (Kelleher, 2005). Alverson et al. (1994) estimated 27.0 million metric tonnes (MMT) of global discards annually, based on a target catch of 77 MMT. Nevertheless, these figures do not include data from freshwater, molluscan and recreational fisheries. Discarding of fish or its components constitutes a loss of valuable food with negative consequences for the environment and biodiversity. Figure 8.2 shows discard weight in the oceans and Mediterranean and Black Sea regions. The discarded catch from Pacific Ocean represents the majority of total world discards and about 55% of them are originated within the Northwest Pacific fisheries. Shrimp fishery accounts for 35% of the global marine discards and the ratio of discarded weight to landed weight is 5.2. However, there are important differences from a gear-specific perspective. Shrimp trawls present the highest by-catch to landed ratio (in weight basis), which was around 11.8; the lowest ratio was recorded for pelagic trawls (less than 0.01). In 2000, more than 60% of total world fishery production suffered some type of processing problems as reported by FAO (2002). The demand for fresh fish has generally increased in the last four decades, but in the 1990s the estimated demand rose from 28 MMT in 1990 to 52 MMT at the end of this decade. Since 1960, the share of frozen fish also increased but canned fish remained constant at around 10% and production of cured fish gradually declined from 20 to 10% of the total amount. The processing industry generates a significant amount of by-products, which depends on the species and type of product. These by-products are mainly used for fish meal production or other low price ingredients, but sometimes are just dumped. This means that by-products together with the by-catch constitute an important percentage of total catch. In the United Kingdom it was estimated that only 43% of the total catches end up as product for human consumption and the

By-catch, underutilized species and underutilized fish parts 173

Fig. 8.2

Estimation of discard weight (%) by oceans (Alverson et al., 1994).

remainder is classified as waste (Archer, 2001). The waste material comes mainly from the on-shore processing sector (35% of the resource) whereas the contribution of discards and processing waste at sea represents 17 and 5% respectively. In Norway, it is estimated that 80±90% of the by-products generated at sea are dumped and only 10% of the by-products from the on-shore fish processing is used in products for human consumption (Sandbakk, 2002).

8.2

Key drivers

The declining of traditional fish stocks coupled with strict management measures have led to the exploitation of other fish species usually considered underutilized. It has been also recognized that discards into the sea are responsible for a variety of biological, ecological, environmental, economic and social costs (Alverson et al., 1994). Thus, the importance of determining the amount and variability of discards has been underlined (Connolly and Kelly, 1996; Blasdale and Newton, 1998; Allain et al., 2003). The development of a commercial market for discarded species may contribute to reducing wastage. The Individual Quota system established in Iceland induced the fleet to utilize unconventional species, which were not covered by this quota system (Valdimarsson, 1998). The utilization of these species was encouraged by guaranteeing a minimum price, which was supported by a so-called By-Catch Bank. This Bank acted as a matchmaker between the fish owners and marketing firms for selling the fish. The development of this project permitted commercial exploitation of some unconventional species including grenadiers, starry rays and deep-sea rosefish among others. The disposal of fish waste at sea from vessels or other marine vessels is also a main concern. For instance, most demersal fish are processed to some extent at

174

Maximising the value of marine by-products

sea before landing and it is estimated that gutting waste represents 16% of the total fish weight (Archer, 2001). In the United Kingdom the processing waste at sea accounted for about 5% (ca. 42 600 tonnes) of the total catches (Archer, 2001). In Norway most of the by-products from fish processing are used as raw material for different purposes but about 150 000 tonnes (ca. 29% of the total by-products) are still dumped into the sea (RUBIN, 2005). The high volume of these by-products, their nutritional value and potential industrial applications, as well as the environmental problems resulting from sea dumping, have led to the introduction of several regulations to control the disposal of such wastes at sea. This is the case of the Food and Environment Protection Act 1985 in UK to control the disposal of waste at sea, and the regulations in Iceland where it is compulsory for vessels to return the fish livers to shore or in Norway where the total fish catch must be landed. Environmental protection is nowadays a major concern for governments, particularly in developed countries. In the European Union, Regulation 1774/ 2002 was adopted laying down health rules concerning animal by-products not intended for human consumption. This new legislation on the disposal of animal waste by-products has major implications for the seafood industry. Important aspects of this legislation are banning the landfill of all untreated animal byproducts and the inclusion of the shell from shellfish in animal by-products.

8.3

Using the by-catch and underutilized species

The by-catch is usually a combination of many species, particularly that from tropical shrimp fisheries, which could attain 200 different species. In the tropical and subtropical regions, for instance, the main discards from the shrimp fisheries include species from the Carangidae, Mullidae, Synodontidae, Gerreidae, and Nemipteridae families, among others (Alverson et al., 1994). The fish size is also quite variable and may include large and medium size (15±25 cm) marketable species as well as small fish of less than 14 cm in length. The small fish size constitutes as much as 50% of the catch (Allsopp, 1982). Generally, it is estimated that between 24 and 69% of the fish from by-catch have a market, depending on the fishing ground. However, fish that are saleable vary considerably from country to country. The large species do not usually represent a problem because they can be sold in the traditional forms. Thus, the main difficulties arise from the smaller species due to the large amounts of fish caught, the species variability, the range of sizes and the low value. This last factor normally determines the final utilization of the by-catch, which is constituted by a large proportion of underutilized species. However, the designation of underutilized species is quite dependent on the consumers' food habits in a given population. Thus, one fish species could be underutilized in a certain region but the main fish supply in another one. For instance, black scabbard fish (Aphanopus carbo) is considered an underutilized species in Ireland, but it is the dominant fish species consumed in the Portuguese

By-catch, underutilized species and underutilized fish parts 175 island of Madeira. In the Pacific artisanal and subsistence fisheries, the fish discards are very few because almost all the catch is used for consumption. In Africa the consumption of sun-dried tiny species of less than 4 cm long is quite frequent. Another example is red fish (Sebastes spp.), which was a by-catch of cod (Gadus morhua) fisheries in the Northwest Atlantic in the 1950s. However, after the introduction of improved freezing facilities on board, this species is nowadays a very much-appreciated species in the European market. There are also examples of very-low priced species that became very expensive such as monkfish (Lophius spp.). The underutilized species are often discarded and only the last catch of the trip is kept on board where the holding facilities are primarily used for the targeted species. This also means that they are not well preserved, thus reducing the quality and the price as a consequence. These species pose several problems related to recovery, handling and preservation on board and later on their processing. Regarding the fish recovery, several approaches have been tried to minimize the by-catch. The reduction of fishing effort and time, the area closures of fishing grounds; and enforced prohibitions on discharges are some management measures introduced. Another type of measure involves the utilization of more selective gears. A few examples are: (i) the introduction of gear modifications (raised footrope, cutaway trawl); (ii) changes of the cod end characteristics (mesh size, twine thickness, number of mesh in the circumference); (iii) the increase of mesh size or the inclusion of grids, square mesh panels, and separator mesh panels; and (iv) the installation of sound emissions devices or use of electric pulse fields. All these devices permit the fish to escape due to its size, mobility or response to electric pulses. Nowadays, the grids are used in fish or crustacean bottom trawl in several European, American, and Australian fisheries (Fonseca et al., 2005a,b). The fish sorting and grading after catching are critical steps, which may represent a strong workload for the crew if it is done manually. However, a wide variety of equipment has been developed allowing mechanization of some of those operations. Olsen (1992) reported that Danish industrial fish trawlers are equipped with a rotating sorter for separating white fish by-catch from the industrial fish. There are also several types of graders based on the fish thickness available in the market and well adapted to handle pelagic species (Sùrensen and Mjelde, 1992; Olsen, 1992). The recent development of equipment based on computer vision represents a powerful tool to address the problems of fish processing industry resulting from the diversity of species, sizes, shapes and colours as well as processing methods. This equipment is intended for cutting portions and fillets, for shape, length and size grading as well as for quality grading. Computer vision for sorting fish was applied by Zion et al. (1999) to three freshwater species. According to these authors the fish species detection was successfully performed regardless of size and orientation. Storbeck and Daan (2001) also applied computer vision and a neural network to fish species recognition. The vision system measured a number of features of fish using a camera perpendicular to a conveyer belt. The

176

Maximising the value of marine by-products

results of classification by the neural network showed that more than 95% of the fish could be classified correctly. For Gunnlaugsson (1997), this equipment was successfully introduced in fish processing plants increasing their efficiency and productivity. The use of computer vision equipment in portioning machines has presented opportunities for accurate portion preparation of a predefined weight or size, better utilization of the raw material and labour savings. The grading equipment using computer vision can recognize automatically different shapes and forms. Shape graders can be used to grade fresh or frozen products and to separate different sizes of fillets or portions as well as individual shrimp or prawns headon or headless. In the case of quality grading, the vision equipment has been used in the shrimp industry to separate unpeeled or not fully peeled shrimp from properly peeled shrimp. It has also been used to grade salted fish according to colour shades, blood spots, cuts and size. Another type of product is herring and salmon fillets, which are graded according to the colour of flesh and silver mirror on skin side for herring or the colour uniformity of smoked salmon slices. Sensor technology was also developed for quality inspection and grading of seasonal herring roe sacks to be implemented in a prototype-grading machine (Croft et al., 1996). In a recent paper, Brosnan and Sun (2004) reviewed the use of computer vision to a large range of food products inspection. Another important aspect is fish preservation on board, which presents special problems when dealing with small-sized species caught in very large quantities and high temperatures as is the case in the tropical countries. Fish deteriorates more rapidly than other animal products, but the spoilage of small fish tends to be faster than large fish (Burt and Hardy, 1992). Thus, there is a need to efficiently cool the fish as quickly as possible just above the freezing point. Icing fish in boxes is a widespread practice for storing and transport. There are many types and shapes of boxes and the materials of construction include wood, aluminium, plastic (HD-polyethylene), fibreboard, and expanded polystyrene (styrophor). Their utilization depends on tradition and specific needs and requirements. For instance, fibreboard and styrophor boxes are nonreturnable and frequently used for dispatching fish by air. Boxing has several advantages over other processes of fish storage (bulk, shelves). The use of boxes reduces static pressure on fish and also facilitates the unloading of fish. Despite the advantages of fish boxing there are some key points to consider for their full utilization on board as referred to by Olsen (1992): (i) adequate handling rate of filling in to prevent quality loss due to delays in icing; (ii) sufficient icing to chill the fish to 0ëC and to keep this temperature until unloading; (iii) appropriate hold construction to guarantee safe and easy stacking of the boxes; and (iv) good hold insulation to prevent heat transfer. The installation of a small mechanical refrigeration plant is also recommended. The introduction of bulk chilling systems on board has been indicated as an alternative to the traditional method of icing in boxes. The fixed RSW (refrigerated sea water) and CSW (chilled sea water) tanks have been successfully used for different fish species (Olsen, 1992; Hassan, 2002). These systems are very

By-catch, underutilized species and underutilized fish parts 177 fast and effective chilling methods, but their installation on board has to be carefully considered in every fishery and adapted to each vessel. For RSW systems, some recommended practices needed to be followed to render the maximum shelf life of the catch. According to Kraus (1992), the temperature homogeneity between ÿ1.5ëC and 0ëC during storage is very important. The input of cooled seawater must be divided across the total bottom tank area and the filling capacity of the tanks has to be taken into account depending on the fish species (80% for mackerel and 70% for herring). It is also recommended that at least one temperature sensor be installed on each tank at the warmest place, which is in the suction box. In the CSW system the seawater is chilled with ice and is a cheaper investment than the RSW system. The main practical difficulty is the stratification of temperature due to differences in specific gravity between seawater and ice and between water of different salinity and temperature. The most popular method used to overcome the temperature stratification is by blowing compressed air introduced at the bottom of the tank. This is the so-called `champagne' method where a rapid heat transfer between fish and ice is achieved. The CSW systems have been used in different types of fishing vessels including small coastal ones. Graham et al. (1993) reviewed all aspects related to the utilization of ice to chill and preserve fish. They also discussed the benefits and the disadvantages of the use of different chilling technologies. At present, the use of RSW and CSW systems on board is scarce, the main criticism being the need for major space in the warehouse without significant improvement in the fish quality. In recent years a new type of ice, known either as slurry ice or binary ice, has been used as one cooling medium for chilling the catch or products, at sea or on shore. Slurry ice is a mixture of ice microcrystals in brine or seawater, which enables quick cooling and can keep a low temperature for a long time. Its faster chilling rate derives from its higher heat-exchange capacity when compared with the traditional flake-ice and RSW (PinÄeiro et al., 2004). The application of slurry ice reduces the physical damage to fish due to the small size of the ice particles. Slurry ice also has the advantage of being pumpable and permits the addition of preservatives such as ozone or chemicals like inhibitors of melanosis (Huidobro et al., 2002). The main disadvantages are the occurrence of cloudiness in the eyes of gilthead seabream (Sparus aurata) and the development of dull colour in shellfish. The initial relatively high investment costs are also another aspect to take into account. The new chilling method using slurry ice has been applied to many species as reviewed by PinÄeiro et al. (2004) and the incorporation of slurry ice systems into fishing vessels has been expanded in recent years. The use of underutilized fish species for consumption as fillets or portions and in the preparation of a range of fish products has gained importance as a result of the great pressure on the exploitation of traditional fish stocks. Thus, several programmes have been developed envisaging the upgrading of many species. The study of each species generally involves different aspects dealing with the size, shape, type of scales, fat content and protein stability among others. Venugopal and Shahidi (1995, 1998) provided a general view on the

178

Maximising the value of marine by-products

upgrading of underutilized species and discussed the various possibilities for their utilization for human consumption. Freezing is also an important preservation technology on board. It is of major importance in the case of long fishing trips, because the chilling techniques do not allow the fish quality to be kept at high standards for long periods. Blast or plate freezing are widely used on board the fishing boats and its choice depends on different aspects such as the fish size or the type of processing. In a study on the utilization of four underutilized species (sand eel, greater sand eel, sprat and Norway pout) of the North Sea, different processing alternatives were followed (Nielsen et al., 1992). Washed mince of sand eel (Ammodytes tobianus) was prepared and used in kroepoek, an expanded snack very popular in Malaysia, and in the case of greater sand eel (Hyperoplus lanceolatus) hot smoked products were prepared. It was considered that the production of good smoked greater sand eel was technologically possible, but it has to compete with the well-established smoked herring production. Sprats (Sprattus sprattus), traditionally processed into canned products in the Nordic countries, were used in the preparation of canned sprat in two new marinade sauces (lemon/oil and Escabeche), which were compared with the traditional smoked sprat canned in oil. The lemon/oil product was considered more adjusted to the market taste but the panellists preferred the traditional product. Norway pout (Trisopterus esmarkii) was used to prepare minces and surimi. The minces obtained under different conditions were found unacceptable as a substitute for white fish mince or fish fingers due to colour and texture. The gel strength of surimi was comparable with the gel of a good quality product but it could not be graded as first quality because of a very light grey colour and tiny spots of membranes. Larsen (1992) reported the studies made for the upgrading of roundnose grenadier (Coryphaenoids rupestris) and greater argentine (Argentina silus), which are by-catch in the shrimp fisheries. The large scales in the trunk and tail of roundnose grenadier have to be removed before skinning or filleting. The heads represent about 35% of the gutted fish and the filleting yield by machine is 22±23% round fish. Many products can be prepared from this species, including fresh or frozen skinned fillets, skin-on fillets as smoked product, as well as skinned fillets cut into squares for use in pizzas, salads, and soups among others. In the case of argentine the presence of two rows of parietal ribs represents a problem because they are difficult to detect and remove. The yield of machine filleting is on average 30% of the round weight after skinning. Argentine has high gel-forming ability and is also very satisfactory for mince and surimi production. Gormley et al. (1991, 1992, 1993, 1994) studied the effect of freezing and thawing on the water-binding capacity of the argentine proteins and considered it best to make products from fresh or frozen whole fish/fillets rather than from frozen mince. Great argentine also afforded highly acceptable fish cakes and enrobed prawn analogues. Maier et al. (1997) evaluated nine underutilized species for the manufacture of consumer fish products. Breaded nuggets of different species were prepared and the results of the taste panel indicated that

By-catch, underutilized species and underutilized fish parts 179 all were favourable compared with cod, but Greenland halibut (Reinhardtius hyppoglossoides) and greater argentine were the most outstanding. Cardinal fish (Epigonus telescopus), Portuguese shark (Centroscymnus coelolepis) and dogfish (Scyliorhinus stellaris) had the most favourable water-holding quality and gel strength, indicating good potential for use in fish products. In general, a larger number of underutilized species has been introduced in products for human consumption. The increasing recognition of the nutritional value of fish together with the development of new types of seafood products have contributed to their utilization. The globalization of the fish market also contributes to upgrading those species.

8.4 Using underutilized fish parts as food and food ingredients The relevance of the actual concept of by-product as all the raw material, edible or inedible, left over during the preparation of the main product (Gildberg, 2002) has become very important. Many approaches have been tried for the upgrading of fish by-products to produce food, feed or for extraction of valuable biomolecules. However, a great variety of fish parts has traditionally been recovered worldwide for human consumption. A number of them have very wide distribution while others are only characteristic of some regions. On the other hand, the current globalization contributes to a generalized knowledge of this type of products, which are considered delicacies in some places. Thus, this section will focus on different products obtained from fish parts intended for human consumption. In Fig. 8.3 the different steps in fish handling and processing and the resulting by-products intended for human consumption are shown. 8.4.1 Fish heads, cheeks and tongues Fish heads are usually discarded or reduced to fish meal. However, there are some markets where they are commercialized fresh or frozen for direct human consumption and in some Asian countries the head is one of the favourite fish parts. They are usually from large fish such as salmon (Salmo salar), meagre (Argyrosomus regius), white grouper (Epinephelus aeneus), wreckfish (Polyprion americanus), conger (Conger conger), tuna (Thunnus spp.), etc. and are sold whole and sometimes with the collar. They can be prepared in a variety of ways including soups, fried and steamed dishes. Salmon heads, for instance, are sold in several countries where the price per kilogram can be the same as the fish itself, such as in the Taiwan market. The heads from tuna fish and from some big size tropical fish are consumed grilled over an open fire or in an oven and are know as kabutoyaki in Japan (Tùnsberg et al., 1996). Other parts of the fish head can also be found in the market such as the jaw of many local species in Taiwan, where they are used in soups, barbecues and fried.

180

Maximising the value of marine by-products

Fig. 8.3

General scheme for the utilization of fish by-products for human consumption.

The neck meat from salmon and trout is quite popular in the Japanese market where it is sold fresh or frozen in small packs. Some neck meat is also smoked or canned. In Iceland, where fish is an important item in the diet, dried fish heads softened in dairy whey are used to prepare various dishes. In Nigeria salted and fermented cod heads are considered a delicacy. The processing of cod heads intended for human consumption has a long tradition. The gill-covers, the

By-catch, underutilized species and underutilized fish parts 181 tongues and the cheeks are manually recovered and salted onboard. Codfish heads contain relatively little meat, but this fish part is a very much appreciated delicacy due to the taste and particularly its texture. The tongues constitute approximately 1±4% of the weight of the head, cheeks 5±10%, collar 15±20%, and upper head meat 5±15% (Arason, 2002). In order to reduce the manpower required for processing these by-products specific equipment has been developed in Iceland. One machine splits fish heads and tears the gills out. It is claimed that it can process all sizes of head without special adjustments. Another processing machine separates cheeks and tongues from the head. Cod tongues are used to prepare a variety of dishes, for instance in Iceland, Portugal and Spain. The processing of cod tongues involves washing thoroughly in clean seawater and trimming of any loose skin or fragments. After draining, the tongues are mixed with salt following a standard procedure. Salting requires on average ten days and after curing, they are rinsed in light brine and repacked in barrels with brine. The tongues gradually harden after nine months in salt, becoming inedible. 8.4.2 Fish roe Fish roe is the common name of fish eggs, particularly when they are in sacks. It is a very common seafood commodity worldwide and it is collected from many different fish species. Whole fresh or frozen fish roe is available in different markets. In England, for instance, fresh cod roes are sold already boiled and they can be eaten cold or sliced and then fried, grilled or used to prepare other dishes (Bannerman, 1977). Another example is the consumption of roe from Southern Atlantic hakes in Uruguay, where it is used to prepare traditional foods (Mendez et al., 1992). As a rule, the roe from large fish is highly priced but those from smaller fish such as menhaden (Brevoortia tyrannus) could also attain good prices in Carteret County (US). Frozen or salted fish roe is also the raw material for preparation of smoked, canned and several kinds of spreads. As reported by Arason (2002), the increased fish roe prices in Iceland led to its utilization from most of the ground fish. After gutting the fish at sea, roe is collected in insulated plastic tubs and preserved with salt or frozen onboard the freezing trawlers. There are many different products, prepared from fish roe, available in the market but they can be grouped into three types: whole sacks, individual eggs and pate or spreads. Caviar, the most famous roe product in Europe and Japan, is salt-cured and preserved individual eggs. The most valued caviar is made from sturgeon caught in the Caspian Sea. Sturgeon caviar may be produced from more than 20 species of sturgeon and the most renowned are produced from the Russian and Iranian beluga, osetra, and sevruga sturgeons (Bledsoe et al., 2003). There are also caviars from the Kaluga sturgeons and Amur River sturgeons. The supplies of sturgeon caviar have been decreasing as a result of the reduction in the availability of Caspian Sea sturgeon, which led to increasing attention to caviar

182

Maximising the value of marine by-products

products from other fish species. However, the caviar produced from other fish or aquatic species than sturgeon must be identified with the indication of the common name of the species used. In the US, the most common source of black caviar is the farmed white sturgeon (Acipenser transmontanus) because the production from wild sturgeon is normally not permitted due to the near extinction of certain sturgeon species and subspecies. Black caviar with very high quality and reasonably good price is also produced from the roe of fresh water sturgeons (Acipenser sp.), shovel-nose catfish (Hemisorubim platyrhynchos), and paddlefish (Polyondon spathula). Salmon roe is commonly processed into individual eggs (salmon caviar) or as a whole sack. Salmon caviar is known as `red caviar' and is prepared from pink (Oncorhynchus gorbuscha), silver (Oncorhynchus kisutch) and chum salmon (Oncorhynchus keta). For the production of salmon caviar (ikura in Japanese) the eggs must be absolutely fresh, free from blood and of clear colour and with good consistency. After separation from sack membranes the eggs are cured in brine following slightly different processes according to local practices. The brining time varies with species, season, temperature, size, and degree of maturity. The brined eggs are placed on wire-meshed screens and usually drained overnight. After draining they are packed and stored at refrigerated temperatures. The screening, i.e., the separation of eggs from each other and from the sack membrane, is normally a manual and time-consuming process. As an alternative, different enzyme preparations for splitting the linkages between eggs and the connective tissue of fish roe sack have been tried. Collagenase from crab hepatopancreas or proteolytic enzymes extracted from fish viscera were tried in the production of salmon caviar. The yields of recovery achieved were around 80% using the former enzyme and ranged between 84 and 93% for fresh roe and 76 and 87% for frozen roe when the viscera enzymes were used (Bledsoe et al., 2003). The product obtained with proteolytic enzyme from fish viscera had better quality than that obtained with the crab collagenase, which partially hydrolyzed the egg sheath resulting in caviar softer than that obtained by the traditional mechanical screening process. The enzyme treated roe also presented faster salt uptake than fresh fish roe. The traditional production of cured whole salmon roe (sujiko) is made from freshly caught Pacific salmon. The main species used for its production is sockeye salmon (Oncorhynchus nerka), but chum and pink salmon are also used. The intact roe sacks from salmon are separated from the other viscera and sorted for quality. The salmon roe is then immersed in saturated brine containing nitrites, polyphosphates and other additives and seasonings and gentle agitated for about 20 min (Bledsoe et al., 2003). The product is then packed in layers into poly-lined wood or plastic containers. Fine granular salt is applied between layers and the product then cures at ambient temperature for several days after packing. The finished product exhibits salt content ranging normally from 7 to 10% and is sold in frozen or refrigerated state. The consumer may use it without any further processing or preparation. Sujiko is often prepared with a special soy sauce based marinade. Barako is the designation in Japanese of a by-product of

By-catch, underutilized species and underutilized fish parts 183 sujiko production, which is constituted by singled-out eggs from broken or rejected sacks of sujiko. Lumpfish (Cyclopterus lumpus) roe is another source for production of moderately priced caviar. The individual eggs are mechanically separated from the roe sack and salted to 3±5% sodium chloride and immediately packed. In another alternative process the eggs are salted to 10±14% and then delivered for reprocessing, desalting, and repacking in retail packages (Sternin and Dore, 1993). Lumpfish caviar is generally black or red coloured and dyeing is part of or after the curing process. The final product is vacuum packaged and kept refrigerated. The salt concentration in brine of commercial lumpish caviar varies between about 4.2 and 12.9% (Bledsoe et al., 2003). Alaska pollock (Theragra chalcogramma) roe is commonly processed into mentaiko (or mentiko) and tarako. In 2000, the total production of mentaiko and tarako was around 30 470 and 25 970 tonnes, respectively. Mentaiko is whole, a matched pair of sacks and its preparation involves brining and curing. It may be dyed and/or seasoned with salt, sugar, monosodium glutamate, garlic and other spices, sesame, chilli or other flavoured oils, soy sauce or sake. Tarako is literally the name of cod roe in Japanese but now designates smaller salted roes. It is actually produced mostly from pollock roe, which is a by-product of the surimi production. The roe for the preparation of tarako is removed, washed with seawater and soaked in about 15% sodium chloride solution for 40±60 min and drained for about 1 hour (Chiou et al., 1989). The fat content depends on the raw material but could be between 2 and 4%. The ash content is relatively high (6.9%) due to salting. Pacific cod roe is also used to prepare tarako in a similar manner. Botargo or the Mediterranean caviar is a delicatessen product prepared from dried salted roe from mullet or tuna. It is very much appreciated in the Mediterranean countries (Southern Europe and North Africa) as well as in Japan where it is called karassumi. There is also some production of dry-salted mullet roe in the United States, where it is believed that the method of preparation was introduced in colonial times by Englishmen who discovered it in Greece (Jarvis, 1950). The roe for the production of botargo must be fresh, of good colour and the skin of the roe sack not broken. It must be neither over-ripe nor too underdeveloped. The egg sack is taken out of the fish as soon as it is landed. The lobes should be freed from blood, gall bags, bits of intestine, and black skin and then washed thoroughly and allowed to drain for further salting. After size grading, the lobes of roe are rolled in fine salt, kept with salt for 6 to 12 hours and rinsed to remove the excess of salt. The sacks are then placed between two wooden boards and compressed slightly for 2 to 3 days until most of the liquid drains from the roe. A small piece of the muscle is left in the roe in order to avoid its emptying and also to be used for hanging them during drying or smoking. The drying process takes 6 to 10 days and is completed when the roe is reddishbrown in colour and feels hard. The moisture content of the dried product is typically between 28 and 32%. The colour is amber honey and the texture very soft. During storage the colour becomes dark and more dried and hard.

184

Maximising the value of marine by-products

The traditional product presented the original and transparent sack skin, but nowadays it is covered with food grade paraffin to improve the shelf life and to prevent lipid oxidation. This product is stored between 5 and 15ëC and its shelf life is around eight months. The utilization of a vacuum preservation method allowed the increase of shelf life of botargo, thus opening its commercialization to new markets. Tuna roe, mainly from Thunnus thynnus, are also used to produce botargo, which is normally dark brown in colour. The fish roe of small pelagic fish such as sardine (Sardina pilchardus), which is used to produce canned products, is usually not removed from fish and is consumed like the fish flesh. Capelin (Mallotus villosus) is another pelagic species whose eggs are used to produce faux tobiko, the roe from flying fish (Cheilopogon furcatus). The European market for capelin eggs is estimated at 100 tonnes per year but the largest market is Japan. It is a high priced product, normally sold under the term masago, used in sushi cuisine. In the case of herring it can make up 12% of the body weight of the fish. For the European market, herring roe is processed to give separate non-sticky eggs. The fish are machine filleted, the roe sack removed and the small eggs separated from the gut tissue. The eggs are then concentrated and the small pieces of gut tissue discarded. After being immersed in 3±5% brine for up to two hours to reduce stickiness, the eggs are dewatered and packaged. For the Japanese market the whole herring roe is a delicacy and has a much higher price. The highest value cured herring roe, kazunoko in Japanese, is prepared from perfectly matched pairs of skeins. To obtain the highest quality product the herring roe must have no defects, the fish should have reached the exact, desired degree of maturity and the average roe content of 10% by weight. Kazunoko is made from frozen herring; the roe is removed by hand or mechanically and cured by brining process, which involves many steps. This product attains a very high price in the Japanese market and is commonly prepared as sushi or for other dishes. Another high priced product made of herring roe is kazunoko kombu or herring roe on kelp. It is a uniform, dense layer of herring eggs covering both sides of a piece of kelp. The kelp covered with herring eggs can be harvested either in the wild or from kelp suspended in pens where live herring are placed just prior to spawning. The egg-coated kelp is washed, trimmed, cut to market size and packed in brine. The finished product is usually consumed in soups, salads, or as a side dish. Bledsoe et al. (2003) also reported the utilization of fish roe from many other species to prepare caviar-type products. They are consumed alone or mixed with different flavourings, or blended with butter, soft cheese or other spreads. The gonads of some invertebrates are also used to prepare roe-based foods. Among them the roe from sea urchin, sea cucumber, crustaceans, and octopus are important. Sea urchin roe (uni) are collected from several urchin species and may be sold fresh, steamed, baked, sauteÂed, frozen, or canned. When used for sushi, they are brined and treated with alum. Other types of products prepared from sea urchin roe include neri uni, a paste obtained after roe fermentation,

By-catch, underutilized species and underutilized fish parts 185 mizu uni, obtained by a dry cure process, doro uni where the roe is washed with dilute alcohol, drained and mixed with salt. Roe from sea cucumber has a good market in China where it is sold at a price 50 times higher than that of sea cucumber muscle. The roe from crustaceans (shrimp, lobster, and crab) is used to prepare traditional dishes. The salted octopus (Octopus vulgaris) roe is considered a delicacy in Greece, but it is not usually available in the market since it is only produced at an artisanal level. 8.4.3 Fish milt Milt, also known as soft or white roe, is the sperm-containing fluid of the male fish. Milt is sold fresh or frozen, but canned milt, particularly from herring and mackerel, is also commercialised. It can be used cooked or fried and eaten sliced or chopped on canapeÂs or mixed in salads, soups and stews (Rustad, 2003). As reported by Richardsen (1992), the chemical composition of herring milt is 82.5% water, 2% fat, 16.7% protein, and 2% ash for fish with 21% milt. Cod milt has a similar composition (82% water, 1.1% fat, 14.5% protein, and 1.8% ash) (www.fao.org) and is traditionally used in Japanese cuisine. In Japan, it is mostly imported fresh (90%) and can be consumed raw, seasoned with vinegar and few drops of soy sauce or cooked together with meat, cod fillets and vegetables (Tùnsberg et al., 1996). 8.4.4 Fish stomachs Fish stomachs are traditionally consumed in Iceland as well as in Japan, Korea and other Eastern countries (Archer, 2001). In Iceland, the stomach of some species (cod, tusk, Brosme brosme, ling, Molva molva) are removed just after catch and immediately frozen or kept in ice. A popular Icelandic dish is prepared with cod stomach filled with a piece of cod liver. The stomachs and intestines from cod, saithe (Pollachius virens) and haddock (Melanogrammus aeglefinus) caught from the end of January until April are considered to be of good quality for consumption in the northern Norway (RUBIN, 2002). In Japan the fresh stomachs are consumed stewed with vegetables and spices. They are also used after partial hydrolysis to prepare a dish (changi) with a characteristic flavour and texture. High priced fish sauces are produced after full hydrolysis of fish stomachs. The consumption of the Alaska pollock stomach is very popular in Korea where it is called changran. In Taiwan the stomach of some sharks, milkfish (Chanos chanos), and glassfish (Chanda spp.) are used for steaming and sold as a delicacy. Stomach and intestines are also part of some traditional Chinese dishes, usually steamed (Tùnsberg et al., 1996). 8.4.5 Fish maws Dried fish maws means the dried swim bladder from different fish species. The swim bladder is a part usually discarded in European fisheries, although dried

186

Maximising the value of marine by-products

cod maw is available in some niche markets where it is considered a delicacy. However, as reported by Clarke (2004) the foreign trade in fish maws to or through Hong Kong has been very important for many decades. In the Far East dried fish maws are consumed as a food, but it is also believed that they have medicinal properties. Fish maws are produced from a variety of species (Nile perch, Lates niloticus; croaker such as Bahaba taipingensis and Otolithoides brunneus; jew fish, Pseudosciacna sp.; eel, Muraemesox talaboieds, among others). The main characteristics looked for in the fish maws are shape, size, colour (transparency), species and gender. The processing involves splitting open the maw, washing and drying it in the sun. Fish maw is simply boiled with other ingredients to prepare a soup or broth or is cooked with beans. In Hong Kong the smaller fish maws are fried (dim sum) and consumed as a snack food, especially for breakfast. Swim bladder is also a potential source of gelatin (Regenstein, 2004). 8.4.6 Belly flap/trimmings Fish bellies are frequently discarded, but in some markets they are considered a delicacy. The belly of several species (salmon, tuna, milkfish, and blue marlin, Makaira nigricans) is particularly appreciated and consumed in soups or grilled. The reason for high preference of fish bellies is due to the high fat content and softer texture. Canned tuna belly is an example of a softer and more succulent product than the canned tuna flesh. At the beginning of the 20th century, the production of salted salmon bellies was usual in some salteries but it was forbidden under the Alaska fishing regulations because of the wastes resulting from the production method followed (Jarvis, 1950). However, nowadays the modern trimming machines can perform the most difficult trim cuts. In these machines every single fillet is measured individually and trimmed according to the operator's demand. The resulting cut offs may be used to prepare smoked products, which present a good market in Japan. The belly flap of salmon and salmon trout is popular in Taiwan where it is barbecued or fried (Tùnsberg et al., 1996). The belly flaps of milkfish are sold in the Philippines as special parts, which are more expensive than the fillets (Peralta, 2002). In the case of dry salted cod the trimmings and small pieces are recovered and used in the preparation of special dishes. The red muscle of tuna fish species used in the canning industry is usually removed and reduced to fish meal. However, in some markets it is sold as a salted product or used to produce low-priced canned fish. 8.4.7 Fish liver The cod liver oil is a well known product, which was previously recommended as a source of vitamin D and the actual recognition of the beneficial health effects of !3 fatty acids gave a new interest for its consumption. Liver oil is still the main product obtained from fish liver. According to Arason (2002), fish liver oil (medicinal oil and ground fish oil) represented 85.6% of total production of

By-catch, underutilized species and underutilized fish parts 187 different products (4344 tonnes) from the liver of gadoid species in Iceland (2001), whereas the other categories of products from fish liver were: canned liver (5.0%), frozen and fresh liver (5.5%), and other liver products (3.9%). The mean chemical composition of cod liver is 32% moisture, 55.1% fat, 4.6% protein, and 3.6% ash. Fish liver is also consumed such as fish flesh. It is very tender, tasty and highly nutritious but it should be extremely fresh. Hake (Merluccius spp.), cod and monkfish are a few examples of fish species where the liver is very much appreciated to prepare different dishes. Canned monkfish liver (canned ankimo) is consumed in Japan as well as the pateÂ, which is becoming very popular in United States. 8.4.8 Fish frames Fish frames resulting from filleting as well as fish heads and tails present considerable amounts of proteins, calcium and other minerals. As referred by Gildberg et al. (2002) in the production of cod fillets about 60% of the whole fish are by-products and the frames represent about 15% of the fish weight. Furthermore, the flesh attached to the frames is about 85% of the frame weight on a wet basis. For the upgrading of these by-products several approaches have been tried. The flesh can be removed by mechanical separation to afford frame mince, which is much darker than the mince from headed and gutted fish. Frame mince has much higher iron content due to the fish blood present and it is considered to be of the lowest grade. It can be used to make a variety of fish products either for human consumption or pet food manufacture (Arason, 2002). Kim et al. (1991) evaluated the preparation of surimi from catfish frames resulting from filleting. The results obtained indicated that surimi obtained had functional properties, which were feasible for commercial production of different seafood products. This raw material was also used by Hoke et al. (1994) to study the feasibility of producing mince and methods of maintaining the overall quality of the mince during frozen storage. Another alternative for protein recovery of the attached flesh to the frames is on the preparation of protein hydrolysates (Levin et al., 1990; Ferreira and Hultin, 1994; Kim et al., 1997, Jeon et al., 2000; Liaset et al., 2000). The enzymatic hydrolysis of proteins was also used to remove the flesh and recover the fish bones (Kim et al., 2003). Fish bones are recognized as a good source of calcium, especially for people with allergies or intolerance to milk or dairy products, which are the most common source of calcium. Powdered fish calcium produced from skipjack bones (Katsuwonus pelamis), was used in entreÂes as a calcium supplement (Sada, 1984). Fish bones meal prepared from hake (Merluccius sp.) or sole (Solea vulgaris) bone was also used as an ingredient in baby food (Martinez et al., 1998). However, fish bones have traditionally been used in soups, dried (eel) or fried as snack foods. In Japan the canned tailbone of salmon (chum or sockeye salmon) or trout became popular. Canned smoked salmon bone is another version of this product, which is relatively recent in the Japanese market. Deep fried milkfish frames are sold as fish flakes

188

Maximising the value of marine by-products

in Philippine restaurants (Peralta, 2002). Shark bone has a market in Hong Kong where it is consumed as a medicine against cancer. Fish bones have also been used as a source for the extraction of collagen. Nagai and Suzuki (2000) used the bones from skipjack, Japanese sea-bass (Lateolabrax japonicus), ayu (Plecoglosus altivelies), yellow sea bream (Dentex tumifrons), and horse mackerel (Trachurus japonicus) for the extraction of collagen. The yields achieved were between 40.1 and 53.6% and the denaturation temperature ranged between 29.5 and 30.0ëC. According to these authors the bones as well as the skin and fins from the different fish species studied have potential in supplementing the skin of land vertebrates as a source of collagen. The gelatin obtained by Gildberg et al. (2002) from cod bones generally had relatively low molecular weight and as referred by the authors it could be used as a nutraceutical. According to Morimura et al. (2002), the enzymatic hydrolysate obtained from pre-treated fish bone exhibited both high anti-radical activity and high potential for decreasing blood pressure, suggesting that it could be a useful additive in food materials. The inhibitory activity of angiotensin I-converting enzyme of fish peptides prepared from fish viscera or skin was also reported in several works (Matsumura et al., 1993; Byun and Kim, 2001; Bordenave et al., 2002). Antioxidant activity has been also measured in fish by-products and gelatin, which opens good perspectives for their utilization in various seafood products (Shahidi et al., 1995; Amarowicz and Shahidi, 1997; Amarowicz et al., 1999; Kim et al., 2001; Sathivel et al., 2003). The quality of the Alaska pollock bone gelatin obtained by Regenstein et al. (2003) was not as good as the gelatin extracted from the skin but it may have some food applications. 8.4.9 Fish fins and fish skins Fish fins are frequently dumped but are consumed in some countries such as in Japan where the fins from some species are eaten as fried foods, called karaage, and there is a certain market for dried blue shark. In fact, shark fins are much appreciated in the Taiwanese, Chinese and Hong Kong markets. In the latter market they are one of the most expensive seafood delicatessens where the price depends on the grade. Salmon fins are also available in canned form as a snack food (Tùnsberg et al., 1996). Fish skins represent a significant fraction of fish wastes and are frequently discarded but in some countries are converted to value-added products for direct human consumption as snacks. However, several works on the utilization of fish skin for the extraction of collagen or gelatine have been published (Gudmundsson and Hafsteinsson, 1997; Nagai and Suzuki, 2000; Montero and GoÂmezGuilleÂn, 2000; GoÂmez-GuilleÂn and Montero, 2001; FernaÂndez-DõÂaz et al., 2001; GoÂmez-GuilleÂn et al., 2002; Regenstein et al., 2003; Haug et al., 2004). 8.4.10 Fish sauce Fermented fish products are very common in tropical countries where the traditional salting and drying preservation methods are prolonged primarily due

By-catch, underutilized species and underutilized fish parts 189 to the weather conditions. This process leads to the development of special products enhancing the flavour or even masking some taste of food (Saisithi, 1994). This author classified these products into the three groups: (i) fish is fermented by enzymes from fish and bacteria present in the fish/salt mixture; (ii) fish and carbohydrate are fermented mostly by bacterial enzymes present in the fish/salt carbohydrate mixture, and (iii) fish is fermented by fish enzymes and the carbohydrate by yeasts and moulds added in starter cultures. Both small whole fish or shrimp and dressed or whole medium or large fish are used to produce fish sauce depending on the type of product. Fish sauce is the most well known fermented fish product and not only in Southeast Asia region but also in Europe and the United States. In this latter country the value of imported fish sauce was $16.6 million in 2000 (Tungkawachara and Park, 2003). It is a clear brown liquid with a salty taste and a mild fishy flavour, which is consumed on a daily basis as a condiment by a large number of people (Raksakulthai et al., 1986). Lopetcharat et al. (2001) give an overview of fish sauce manufacturing, factors affecting quality, composition and estimation of the fish sauce quality. The long production period required (6 to 12 months) has led to some efforts to accelerate the fermentation period by adding external enzymes, heating or adjusting the conditions to increase the enzyme activities (Gildberg, 2002). The utilization of whole Pacific whiting or a mixture of surimi by-products and whole fish for the production of fish sauce is described by Tungkawachara and Park (2003). The sauce obtained had a positive consumer acceptance, good quality and low cost and could potentially replace fish sauce from other sources. Gawborisut et al. (2003) described the preparation of fish sauce from catfish nuggets with bromelain added. Based on chemical results these authors claimed that a catfish nugget sauce could be made in 14 days with 11% salt and a maximum of 0.15% bromelain.

8.5

Future trends

An increasing number of new fish species has been introduced in the market in recent years. It is expected that this tendency will continue as a result of the market demand and the pressure on traditional fish stocks. There are also good prospects of increasing the value of by-products from fisheries and aquaculture as pointed out by Gildberg (2002) as was registered in recent years. On the other hand, the legal requirements for fish waste disposal represent a challenge to find alternatives for better utilization of seafood by-products. Another important aspect is that fish and fish products will continue to be the most internationally traded of all foodstuffs on the global market in the near future (Valdimarsson and James, 2001). The general trends mentioned above indicate that the introduction of management measures to reduce discarding will be reinforced. Equally important is the improvement of gear selectivity, which may represent a significant contribution to reducing the by-catch. The improvement of sorting, handling and preservation

190

Maximising the value of marine by-products

methods on board to improve the quality of fish and by-products when landing will be in line with the advances recorded in last decade. A better knowledge of the chemical composition, seasonal changes and technological characteristics of unconventional or underutilized species and by-products is also needed. The recent development of new equipment for fish processing and the increasing demand for ready-to-eat and heat-and-eat products seem set to continue in the future. The expanding international fish market also requires a reliable traceability system in order that the entire story of fish along the value chain is recorded and can be available. The development of the telemarket also opens new perspectives for a global market of fish products and for increasing the revenue of fishermen.

8.6

Sources of further information and advice

In recent years a number of books on fish processing, nutritional value of fish, safety, and quality assurance have been published. In parallel, interest in the upgrading of fish by-products has gained special relevance, particularly in the area of biotechnology. The following recent books are also recommended: 2000. R. E. Martin, E. P. Carter, G. J. Flick Jr, G.J. and L. M. Davis. Marine and Freshwater Products Handbook. Technomic Publishing Co., Lancaster, USA, 963 pp. 2000. N. F. Haard and B. K. Simpson (eds), Seafood Enzymes Utilization and Influence on Postharvest Seafood Quality. Marcel Dekker Ltd, New York, USA, 681 pp. 2002. M. Fingerman and R. Nagabhushanam (eds), Recent Advances in Marine Biotechnology. Vol. 7: Seafood Safety and Human Health. Science Publishers Inc, Enfield, USA, 315 pp. 2002. C. Alasalvar and T. Taylor (eds). Seafoods ± Quality, Technology and Nutraceutical Applications. Springer-Verlag, Berlin, Germany, 252 pp. 2002. H. A. Bremner (ed.), Safety and Quality Issues in Fish Processing. Woodhead Publishing Limited, Cambridge, England, 520 pp. 2003. P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference Proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, Fairbanks, 566 pp. 2004. M. Sakaguchi (ed.), More Efficient Utilization of Fish and Fisheries Products. Elsevier Science Publishing Company, London, England, 464 pp. 2005. Y. Le Gal and R. Ulber (eds) Marine Biotechnology I. Advances in Biochemical Engineering/Biotechnology. Vol. 96. Springer-Verlag, Berlin, Germany, 288 pp. However, the Internet has become a very rich and easy source of information about this subject and the following websites are suggested:

By-catch, underutilized species and underutilized fish parts 191 www.fishbase.com, provides a vast amount of information on fish species worldwide. www.fao.org, is a solid base on fish and aquaculture statistics and diversified information on fisheries in general. www.nfi.org, this is a website with general information related to different aspects on fish, aquaculture and fish consumption. www.onefish.org, this website is a wide source of information of the fishing sector, giving particular emphasis on developing countries.

8.7

Acknowledgement

The author would like to thank Dr Maria Leonor Nunes and Eng. Carlos Cardoso for their help with the chapter and the fruitful discussions.

8.8

References

and KERGOAT B, 2003. Preliminary estimates of French deepwater fishery discards in the Northeast Atlantic Ocean. Fish Res, 60: 185±192. ALLSOPP W H L, 1982. Use of Fish By-Catch from Shrimp Trawling: Future Development. In Fish by-catch ± bonus from the sea: report of a technical consultation on shrimp by-catch utilization held in Georgetown, Guyana, 27±30 October 1981, pp. 29±36. Ottawa, Ont., IDRC. ALVERSON D L, FREEBERG M H, MURAWSKI S A and POPE J G, 1994. A global assessment of fisheries bycatch and discards. FAO Fish Technical Paper, 339, 233 p. AMAROWICZ R and SHAHIDI F, 1997. Antioxidant activity of peptide fractions of capelin protein hydrolysates. Food Chem, 58(4): 355±359. AMAROWICZ R, KARAMAC M and SHAHIDI F, 1999. Synergistic activity of capelin protein hydrolysates with synthetic antioxidants in a model system. J Food Lipids, 6: 271± 275. ARASON S, 2002. Utilization of Fish Byproducts in Iceland. In P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference Proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, Fairbanks, pp. 43±62. ARCHER M, 2001. Fish Waste Production in the United Kingdom ± The Quantities Produced and Opportunities for Better Utilisation. Seafish Report No. SR537. Sea Fish Industry Authority, Seafish Technology, 57 p. BANNERMAN A MCK, 1977. Processing cod roes. Torry Advisory Note No. 18, 8 p. BLASDALE T and NEWTON A W, 1998. Estimates of discards from two deepwater fleets in the Rockall trough. ICES CM, O:11, 18 pp. BLEDSOE G E, BLEDSOE C D and RASCO B, 2003. Caviars and fish roe products. Crit Rev Food Sci and Nutr, 43(3): 317±356. BORDENAVE S, FRUITIER I, BALLANDIER I, SANNIER F, GILDBERG A, BATISTA I and PIOT J M, 2002. HPLC preparation of fish waste hydrolysate fractions. Effect on guinea pig ileum and ACE activity. Prep Biochem and Biotech, 32(1): 65±77. BROSNAN T and SUN D-W, 2004. Improving quality inspection of food products by computer vision ± a review. J Food Engng, 61: 3±16. ALLAIN V, BISEAU A

192

Maximising the value of marine by-products

and HARDY R, 1992. Composition and deterioration of pelagic fish. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp 115±141. Fishing News Books, Oxford. BYUN H G and KIM S K, 2001. Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska Pollack (Theragra chalcogramma) skin. Process Biochem, 36: 1155±1162. CHIOU T-Z, MATSUI T and KONOSU S, 1989. Comparison of extractive components between raw and salted Alaska pollack roe (`Tarako'). Nippon Suisan Gakkaishi, 55(3): 515±519. CLARKE S, 2004. Understanding pressures on fisheries resources through trade statistics: a pilot study of four products in the Chinese dried seafood market. Fish and Fisheries, 5: 53±74. CONNOLLY P L and KELLY C J, 1996. Catch and discards from experimental trawl and longline fishing in the deep water of Rockall Trough. J Fish Biol, 49 (Supplement A): 132±144. CROFT E A, DE SILVA C W and KURNIANTO S, 1996. Sensor technology integration in an intelligent machine for herring roe grading. IIEE/ASME Transactions on Mechatronics, 1(3): 204±215 FAO, 2002. The State of World Fisheries and Aquaculture (SOFIA), 150 p. Â NDEZ-DIÂAZ M D, MONTERO P and GO Â MEZ-GUILLEÂN M C, 2001. Gel properties of FERNA collagens from skins of cod and hake and their modification by the coenhancers magnesium sulphate, glycerol and transglutaminase. Food Chem, 74: 161±167. FERREIRA N G and HULTIN H O, 1994. Liquefying cod frames under acidic conditions with fungal enzyme. J Food Process Preserv, 18: 87±101. FONSECA P, CAMPOS A, MENDES B and LARSEN R B, 2005a. Potential use of a Nordmùre grid for by-catch reduction in a Portuguese bottom-trawl multispecies fishery. Fish Res, 73: 49±66. FONSECA P, CAMPOS A, LARSEN R B, BORGES T and ERZINI K, 2005b. Using a modified Nordmùre grid for by-catch reduction in the Portuguese crustacean trawl fishery. Fish Res, 71: 223±239. GAWBORISUT S, SILVA J L and CHAMUL R S, 2003. Fish Sauce from Catfish (Ictalurus punctatus) Nuggets as Affected by Salt Content and Enzyme Addition. In P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference Proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, Fairbanks, pp. 333±342. GILDBERG A, 2002. Enhancing returns from greater utilization. In Safety and Quality Issues in Fish Processing (Ed. by H. A. Bremner), pp. 425±449. Woodhead Publishing Limited, Cambridge. GILDBERG A, ARNESEN J A and CARLEHOG M, 2002. Utilization of cod backbone by biochemical fractionation. Process Biochem, 38: 475±480. Â MEZ-GUILLEÂN M C AND MONTERO P, 2001. Extracting conditions for megrim (LepidoGO rhombus boscii) skin with several organic acids. J Food Sci, 66(2): 213±216. Â MEZ-GUILLEÂN M C, TURNAY J, FERNA Â NDEZ-DIÂAZ M D, ULMO N, LIZARBE M A and MONTERO P, GO 2002. Structural and physical properties of gelatine extracted from different marine species: a comparative study. Food Hydrocolloids, 16: 25±34. GORMLEY T R, WARD P and SOMERS J, 1991. Silver Smelt: A valued non-quota fish? Farm and Food, 1: 8±10. GORMLEY T R, WARD P and SOMERS J, 1992. Preparation and evaluation of gels from Silver Smelt (Argentinus silus). Irish J Agric and Food Res, 31: 212. BURT J R

By-catch, underutilized species and underutilized fish parts 193 and SOMERS J, 1993. A note on the effect of long-term frozen storage on some quality parameters of Silver Smelt (Argentinus silus). Irish J Agric and Food Res, 32: 201±204. GORMLEY T R, CONNOLLY P L and WARD P, 1994. Evaluation of deep-water fish species. Farm and Food, 4(1): 8±11. GRAHAM J, JOHNSTON W A and NICHOLSON F J, 1993. El hielo en las pesquerõÂas. FAO Documento TeÂcnico de Pesca No. 331. Roma, FAO, 95 p. GUDMUNDSSON M and HAFSTEINSSON H, 1997. Gelatin from cod skins as affected by chemical treatments. J Food Sci, 62(1): 37±39, 47. GUNNLAUGSSON G A, 1997. Vision technology: intelligent fish processing systems. In Seafood from Producer to Consumer, Integrated Approach to Quality. (Ed. by J. B. Luten, T. Bùrresen and J. OehlenschlaÈger), pp. 351±359. Elsevier, Amsterdam. HASSAN A R, 2002. The effects of different cooling techniques on quality parameters of herring in relation to Malaysian fisheries and design of refrigeration system suitable for Malaysian vessels. UNU ± Fisheries Training Programme, 40 p. HAUG I J, DRAGET K I and SMIDSRéD O, 2004. Physical and rheological properties of fish gelatin compared to mammalian gelatin. Food Hydrocolloids, 18: 203±213. HOKE M E, JAHNCKE M L, SILVA J L and HEARNSBERGER J O, 1994. Stability of frozen mince from channel catfish frames. Proceedings of the 19th Annual Tropical and Subtropical Fisheries Technological Conference of the Americas, 174±185. Â PEZ-CABALLERO M E and MENDES R, 2002. Onboard processing of deepHUIDOBRO A, LO water shrimp (Parapenaeus longirostris) with liquid ice: effect on quality. Euro Food Res Tech, 214: 469±475. JARVIS N D, 1950. Curing of fishery products. Research Report 18, 271 p. JEON Y J, BYUN H G and KIM S K, 2000. Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes. Process Biochem, 35: 471± 478. KELLEHER K, 2005. Discards in the world's marine fisheries. An update. FAO Fisheries Technical Paper. No. 470, Rome, FAO, 131 p. KIM J M, LIU S, JAHNCKE M, VEAL C D, HEARNSBERGER J O and EUN J B, 1991. Evaluation of catfish surimi prepared from frames after filleting. Proceedings of the 16th Annual Tropical and Subtropical Fisheries Technological Conference of the Americas, 197±202. KIM S K, JEON Y J, BYUN H G, KIM Y T and LEE C K, 1997. Enzymatic recovery of cod frame proteins with crude proteinase from tuna pyloric caeca. Fisher Sci, 63: 421±427. KIM S K, KIM Y T, BYUN H G, NAM K S, JOO D S and SHAHIDI F, 2001. Isolation and characterization of antioxidative peptides from gelatin hydrolysates of Alaska Pollack skin. J Agric Food Chem, 49: 1984±1989. KIM S K, PARK P J, BYUN H G, JE J Y, MOON S H and KIM S H, 2003. Recovery of fish bone from hoki (Johnius belengeri) frame using a proteolytic enzyme isolated from mackerel intestine. J. Food Biochem, 27: 255±266. KRAUS L, 1992. RSW treatment of herring and mackerel for human consumption. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp. 73±81. Fishing News Books, Oxford. LARSEN E P, 1992. Development of a new fishery in Denmark. Catching, handling and utilisation of roundnose grenadier and greater argentine. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp. 278±284. Fishing News Books, Oxford. LEVIN R E, FAGERSON I S, KARP D, PARK Y W, KIM J and GOLDHOR S H, 1990. Optimization of GORMLEY T R, WARD P

194

Maximising the value of marine by-products

papain hydrolysis of cod frames. In Advances in Fisheries Technology and Biotechnology for Increased Profitability (Ed. by Voigt M N, Botta J R), pp. 2516± 2521. Lancaster, PA, Technomic Publ. Co. Inc. LIASET B, LIED E and ESPE M, 2000. Enzymatic hydrolysis of by-products from the fishfilleting industry chemical characterisation and nutritional evaluation. J Sci Food Agric, 80: 581±589. LOPETCHARAT K, CHOI Y J, PARK J W and DAESCHEL M, 2001. Fish sauce products and manufacturing: a review. Food Rev Internat, 17(1): 65±88. MAIER K, GORMLEY T R, CONNOLLY P L and AUTY M, 1997. Assessment of underutilised fish species. Farm and Fish, 7(2): 30±34. MARTINEZ I, SANTAELLA M, ROS G and PERIAGO M J, 1998. Content and in vitro availability of Fe, Zn, Mg, Ca, and P in homogenized fish-based weaning foods after bone addition. Food Chem, 63: 299±305. MATSUMURA N, FUJII M, YASUHIKO T, SUGITA K and SHIMIZU T, 1993. Angiotensin Iconverting enzyme inhibitory peptides derived from bonito bowels autolysate. Biosci Biotech Biochem, 57(5): 695±697. MENDEZ E, FERNANDEZ M, PAZO G and GROMPONE M A, 1992. Hake roe lipids: composition and changes following cooking. Food Chem, 45: 179±181. Â MEZ-GUILLEÂN M C, 2000. Extraction conditions for megrim MONTERO P and GO (Lepidorhombus boscii) skin collagen affect functional properties of the resulting gelatine. J Food Sci, 65: 434±438. MORIMURA S, NAGATA H, UEMURA Y, FAHMI A, SHUGEMATSU T and KIDA K, 2002. Development of an effective process for utilization of collagen from livestock and fish waste. Process Biochem, 37: 1403±1412. NAGAI T and SUZUKI N, 2000. Isolation of collagen from fish waste material ± skin, bone and fins. Food Chem, 68: 277±281. NIELSEN J, RéNSHOLDT B, JENSEN N C and ALSTED N, 1992. Utilisation of sand eel (Ammodytes tobianus), greater sand eel (Hyperoplus lanceolatus), sprat (Sprattus sprattus) and Norway pout (Trisopterus esmarkii) for human consumption. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp. 285±290. Fishing News Books, Oxford. OLSEN K B, 1992. Shipboard handling of pelagic fish with special emphasis on fast handling, rapid chilling and working environment. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp. 55±69. Fishing News Books, Oxford. PERALTA J P, 2002. Process Accounting (PA) Applications to Milkfish Processing. In P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference Proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, Fairbanks, pp. 393±401. Ä EIRO C, BARROS-VELAÂSQUEZ J and AUBOURG S P, 2004. Effects of newer slurry ice PIN systems on the quality of aquatic food products: a comparative review versus flakeice chilling methods. Trends Food Sci & Technol, 15: 575±582. RAKSAKULTHAI N, LEE Y Z and HAARD N F, 1986. Effect of enzyme supplements on the production of fish sauce from male capelin (Mallotus villosus). Can. Inst. Food Sci Technol, 19: 111±114. REGENSTEIN J M, 2004. Total utilization of fish. Food Technol, 58(3): 28±30. REGENSTEIN J, GOLDHOR S and GRAVES D, 2003. Increasing the value of Alaska Pollock byproducts. In P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference Proceedings. Alaska Sea Grant College Program, University of Alaska

By-catch, underutilized species and underutilized fish parts 195 Fairbanks, Fairbanks, pp. 459±482. 1992. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp. 299±306. Fishing News Books, Oxford. RUBIN, 2002. Rapport nr 4203/100: Karakterisering av marine biprodukter til konsum. Steds- og sesongmessinge variasjoner. RUBIN, 2005. Bioprodukter fra fiskerinñringen: fra utkast til inntekt. Stiftelsen RUBIN. (www.rubin.no). RUSTAD T, 2003. Utilisation of marine by-products. Elec J Env, Agric and Food Chem, 2(4). SADA M, 1984. Fish calcium. Infofish Mktg Digest, 3: 29±30. SAISITHI P, 1994. Traditional fermented fish: Fish sauce production. In: Fisheries Processing: Biotechnological Applications (Ed. by A.M. Martin), pp. 111±131. Chapman and Hall. London. SANDBAKK M, 2002. Handling of by-products from cod-fish a state of the art report from selected countries. Sintef report. 2002. STF80 A0405038. SATHIVEL S, BECHTEL P J, BABBITT J, SMILEY S, CRAPO C, REPPOND K D and PRINYAWIWATKUL W, 2003. Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates. J Food Sci, 68(7): 2196±2200. SHAHIDI F, HAN X-Q and SYNOWIECKI J, 1995. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem, 53(4): 285±293. SéRENSEN N K and MJELDE A, 1992. Preservation of pelagic fish quality for further processing on board and ashore. In Pelagic Fish. The Resource and its Exploitation (Ed. by J. R. Burt, R. Hardy and K. J. Whittle), pp. 38±54. Fishing News Books, Oxford. STERNIN V and DORE I, 1993. Caviar: The Resource Book. Cultura Enterprises, Stanwood, WA. STORBECK F and DAAN B, 2001. Fish species recognition using computer vision and a neural network. Fish Res, 51: 11±15. TéNSBERG T, WONG S, HONG L J and TANGEN G, 1996. Preliminary Study on the Market for Fish By-Products for Consumption in Asia ± Taiwan, Japan, China and Hong Kong. A Research Project for Stiftelsen Rubin. Rapport nr. 314/56. Norwegian Trade Council, 43 p. TUNGKAWACHARA S and PARK J W, 2003. Development of Pacific Whiting Fish Sauce: Market Potential and Manufacturing in the United States. In P. J. Bechtel (ed.), Advances in Seafood Byproducts: 2002 Conference Proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, Fairbanks, pp. 321±331. VALDIMARSSON G, 1998. Developments of fish food technology ± implications for capture fisheries. J Northw Atl Fish Sci, 23: 233±249. VALDIMARSSON G and JAMES D, 2001. World fisheries ± utilisation of catches. Ocean & Coast Mgmt, 44: 619±633. VENUGOPAL V and SHAHIDI F, 1995. Value-added products from underutilized fish species. Crit Rev Food Sci and Nutr, 35(5): 431±453. VENUGOPAL V and SHAHIDI F, 1998. Traditional methods to process underutilized fish species for human consumption. Food Rev. Int., 14(1): 35±97. ZION B, SHKLYAR A and KARPLUS I, 1999. Sorting fish by computer vision. Comps and Elecs in Agric, 23: 175±187. RICHARDSEN R,

9 Mince from seafood processing byproduct and surimi as food ingredients J.-S. Kim, Gyeongsang National University, South Korea and J. W. Park, Oregon State University, USA

9.1

Introduction

In the past, fresh fish was purchased at retail market and prepared at home. Fresh fish, however, undergoes rapid quality changes and when compared to other muscle foods may need pre-treatment.1 In recent years, consumers have also made major changes in their food purchasing habits. As social patterns changed, consumers began to purchase processed seafood to avoid dealing with fish odor and bones.2,3 However, the more the fish is processed for consumer convenience, the higher the price. The goal should then be to provide processed seafood at a reasonable price. Washed fish mince/surimi is an inexpensive intermediate material for manufacturing various seafood products.4±6 Unwashed fish mince also offers nutritional advantages (containing water-soluble vitamins, minerals, and lipids), economic benefits (lower processing costs and higher yield of protein) as well as functional advantages (exhibiting meat-like texture) compared to the other intermediate materials.7,8 Higher yields in fish mince are especially important now as many conventional fishery resources in the world continue to decline and there is a shift toward higher utilization of harvest. Fish mince can also be successfully used directly in various food systems and in a physically or chemically altered form to produce an array of nutritional and functional products.9 Most intermediate materials used for manufacturing various seafood products are frozen to keep the quality good for a relatively long period. However, frozen stability of fish mince is poor, particularly for cold water species like Alaska pollock, due to the higher content of active enzyme systems (i.e., trimethylamine

Mince from seafood processing by-product and surimi as food ingredients

197

oxidase),10,11 and their substrates,12 heme-containing compounds (imparting undesired colour and catalyzing lipid oxidation),13±16 lipids containing n-3 polyunsaturated fatty acids,17 and trimethylamine.18,19 Consequently, the surimi processing method was developed20,21 in Japan to extend the shelf life. For longer frozen shelf life of surimi, minced flesh is repeatedly washed using chilled water (5±10ëC) which removes unnecessary components that promote protein denaturation during frozen storage.22,23 Surimi is commonly mixed with cryoprotectants (4% sugar, 4±5% sorbitol, and 0.2±0.3% polyphosphates). Surimi has excellent functional properties, such as gelling, emulsifying, and water-binding properties.24,25 The high concentration of myofibrillar protein enables the product to gel upon heating to produce a chewy, elastic texture.26,27 Under constant low temperature storage (below ÿ20ëC), frozen surimi can usually be stored up to 1±2 years without significant changes in functional properties.23,28±30 Commercial surimi blocks are processed with the label `good if used within 2 years'. Due to its unique characteristics, surimi is used as an intermediate raw material for processing kamaboko31 and surimi seafood products with various types and flavours of crabmeat,32,33 lobster meat,34,35 and shrimp meat. In addition, its application has been extended beyond shellfish analogs. These include fish nuggets,36 frankfurters,36 and fish patties.37,38 The ratio of supply to demand of fish in the world has decreased over recent decades due to a rise in population, maximization of harvest limits, and a positive change in consumer attitudes towards the consumption of seafood.39 Today's fishing industry, therefore, faces increasing demands for better utilization of all the available raw materials. Unfortunately, seafood processors producing fish fillets and surimi utilize only 15±30% of the harvest23,40,41 and the remainder is discarded as processing by-product or waste.42 Improved utilization of fish fillet or upgrade of surimi waste as food not only resolves many of the environmental concerns that the fish processors face, it also serves as a means of producing value-added products,43±46 and more profit. This chapter will discuss manufacturing methods and machinery for fish mince/surimi, characteristics of mince/surimi processing by-products, functional and nutritional properties of mince/surimi, and its utilization.

9.2

Manufacturing of fish mince/surimi

Fish mince can be defined as deboned and unwashed fish flesh from fillets or frames and is produced at the initial step of surimi manufacturing. When compared to surimi, fish mince can be obtained at a significantly higher yield with much less capital investment. Fish mince as a food ingredient also has a functional advantage exhibiting a more meat-like texture than mechanically deboned mammalian meat. However, it has inferior frozen stability due to the higher content of active enzymes and their substrates,12 metals, lipids, and trimethylamine. As a user-friendly ingredient, fish mince should be available in a form that can be stored in a stable state for a reasonable period of time prior to

198

Maximising the value of marine by-products

Table 9.1

Definition and characteristics of fish mince/surimi

Items

Mince

Surimi

Definition

· Deboned fish flesh separated from fillets or fish frames that have not been washed

· Deboned fish flesh that has been washed with cold water and mixed with cryoprotectants

Advantage

· · · · ·

· Good frozen stability · High functional property

Disadvantage

· Poor frozen stability

High yield High nutritional property High functional property Simple processing Very low water consumption

· Low yield · Complicated processing · Very high water consumption

use. In that sense, surimi was developed to improve frozen stability of fish mince by washing and adding cryoprotectants. Surimi can, therefore, be considered stabilized fish mince. Fish mince and surimi have their own advantages and disadvantages based on the nature of processing and the subsequent functional characteristics (Table 9.1). 9.2.1 Preparation of mince Interest in white fish mince has increased in recent years and its production volume has been similar to that of surimi. Fish mince is mechanically separated fish flesh that has not been washed and, therefore, processed with a significantly higher yield (Fig. 9.1). For the preparation of fish mince, H&G (headed and gutted) fish is typically subjected to mechanical deboning, while some are processed using fish fillets. In the mechanical deboner, the fish flesh is forced by means of a rubber conveyor belt through a perforated drum. The skins and bones from the fillets remain on the outside of the drum, while the fish mince is collected from the inside. As shown in Fig. 9.1, fish mince can be processed further into surimi using extensive washing, dewatering, refining, and screw pressing consecutively. 9.2.2 Preparation of conventional surimi For preparation of conventional surimi, fish mince is repeatedly washed with chilled water (5±10ëC) until it becomes odorless and colorless. Washing cycles, water volume, and water temperature will vary depending on fish species and the condition of the fish upon processing. The washing cycles and total water to meat ratios (v/w) commonly used are 2 to 4 times and 12:1 to 24:1 for on-shore processors,20,47,48 respectively. However, with the recent efforts made by US surimi processors, this ratio has decreased significantly to two washing cycles

Mince from seafood processing by-product and surimi as food ingredients

Fig. 9.1

199

Processing flow of fish mince/surimi and waste.45

and 2:1 to 3:1 for at-sea vessel processing and 5:1±10:1 ratio for on-shore processing, respectively.23 Washing and dewatering removes lipids and water-soluble sarcoplasmic proteins such as blood, enzymes, and heme compounds which cause lipid oxidation. As a result, this process concentrates myofibrillar proteins. Washed fish mince is then pumped to a refiner to remove connective tissues and small pin bones. As a final dewatering step, washed meat is subjected to a screw press before mixing with cryoprotectants. Cryoprotectants commonly used in a commercial application for cold water species are 4% sugar, 4±5% sorbitol, and 0.2±0.3% polyphosphate with approximate moisture contents at 74±76%.23 However, for surimi from warm water species such as threadfin bream manufactured in SE Asia and India, only 6% sugar and 0.2±0.3% polyphosphates are used without sorbitol. Even though fish proteins from warm water species have better frozen stability, the addition of equal or similar amounts of cryoprotectants is desired to maintain the consistency in sweetness and longer shelf life. Finally surimi is stuffed into 10 kg plastic bags before subjecting to a plate freezer. After freezing blocks with their core temperature at ÿ20ëC, two blocks are packed in a carton box for frozen storage. 9.2.3 Preparation of surimi by new technology In conventional surimi processing, washing and dewatering are repeatedly required to concentrate myofibrillar proteins. Consequently, relatively lower protein recovery and large quantities of wastewater are environmental and economic challenges. Fish protein isolate processing by new technology, such as acid- or alkali-aided method, demonstrates a high potential to provide good protein recovery with acceptable functional properties. Unlike the conventional

200

Maximising the value of marine by-products

Fig. 9.2 Solubility of Pacific whiting mince as affected by pH (modified from Ref. 50).

method, the new method utilizes protein charges and isolates the protein by shifting pH prior to centrifugation. As a result, water-soluble proteins are retained in the final products. The solubility of fish mince at various pH values is very important for the recovery of fish proteins by this new technology.49 The effect of pH on solubility of Pacific whiting mince is shown in Fig. 9.2. The solubility of Pacific whiting proteins was very low at pH 4 to 5 and increased as the pH is shifted to acid or alkali. A sharp increase in solubility occurred at a pH from 9.5 to 10.5 in the alkaline side and from 3.0 to 1.5 on the acidic side. These results show that shifting pH away from the isoelectric point of fish muscle proteins, around pH 5.0, results in increased solubility. This indicates that for the surimi processing, the fish muscle protein has to be solubilized at about pH 2.0 for the acid-aided method and about pH 10.5±11.0 for the alkali-aided method, and then precipitated at pH 5.0 for high recovery of protein.50±53

9.3

Machinery for preparation of fish mince/surimi

9.3.1 Fish header/gutter The header/gutter is a machine for removing the head, viscera, and a major part of the backbone from the raw material before filleting. Most of the microorganisms and enzymes found in the viscera and gills are removed at this processing stage. This step also influences both the quality and yield of fish mince. If the position of the head cut is too far forward, the gills and heart remain and the product quality decreases. If the position of the cut is too far to the rear, the yield decreases.

Mince from seafood processing by-product and surimi as food ingredients

201

9.3.2 V cut The V cut is used for preparing a boneless fillet by removing the pin bones, which generally accounts for 5±7% loss based on the original weight. The fillet prepared by using a V cut is a very high-quality fillet material. Water knife or laser cutting methods completely remove the bone portion. 9.3.3 Belt-drum type meat separator A belt-drum type separator (Fig. 9.3) is the most common meat separation technique for the mincing/deboning operation.23 For preparation of fish mince/ surimi, fish are fed into a hopper and transferred by a rubber conveyor belt to a drum perforated with openings.54 Most mechanical deboners have perforated holes of 3 or 5 mm depending on the size of the fish. However, 5 mm is probably the most common size because it yields a more textured final product as a fish mince compared to a smaller hole.23,55 Conversely, a larger hole may allow an unacceptable level of bone. The size and texture of fish are also factors in selecting the right meat separator for optimal recovery and quality. Fish of smaller size or firmer texture would benefit from a smaller opening diameter. In meat separation processing of smaller fish, the use of a large opening would generate more bone fragments and/or broken skin in the resultant mince.23 The higher the belt pressure, the higher the yield of minced fish forced through the holes. However, elevated belt pressure can lead to lower frozen storage stability.56 The effects of pressure, perforation size, and perforation area on bone content, protein functionality, discoloration, and lipid stability can result in a compromise for optimal deboner/mincer conditions.57

Fig. 9.3

Belt-drum type meat separator.

202

Maximising the value of marine by-products

9.3.4 Rotary screen dehydrator Fish mince mixed with wash water is strained through large rotary screens. The rotary screen dehydrator is used for intermediate dewatering. The screens are constructed with stainless steel mesh or punched plate and selected to obtain fine meat particles. The holes in the screens are commonly about 0.5 mm in diameter. Decreasing the opening diameters of screens from the front end to the rear end enhances dewatering action. However, fine particles lost through screens account for about 8% of the starting mince meat weight. For these reasons, in recent years, most surimi plants have employed a decanter centrifuge to recover protein particles in the wash water. The moisture content of the material passed through a rotary screen is commonly reduced from 99.0% to 93.3%. 9.3.5 Refiner Before final dewatering under a screw press, a refiner is used to refine the mince.23 The washed fish mince passes through a refiner to remove refiner discharge, which consists of connective tissue, skin, bone and scale.58 The refiner is commonly a rotating drum (Fig. 9.4). Running the refiner at a slower speed with a smaller screen size will result in cleaner surimi with less recovery. On the other hand, running the refiner at a faster speed with a larger screen size will enhance recovery but with a risk of higher impurities. Screen sizes of 1.5±1.7 mm are commonly used in commercial applications.23 When washed mince is fed into the machine, soft flesh is forced through the perforations under compressive force generated by the rotor. The flesh emerges from the front part to the rear part. The material passed through a refiner contains about 90% moisture. 9.3.6 Screw press Screw press removes a significant amount of water at the final dewatering stage before incorporating cryoprotectants and freezing in blocks. The length and speed of the screw press, the volume reduction ratio, and the perforation of the

Fig. 9.4

Refiner and its close view.

Mince from seafood processing by-product and surimi as food ingredients

203

screens determine the effectiveness of water removal. Screens with 0.5 to 1.5 mm perforations are commonly used in the industry. In addition, screens with smaller perforations are usually placed at the end section to preserve recovery.23 The material passed through a screw press commonly contains about 80±84% moisture. 9.3.7 Decanter centrifuge The use of a decanter centrifuge results in improvements in surimi yield because of greater solid material (more than 80% insoluble solids) recovery during centrifugation compared to traditional screen techniques.59 The main part of decanter centrifuge is the rotating bowl, which consists of a cylindrical part and a conical part.23 Inside the bowl a conveyor moves the solids toward the solid discharge port. A stationary inlet tube is inserted into the centre of the conveyor. The bowl is enclosed in a vessel with discharge arrangements and mounted on a base frame. Three main factors influence the separation of mince and water in a decanter centrifuge: the design of the decanter (geometrical configuration, bowl diameter, length, and speed, differential speed of the conveyor relative to the bowl, and conveyor type); the composition of the liquid and particles to be separated (density, viscosity, size, distribution, configuration, and concentration of the particles); and process-related aspects (temperature and feed rate).23 A large bowl diameter increases the solids handling capacity but also dictates a lower main speed, so as not to exceed mechanical limitations. Increasing the bowl length generally improves the liquid clarification as the residence time in the gravitational field increases. In addition, the higher the bowl speed, the higher the gravitational force, which results in better clarification and a drier solid cake. The differential speed between the bowl and the conveyor also affects the separation of solid and liquid and must be carefully adjusted. 9.3.8 Silent cutter/mixer A silent cutter or ribbon blender is commonly used in surimi plants to incorporate cryoprotectants relatively uniformly in a short period of time. Uniform mixing without generating heat will assure better quality throughout frozen storage of mince or surimi. 9.3.9 Freezer Surimi is frozen in rectangular blocks (10 kg) in a plate freezer. Before loading the freezer, the freezer plates should be cleaned. The blocks should be loaded evenly to maintain good contact between blocks and plates. Blocks are held for approximately 2.5 h or until the core temperature reaches ÿ25ëC.23 To have good shelf-life, frozen surimi (Fig. 9.5) should be kept with a minimum temperature fluctuation in cold storage at ÿ18ëC.

204

Maximising the value of marine by-products

Fig. 9.5

9.4

Frozen surimi.

Mince/surimi processing by-products

Fish mince/surimi manufacturing generates huge amounts of by-products, such as surimi wash water and solid by-products.60,61 Fish mince/surimi processing by-products include fish frame generated from the filleting step and surimi wash water generated from the mince washing step, which contain a significant amount of soluble proteins, including myofibrillar protein.62 Addition of sarcoplasmic protein and myofibrillar protein recovered from fish mince/surimi processing by-products back into fish mince/surimi is an important issue for the fish mince/surimi processing industry. We briefly discuss the various kinds of fish mince/surimi processing by-products, technologies for recovering soluble protein, and myofibrillar protein from surimi wash water and fish frame, and their application in this section. 9.4.1 Fish mince/surimi by-products As shown in Table 9.2, fish mince/surimi manufacturing generates various byproducts, such as viscera in the gutting step, heads in the deheading step, frames in the filleting step, skin and bone in the mincing step, surimi wash water in the washing step, and refiner discharge in the refining step. The major components of fish mince/surimi processing by-products are enzyme, lipid, and protein in viscera, extractives, meat and bone in fish frame, collagen and mineral in skin and bone, soluble protein in surimi wash water and collagen in refiner discharges.

Mince from seafood processing by-product and surimi as food ingredients Table 9.2

205

By-products generated during mince/surimi processing

Processing steps

Processing by-products

Gutting Deheading Filleting Mincing Washing Refining

Viscera Head Frame Skin/bone Wash water Connective tissue

Proportion of whole fish (%) 15±30 14±20 17 8±10 14±16 4±8

Valuable components Enzyme, lipid, protein Mineral Protein, mineral Collagen, mineral Soluble protein, enzyme Collagen

9.4.2 Protein recovery methods Water-soluble protein Myofibrillar proteins constitute approximately 66 to 77% of the total protein in fish flesh. However, in a typical surimi operation, only 50 to 60% of the myofibrillar proteins are retained through the washing and dewatering process. Approximately 40 to 60% of the myofibrillar proteins are lost in the soluble or insoluble forms due to factors such as changes in pH and ionic strength, proteolysis, and mechanical forces in mincing, washing, screening, and screw pressing.45 Surimi wash water can be treated by chemical and physical methods. Chemical methods apply chemical agents to coagulate soluble and insoluble materials from the waste water by shifting pH. Physical methods use screens, membrane filters, and other mechanical actions to remove the solids. Ultrafiltration can be a primary means for recovering protein from surimi wash water,45,63,64 but it has never been successfully applied in a commercial scale due to the nature of protein fouling. However, decanter technology has been successfully applied and made a significant difference in surimi processing. (a) pH-shifting method: Nishioka and Shimizu63 developed means of precipitating proteins from surimi waste water by shifting pH. This method is based on myogen-aggregation phenomenon, which occurs when a solution of sarcoplasmic protein is acidified or alkalified beyond the critical pH zone of 4±5 or 9±11, respectively, and then neutralized. The best condition for pH setting is at first from pH 7 to <4 and then from <4 to 7±9. The maximum amount of precipitation is obtained by changing the pH from 7 to <4 and then to a value between 7 and 9, or from 7 to >12 and then to a value between 6 and 7. The precipitates are easily collected by centrifugation at a low centrifugal force of 300  g. This method is simple and low cost for pretreating waste water but results in complete denaturation of the proteins and no gelling properties. (b) New fish protein isolate using pH shift: As briefly discussed earlier, this new process is similar to Nishioka's method.63 However, there is a distinct difference of eliminating lipids in the finished product. The old method separated protein precipitates at 300  g, while the new method at 10 000  g,

206

Maximising the value of marine by-products

which removes membrane lipids.53 Fish protein isolates show good gel values even though the protein is denatured during processing. (c) Membrane filtration: Membrane filtration applies a positive pressure as a driving force to push the liquid phase through membrane pores, without involving a phase change.64 It is the most suitable method for recovering and concentrating thermally sensitive components, such as protein, in the food industry. When compared to other recovery methods, membrane filtration has the advantage that no supplementary additives are used to aid separation and enables recovery of pure soluble and insoluble proteins. Functional properties of recovered soluble and insoluble proteins are preserved and present utilization and new product opportunities for food manufacturers. When wash water is pumped across the membrane, water, and particles with sizes smaller than the membrane pores pass through it, while particles with sizes larger than the membrane pores are retained. With removal of water and smaller particles, solids larger than the membrane pores are recovered. (d) Centrifugation using decanter centrifuge: Decanter centrifuges have also been adopted by the surimi industry to recover large particles from the waste streams. Typically, a range of 1700 to 3000  g centrifuge force is used for recovering protein particles from surimi wastewater streams. Swafford et al.65 reported the use of a decanter centrifuge to recover insoluble solids from rinsing and dewatering wastewater. Up to 80% insoluble solids were recovered. Surimi produced through a decanter is assigned as recovery-grade surimi. Recovery-grade surimi possesses fairly good color and low impurities, but is usually of lower gel strength.45 Fish frame protein Fish frames are important by-products of the fillet industry because of their significant volume. Frames consist of backbone, tail, and dorsal fins connected by muscle not removed during filleting, which are usually discarded or rendered into fish meal. However, a large volume of minced fish can be recovered during the deboning step (Table 9.3). High levels of bones and parasites, bacterial contamination and off-colors from skin, backbone and gut cavity limit the appeal of frame mince prepared by mechanical deboning. It is considered low quality when compared to minced fillets and fillet trimmings.15 Improved methods for separating fish mince from frame are needed to produce frame Table 9.3

Yields of mince from various fish frames

Frame source Pacific whiting Alaska pollock Channel catfish

Yields (%) 42.8 31.4 57.4

Yield (%) = (unwashed mince weight/frame weight)  100

Reference Wendel et al.41 Crapo and Himelbloom43 Suvanich et al.44

Mince from seafood processing by-product and surimi as food ingredients

207

mince with acceptable characteristics. Wendel66 reported that washing frame meat or mince contaminated with kidney tissue can be more effective than removing the kidney tissue before deboning to decrease textural degradation and protein denaturation. For effective utilization of frame mince, one approach is to develop processing techniques, such as backbone removal, to reduce high defect levels. If kidneys are removed from frames without contaminating meat, higher quality mince can be obtained. Another approach is to blend minced frame flesh with minced fillets and trimmings to produce an acceptable quality product. The recovery methods of fish mince from frame are discussed in this section. (a) Mechanical deboning: Mechanical deboning is the conventional method for separating mince from frame. In mechanical deboning, fish frames are deboned with a drum with 5 mm diameter perforations. (b) Water jet deboning system: When compared to mechanical deboning, a water jet deboning system is superior in mince quality, although inferior in yield.66 The dewatering is more difficult in frame mince separated by water jet deboning than that separated by mechanical deboning. It is probably because particle size is smaller from the water jet deboning system than from the mechanical deboning.67 Frames in water jet deboning system are conveyed through high pressure oscillating water jets to remove the flesh. The recovered flesh then goes to a rotary screen to remove broken bone and skin particles. The system results in a white fish mince. However, it consumes a large quantity of water and is difficult to dewater. 9.4.3 Characteristics of by-products recovered from surimi wash water and frame Surimi wash water Insoluble particulates in surimi wash water are recovered by a rotary screen and further concentrated by microfiltration. In the surimi industry, the microfiltration method is applied for recovering protein from surimi wash water, reducing wastewater impact on the environment, and exploring potential for recycling process water. The moisture of discharged water is around 99%, and is reduced to around 93% by the rotary screen and then to around 85% by the microfiltration system. The solids are increased by 6.7 fold and 15.5 fold, respectively. As shown in Fig. 9.6, the microfiltration-treated solids are concentrated to around 16% (protein: around 15%). However, that is slightly lower than around 20% of commercial surimi (protein: around 19%). The myosin heavy chain content in rotary screen-treated protein and microfiltration system-treated protein are 25% and 62%, respectively, as shown in Fig. 9.7. These results indicate that most of the myosin lost in the form of particulate from the screw press is recovered by the rotary screen. However, the myosin heavy chain content of microfiltration system-treated protein is low when compared to commercial surimi (100%).

208

Maximising the value of marine by-products

Fig. 9.6 Moisture and crude protein contents of wash water, rotary screen-treated protein (RS), microfiltrated protein (MF), and commercial surimi (CS) (modified from Ref. 62).

Fig. 9.7 Quantity comparison of myosin heavy chain from rotary screen-treated protein (RS), microfiltration-treated protein (MF), and commercial surimi (CS) (modified from Ref. 62).

Mince from seafood processing by-product and surimi as food ingredients

209

Frame mince Mechanically deboned frame mince generally contains bone fragments and reddish colored kidney juice, which contains high concentrations of the enzyme, trimethylamine oxide (TMAO) demethylase. Therefore, the color and flavor quality of mechanically deboned frame mince containing kidney is low compared to commercial surimi.4 Mechanically deboned frame mince is also low in salt extractable protein and high in bone, parasite and bacterial counts, impurities, and off-flavor compared to commercial surimi. Frame mince is generally much darker than commercial mince, as shown in Fig. 9.8. Color can be improved by blending frame mince with commercial mince. 9.4.4 Utilization as a surimi resource Water-insoluble proteins Surimi wash water contains a significant amount of water-soluble and insoluble protein. Lin et al.62 attempted to recover water-insoluble proteins using microfiltration and to use recovered proteins as an extending agent of surimi. The result indicates that surimi with 10% replacement of microfiltration-treated protein has the same gel quality as a commercial surimi with respect to gel hardness, elasticity, water retention, and color. Recovered protein has a lower shear stress, but the same shear strain values as a commercial surimi. Pedersen67 also reported adding up to 10% protein recovered from surimi wash water resulted in no measurable decrease in gel strength of surimi. The results indicate the recovered

Fig. 9.8 Lightness (L*) of Alaska pollock minces (modified from Ref. 43). 20/80 and 10/90 indicate the mixture of 20% frame mince/80% commercial mince and 10% frame mince/90% commercial mince.

210

Maximising the value of marine by-products

Fig. 9.9 Texture of cooked Alaska pollock minces (modified from Ref. 43). 20/80 and 10/90 indicate the mixture of 20% frame mince/80% commercial mince and 10% frame mince/90% commercial mince.

protein from surimi wash water by microfiltration system can be used as an extending agent of surimi. Lin et al.62 also reported that the surimi production yield could be increased by 1.7% by adding microfiltration-treated protein back into the production line without diminishing surimi functional property. Fish frame protein Fish frame generated in fillet processing contains a significant amount of flesh. However, frame flesh has some defects, such as poor texture and color. The defect of frame mince can also be improved by blending frame mince with commercial mince, as shown in Fig. 9.9. Surimi gel texture prepared from commercial mince has firmer texture than surimi prepared by frame mince. Surimi gel texture prepared from blended minces (10/90 and 20/80) are as firm as gels prepared from commercial mince. Two approaches can be proposed to utilize frame meat: one is to develop processing techniques to reduce the high defect levels and the other is to blend minced flesh with minced fillets and/or trimmings to produce an acceptable quality product. Wendel66 also reported that water jet deboning was a good method for separating flesh from frame and it can avoid the low quality problem of conventional frame mince.

9.5

Functional properties of fish mince/surimi

Functional properties of proteins indicate their ability to make gels stronger, holding more water or oil, or looking whiter. These include protein solubility, water absorption capacity, water binding ability, viscosity, gelation, swelling ability, emulsifying capacity and emulsion ability.68 Among them, the most

Mince from seafood processing by-product and surimi as food ingredients

211

important functional property in fish mince/surimi is the gelling property.69 Gelation of fish mince is thermo-irreversible.8 Fish myofibrillar protein, which is responsible for the gelation property, can form a strong and elastic gel upon heating. Unlike myofibrillar proteins of land animals, fish myofibrillar proteins can be heated to higher temperatures without sacrificing gel strength or waterholding capacity.70 The functional property of fish mince/surimi can greatly be affected through the biological characteristics of the fish, such as species,9,23,30,71 seasonality,23,72 sexual maturity,23 freshness/rigor71,72 and extrinsic factors, such as harvesting and onboard handling, processing water,47,48,73 time/ temperature,15,74±76 washing cycle,21,77,78 pH,51,52 salinity,52,79 and functional ingredients.19,28,37,80,81 A brief discussion will also be provided in this section on factors affecting functional properties of both conventional surimi and fish protein isolates prepared by new technology, such as acid- and alkali-aided methods. 9.5.1 Fish species In addition to Alaska pollock and Pacific whiting, there are a number of species that are utilized as raw materials for commercial mince/surimi processing. Functional properties of the final fish mince/surimi products vary with raw material due to the different biological properties of fish, such as intrinsic enzymes, lipid composition, the proportion of red flesh, and rigor status (Table 9.4). When fish mince/surimi from some fish, such as Pacific whiting,82 arrowtooth flounder,83 threadfin bream,84 lizardfish,23 Atlantic menhaden,85 white croaker, and oval filefish,86 are converted into mince/surimi-based seafood with functional properties, enzyme inhibitors are required or rapid cooking is necessary to minimize functional deterioration caused by heat-stable enzymes. Fish mince/surimi is also prepared from oily/red-fleshed fish. However, the quality of surimi made from these species varies according to the whiteness, trimethylamine oxide (TMAO) and fat contents of the fish.87 To make mince/ surimi with functional properties and storage stability from oily/red-fleshed fish, such as mackerel, sardine, and salmon, certain steps must be applied to negate the effects of the oil and heme proteins. Heme proteins, such as myoglobin and hemoglobin, account for the red color of dark muscle. In addition, fat oxidation in the dark muscle is promoted by heme proteins, which cause an offensive, Table 9.4

Lipid, protein, and dark flesh composition in fish species

Sardine Mackerel pike Mackerel Cod Pacific whiting

Lipid (%)

Protein (%)

Dark flesh (%)

6.0 8.4 4.0 0.6 1.5

17.5 20.0 18.0 16.6 16.8

31.1 23.3 18.1 <1 <1

212

Maximising the value of marine by-products

rancid odor.14 To utilize red fleshed-fish as a mince, vacuum packaging is used or antioxidants can be added to prevent lipid oxidation. To utilize the red fleshed-fish as a surimi source, 0.1±0.5% NaHCO3 is used in the first washing solution and a decanter can be used to remove excess oil. The addition of 0.05± 0.1% sodium pyrophosphate and the use of vacuum during washing are also recommended to remove heme proteins. 9.5.2 Seasonality and sexual maturity Fish muscle is subjected to seasonal variations in parameters, such as pH, fat, protein, and water content, which influence the functional properties of the final products. The composition of Alaska pollock varies between seasons so protein contents are highest (16.9%) in the fall and lowest (15.6%) in the spring, whereas moisture contents are highest (82.7%) in the fall and lowest (80.9%) in the spring.23 Bandarra et al.88 and Leu et al.89 reported seasonal changes of the fat contents of sardines harvested off the Portuguese coast and mackerel harvested in the Southern Rhode Island waters, Nantucket Sound, respectively. The fat content of sardine was as high as about 18% in August±October and lowest in March at 1.2%. The fat content of mackerel was as high as about 22.6% in November and lowest in March at 5.1%. Consequently, to manufacture mince with good frozen storage stability from sardine harvested in summer and mackerel harvested in winter, vacuum package technology must be applied because of the higher fat content. To manufacture surimi with good functional properties and whiteness from sardine harvested in summer and mackerel harvested in winter, special technology using NaHCO3 and a decanter must be applied because of the higher fat content. Ingolfsdottir et al.72 reported on seasonal variations in functional properties of cod mince. The results indicated that both hardness and cohesiveness showed a drop from March to May and another drop was observed for cohesiveness during the autumn months compared to mince prepared from cod harvested in the winter. Surimi prepared from fish harvested in the feeding period commonly has the highest functional properties. It is probably because fish muscle has the lowest moisture content, as well as the highest total protein during the feeding period. 9.5.3 Freshness or rigor The functional properties of fish mince/surimi are influenced by freshness of fish. It is probably because fish freshness affects biochemical properties of fish muscle. Freshness of fish is primarily time/temperature-dependent. The processing of Alaska pollock occurs within 12 h on at-sea vessels, whereas at shore-side operations processing occurs within 24±48 h.23,59 However, due to recent regulations of salmon by-catch and sea lion habitat, it is not uncommon to see pollock processed 100 h after harvesting. Fish freshness is also affected by harvesting conditions and methods used for capture, such as weather conditions

Mince from seafood processing by-product and surimi as food ingredients

213

at sea, size of tow, length of tow, salt uptake, and temperature of fish after capture, as well as on-board handling methods and vessel storage conditions. Development of rigor mortis varies among fish species, and is dependent upon post-mortem temperature, and ante-mortem conditions, such as stress, method of capture, handling conditions, and seasons.90 9.5.4 Time and temperature of processing Time and temperature for fish capture are the most important factors for the functional properties of final mince/surimi. With prolonged storage/processing time and increased storage/processing temperature, severe proteolysis could occur in finished products, mince or surimi. If the fish is a proteolytic enzymeladen species and held for more than 24±48 h even at 5ëC, good functional properties would not be expected due to proteolytic degradation. Consequently, to prepare mince/surimi with good functional properties, fish should be processed promptly on landing or kept at around 0ëC if holding is necessary. Degradation of myosin heavy chain of Pacific whiting increased as the washing cycles increased (Fig. 9.10).91 The trend of actin degradation was similar to that of myosin heavy chain, but to a lesser extent.47,92 Actin degradation increased as storage time and temperature increased. At 0ëC, actin was degraded, the extent increased substantially with storage time. After 14 h at 0ëC, about 20% of actin was degraded. Raw fish for mince/surimi preparation is commonly kept in holding tanks (about 0ëC) up to 14 h before fish are subjected to processing. Similar to myosin heavy chain degradation, actin degraded more rapidly at 5ëC than at 0ëC. As temperatures and time increased further, degradation of actin occurred more rapidly.23

Fig. 9.10

Loss of myosin heavy chain (MHC) at various washing cycles (modified from Ref. 91).

214

Maximising the value of marine by-products

9.5.5 Effects of wash water and washing conditions In conventional surimi making, washing is one of the most critical steps for preparing surimi with good functional properties. A large amount of water is used to remove sarcoplasmic proteins, blood, fat, and other nitrogenous compounds from minced fish flesh. Functional properties of the final product are greatly improved as the myofibrillar protein is concentrated by removal of impurities during washing. The important factors associated with water for preparing surimi with good functionality are temperature, mineral content, pH, and salinity, quantity of water used and washing cycles. Water must be refrigerated to a temperature below which the fish muscle proteins can retain their maximum functional properties. Considering the change in air temperature during processing, the recommended water temperature for obtaining maximum quality is 5ëC or lower. Even though warm water species are not as sensitive as cold water species, cold water temperature, in general, results in better quality of finished products. Soft water with minimum levels of minerals, such as Ca++, Mg++, and Fe++, is recommended for washing. Hard water causes deterioration of texture and color of fish mince/surimi during frozen storage. In addition, Ca++ and Mg++ are responsible for gel texture property changes, whereas Fe++ is responsible for color changes.59 The pH of water must be maintained at approximately that of prerigor fish muscle tissue (pH 6.8±7.0) to obtain better functional property of surimi. Before washing, the salinity of fish mince is approximately 0.7%. Too much salt (>0.3%) in the wash water could cause solubilization of myofibrillar proteins resulting in low yield, and accelerate denaturation during frozen storage. The degree of washing required to produce good functional surimi depends on the type, composition, and freshness of the fish. Water to meat ratios ranging from 4:1 to 8:1 are often used by processors. This washing process is often repeated three or four times to ensure sufficient removal of sarcoplasmic proteins. However, for the last 10 years, the US surimi industry has made an effort to reduce the water to meat ratio to 2:1 and the washing cycle to 2, resulting in a significant yield increase from 15% to 30%. Adu et al.76 reported that most sarcoplasmic proteins are fairly soluble and removed during the initial washing steps. Consequently, after the sarcoplasmic proteins are completely removed, further washing causes a severe loss of myofibrillar proteins. Lin and Park23,48 investigated minimizing water usage for leaching by reducing the water/meat ratio and increasing the wash cycles and wash time. Increased wash time did not enhance the removal of sarcoplasmic proteins once equilibrium was reached. However, increased wash cycles continuously removed residual sarcoplasmic proteins from the mince. At a low water to meat ratio (2:1 or 1:1), regardless of wash cycles and wash time, no significant loss of myofibrillar proteins occurred. Myosin heavy chain content, water retention, and whiteness of the washed mince, however, decreased when the water to meat ratio was reduced. Increasing wash time and/or wash cycles, however, enhanced functional properties but also resulted in higher moisture content.

Mince from seafood processing by-product and surimi as food ingredients

215

9.5.6 Fish protein isolates by new technology This new approach, initiated at the University of Massachusetts,49 has opened up a new way of isolating functional fish protein isolate. New technology for preparing fish protein isolates (acid- or alkali-aided methods) shows significant potential as a new method for maximal protein recovery and results in surimi with commercially acceptable (better with alkaline treatment) functional properties. Unlike the conventional method of surimi manufacturing, no washing or dewatering steps are continuously involved, which significantly reduces waste and water consumption. Kim51 investigated the functional properties of fish protein gels prepared after various treatments (Fig. 9.11). The results indicated that the best functional properties are obtained from fish proteins treated at pH 11 (alkali-aided) and pH 2 (acid-aided). Surface SH content is critical for the formation of disulfide bonds. Alkali conditions, especially pH 10.5 and pH 11.0, are favored for disulfide bond formation. However, pH 10.5 treatment does not give high texture values. It is probably because of the high cathepsin L-like activities, which interfere with gel formation.51 In addition, at pH 11, extensive thiol oxidation and disulfide interchange reactions occur and more disulfide bonds contribute to strong gel formation. Another interesting way of utilizing fish protein isolates from seafood processing by-products has been explored at several universities.53 Using primarily fish frame, fish protein can be isolated using the alkaline method followed by centrifugation. This isolate is homogenized with water as a milky

Fig. 9.11 Textural properties of gels made from fish protein isolates after various pH treatments (modified from Ref. 51). Samples were treated at various pH conditions during protein recovery, and then adjusted to pH 7.0. Different alphabetical letters indicate a significant difference ( p < 0:05). Gels were prepared with 1.5% beef plasma protein.

216

Maximising the value of marine by-products

slurry after mixing one part isolates and three parts water. This pure protein slurry is injected into fillets like salmon, halibut, catfish, and other valuable species. It enhances the yield and improves frozen stability of fish fillets. A different attempt was also made to utilize fish proteins isolates from byproducts. When milky slurry was applied as a dipping solution for fish fingers and patties before battering or breading, the quantity of oil absorbed in fried products was significantly reduced. Fish protein isolate may form a protein film and act as a fat blocker.93

9.6

Nutritional characteristics

Mince is flesh separated in a comminuted form from the skin, bones, scales, and fins. Therefore, the nutritional properties of unwashed fish mince are similar to those of the raw material, while superior to those of surimi. It is probably because of the difference between washing and unwashing. Therefore, fish mince contains a high level of water-soluble vitamins, mineral and fats compared to surimi. Adu et al.76 also reported the effect of washing on the nutritional characteristics of minced rockfish flesh. The results indicated that unwashed rockfish mince was higher in ash and lipid contents based on dry weight than washed rockfish mince. There was no difference in amino composition between washed- and unwashed-rockfish minces. There was a difference in mineral composition between washed- and unwashed-rockfish minces. The mineral composition of the minced fish was greatly changed by washing treatment. Phosphorus, potassium, and sodium levels were reduced, while the iron, copper, zinc, and chromium levels increased in the washed fish mince when compared to unwashed fish mince. Calcium content is commonly high in fish mince compared to fish fillets, due to small bone fragments in the mince as a result of the mechanical deboning.57 Babbitt and Reppond33 compared trimethylamine oxide (TMAO) content between washed mince and unwashed mince from Alaska pollock. Washing reduced the TMAO content from 71.3 to 8.7 mg/100 g. It is well known that fish oils, rich in polyunsaturated fatty acid, have potential for prevention of heart disease, cancer and other diseases. Therefore, surimi is lower in calories and oil including omega-3 fatty acids. It is also bland in taste/odor and white in color due to removal of water-soluble components by washing. However, the nutritional values of surimi-based seafood can be controlled by combination with other nutritious food and/or nutritional fortifiers. Pedersen67 and Lin et al.62 reported the protein recovery of 80% solids in surimi manufacture from Alaska pollock and Pacific whiting using membrane filtration. Comparing ash contents of before (about 17% of the solid) and after (about 3% of the solid) membrane filtration treatment of wash water, membrane filtration served to remove mineral from the process water. There was no difference in amino acid composition among raw materials, its primary product (surimi) and the recovered protein concentrate from surimi wash water.

Mince from seafood processing by-product and surimi as food ingredients

217

Ohshima et al.6 examined the chemical scores for essential amino acids in surimi, beef, pork, chicken and turkey. The high level of nutritional quality in surimi gel blended with recovered protein concentrate from surimi wash water and commercial surimi matches that of land animal meats. Surimi gel, which is blended with the recovered protein concentrate from surimi wash water and commercial surimi, is higher in isoleucine, leucine, lysine, threonine and tryptophan content than chicken, while lower in histidine and valine content. The amino acid score of surimi is also that of a high quality protein, and is similar to beef, chicken, and turkey.

9.7

Storage stability

Fish mince/surimi are commonly frozen for attaining longer shelf life. Utilization of frozen fish also provides a more constant supply of raw material independent of yearly variation and thereby facilitates consistent production planning. Frozen storage stability of fish mince/surimi can be greatly affected through biological characteristics of raw material (fish species, seasonality, sexual maturity, and freshness), harvesting, onboard handling, and processing conditions (water, time/temperature, washing cycle, pH, salinity, and functional ingredients). A brief discussion of factors affecting frozen storage stability of fish mince/surimi is provided in this section. 9.7.1 Factors affecting storage stability Fish species Various fish species are used as raw material for fish mince/surimi processing. The frozen storage stability of fish mince/surimi varies with different biological factors of fish, such as intrinsic enzymes, lipid composition, the proporation ratio of redflesh, and development of rigor mortis90 (Table 9.4). Usually, tough texture is developed in gadoid fish muscle, such as cod, haddock, Pacific whiting, Alaska pollock, during frozen storage. Such textural changes are caused by trimethylamine oxide (TMAO) demethylase. Formaldehyde is hypothesized to be a cross-linking agent in muscle proteins and may thus cause textural deterioration in frozen fish mince.11,21,69 Dark-fleshed fish minces, such as mackerel, sardine, and salmon, also undergo quality deterioration during frozen storage, mainly through lipid oxidation. To improve frozen storage stability of mince/surimi from dark-fleshed fish, certain steps must be applied to negate the effects of oil and heme proteins. Heme proteins, such as myoglobin and hemoglobin, account for the red color of dark muscle. In addition, fat oxidation in the dark muscle is promoted by heme proteins, which causes denaturation of protein during frozen storage.14 Washing Washing using chilled water of 5±10ëC before freezing removes denaturation promoters, such as active enzymes and their substrates, metals, lipids containing

218

Maximising the value of marine by-products

n-3 polyunsaturated fatty acids and trimethylamine oxide, from fish mince.21 This affects the stability of the remaining proteins (primarily myofibrillar proteins) during frozen storage. If protein cross-linking induced by formaldehyde is the main cause of textural hardening during frozen storage, the textural change should be minimized by washing, since most trimethylamine oxide in fish mince can be removed by washing. Consequently, to improve frozen stability in fish mince, the formation of dimethylamine and formaldehyde should be reduced. Functional ingredients Washed fish mince is generally mixed with cryoprotectants, such as sugar, sorbitol, and polyphosphates, in order to stabilize the fish proteins from freeze denaturation. The use of high levels (>5%) of sugar made surimi too sweet and caused a brown color during frozen storage. Therefore, sorbitol is used to reduce the sugar content and subsequently, the level of sweetness and discoloration. Sugar and sorbitol protect protein from frozen denaturation by solute exclusion from the surface. As sugar or sorbitol is introduced to a system of proteins and water, sugar/sorbitol is excluded from the protein-water system and destabilizes water molecules. As water molecules are reoriented, the hydrophobic groups hidden in the interior create stronger interactions, resulting in a stabilized protein structure. Sugar/sorbitol also increases the surface tension of water. This prevents withdrawal of water molecules from the proteins.4 Noguchi et al.94 reported that pentoses, such as xylose and ribose, have less protective effect for protein denaturation than hexoses, such as glucose and fructose. The difference of protective effect among the monosaccharides is probably because of the difference in the number of OH groups on the molecule. These compounds stabilize the native conformation of the proteins by a solute exclusion mechanism.95,96 The cryoprotective effects of sugar and sorbitol can be enhanced by adding phosphate. The effect of phosphate can be explained based on two facts: one is to raise the pH and the other is to chelate metal ions, which actively promotes oxidation. The average quantity of cryoprotectants added to washed fish mince is approximately 4±6% sugar, 0± 5% sorbitol, and 0±0.3% phosphate.97 Under constant low temperature storage (below ÿ20ëC), frozen surimi with cryoprotectants, such as sugar, sorbitol, and phosphate, can usually be stored up to two years. However, it is important to remember that cryoprotection does not stop, but rather minimizes the freeze denaturation. With its versatile characteristics, surimi can be used as an intermediate raw material for preparing popular surimi seafood, which requires specific textural attributes, such as kamaboko,31 crabstick,26,33,97 lobster analogs,34 shrimp analogs,94 frankfurter,98 nugget,36 and fish patty.38 9.7.2 Comparison of storage stability between fish mince and surimi Fish mince contains a significant amount of water-soluble components, such as active enzymes and their substrates,99 metals, lipids, and trimethylamine. Many

Mince from seafood processing by-product and surimi as food ingredients

219

problems of fish mince, such as color, texture, and odor change, are caused by the water-soluble components. Therefore, surimi made after removing watersoluble components is prepared to improve frozen storage stability of fish mince. The storage stability of fish mince is, therefore, inferior to that of surimi. Crawford et al.99 studied the stability of frozen Pacific whiting mince blocks compared to fillets over a 12-month period and indicated higher levels of oxidative rancidity in minced flesh due to textural deterioration. Regenstein100 examined the stability of frozen mince in cold storage and reported that many problems caused by enzyme action can be minimized by maintaining the storage temperature below ÿ30ëC. However, the storage stability of fish mince is a continuing problem since most commercial storage conditions are well above ÿ30ëC. Abdel-aal13 used antioxidants for extending the shelf life of frozen Nile karmout (Claries lazera), which has many undesirable characteristics such as rapid development of rancid off-flavor. Ascorbic acid and Na2EDTA could retard rancidity development in the frozen karmout mince. 9.7.3 Storage stability of mince from surimi by-products Fish mince from surimi by-products can be used as a surimi yield extender because of its availability in large quantities. However, the unwashed mince recovered from frame has numerous impurities from kidney and bone. If it is not totally removed, residual kidney tissue may be incorporated into mince during deboning. Since kidney tissue can not be totally removed from frame by the conventional method, the chemical and textural changes of recovered fish mince from frame during frozen storage are accelerated due to enzymatic and microbial contamination from the kidney.68 To avoid quality problems associated with trimethylamine oxide (TMAO) demethylase that accelerates dimethylamine (DMA) and formaldehyde formation, Wendel et al.41 used a high pressure water jet deboning system for recovering mince from frame and reported that frozen storage stability of the mince was improved.

9.8

Utilization

Fish mince/surimi can be used as an ingredient in many composite products, such as kamaboko, various surimi seafood (crabstick and other shellfish analogs), seafood patty, seafood nuggets, and frankfurter analog. The mince/ surimi-based products are also very popular in the Asian diet. Due to their healthy nutritional values, functional versatility, and competitive prices in relation to their natural counterparts, the consumption of these products in the West has reached almost 250 000 metric tonnes. Phenomenal growth has also been experienced in the USA and Europe, including Russia, where sales have grown from virtually zero in 1980 to 900 000 metric tonnes and 150 000 metric tonnes in 2004.

220

Maximising the value of marine by-products

9.8.1 Kamaboko Kamaboko is the most typical surimi-based product in Japan. Surimi paste is formed on a wood board before any thermal treatment. Sometimes its surface is coated with colored paste for appearance. The shape and texture of kamaboko varies depending on the geographical region. After its unique shape is formed, the surimi paste is subjected to a low temperature setting process (20±40ëC for 30±60 min), depending on the species. During this process, the gel-forming ability of solubilized myofibrillar proteins is highly enhanced, which yields a strong gel.101 Cooking by either steaming or baking is carried out to complete the gelation of fish proteins. 9.8.2 Crabstick Crabstick is currently one of the most prevalent surimi-based fabricated seafood products in the marketplace.102 The crabstick manufacturing flow is described in Fig. 9.12. First, frozen surimi is either partially thawed or broken before surimi is cut into small pieces. This step is required to reduce the size of particles by avoiding a heavy load to blades or the shaft during chopping. Over-thawing which may induce denaturation must be avoided. Salt is added first into surimi to extract myofibrillar proteins. Then other ingredients (egg white, starch, sugar, sweet rice wine, seasoning and flavors, natural coloring, and water) are commonly added to salted surimi in the silent cutter. Chopping temperatures vary depending on species between 0±5ëC for cold water species and 15±25ëC for warm water species to produce higher gel functionality.101 The comminuted surimi paste is extruded onto a conveyor belt of the cooking machine and then cooked. A common sequence of heating is radiant heat, followed by steam heat, and then radiant heat again. Regardless of the heating method, however, the ambient temperature in the tunnel where the paste is exposed to radiant or steam heat is 90±95ëC. Total cooking time depends on the product specification. A thinly extruded (1±2 mm) sheet undergoes various heating procedures depending on the machinery and production specifications, but in general this primary cooking last 35±60 seconds. This short cooking process induces gelation of the surimi proteins but is not sufficient to swell the starch or gel other protein additives. Immediately on completion of the first cooking step, the product is cooled by air at room temperature or below. Fiberization is accomplished by elongated cuts running lengthwise on the gelled sheet. Slitting is obtained by passing the sheet through two rollers with slitters. The space between the slitters controls the number and width of individual fibers. Bundling is a process that rolls the cooked product sheet tightly into a rope shape. The rope is then passed through the rollers for a conventional color application. A polyethylene plastic film, onto which colored fresh paste is applied, wraps around the product rope, which is then cut to a specified length. The colored paste is then set to a gel by cooking under steam for 15±30 min, followed by rapid chilling. However, co-extruded color application bypasses this laborious processing step.101

Fig. 9.12 A crabstick line with drum cooker (source: Courtesy of Young Nam Machinery, Korea).

222

Maximising the value of marine by-products

Surimi crabmeat may be cut into different dimensions, such as flake, stick, and chunk. The rope is cut diagonally at a 25±30ë angle in 5±8 cm long pieces, tip to tip for flakes. Leg or stick shape is cut straight at a 90ë angle and usually 3, 5, or 8 inches long. Surimi crabmeat is packed in plastic films under full or partial vacuum before going through metal detection followed by pasteurization. Throughout production processing temperatures are important in development of gel structure and resulting textural characteristics of surimi crabmeat. Finished product is strongly influenced by cooking temperature and time.25 The pasteurization step adapted in surimi crabmeat must secure the microbiological quality of products. However, the processing step is not sufficient to sterilize. Pasteurization with proper heat treatment provides a greater advantage over sterilization, which results in serious problems in sensory characteristics, with regard to sensory attributes. The longer the cooking at a higher temperature, the more negative the sensory attributes. For the safety of vacuum-packed surimi seafood against Clostridium botulinum, the US government has recommended the manufacturer to heat the package at 85ëC (internal) for a minimum 15 min, while maintaining 2.4 water phase salt and storage temperature below 3.8ëC. A European guideline of F90 ˆ 10 min is also approved for 6-log microbial reduction.101 The chilling must bring the product temperature to 4ëC within 30 min. Refrigerated products are packaged after chilling to 5ëC. 9.8.3 Seafood patty For preparation of seafood patty, frozen mince is either partially thawed or broken before mixing. Various ingredients (85±87% minced fish, 2±3% soy protein, 2±3% starch, 1.5±2.5% refined salt, a small amount of dried onion, dried tomato, dried pepper, chilli powder, powdered garlic, and allspice, 4±8% sorbitol, 1±2% chicken/beef/pork extracts) are added to thawed or broken mince, and blended in a ribbon mixer. The temperature should be maintained at below 10ëC during mixing. The blend is held at ÿ20ëC for 1 h to facilitate easy forming into patty, and to maintain about 0ëC during forming. Fish patties are formed and dehydrated in a tunnel dryer for 10 h before vacuum packaging. 9.8.4 Seafood nuggets For preparation of seafood nuggets, frozen mince is either partially thawed or broken before mixing. Ingredients (salt, sugar, egg white, oil, and other flavorings) are added to thawed or broken mince and blended in a ribbon mixer. The temperature should be maintained at below 10ëC during mixing. The mixed paste is stuffed, extruded and placed onto trays before chilling overnight. The chilled strands are cut into lengths of about 2.0 cm. The cut product is predusted, battered, and breaded. The resultant nuggets are deep-fried at 350ëC for 45 seconds before cooling and packaging. Seafood nuggets are stored below ±20ëC.

Mince from seafood processing by-product and surimi as food ingredients

223

9.8.5 Frankfurter analog or seafood sausage For the preparation of frankfurter analog, frozen surimi is either partially thawed or broken before surimi is comminuted. The thawed or broken surimi is placed in a silent cutter and chopped for 2 min at high speed with 2.5% NaCl. The temperature should be maintained at below 10ëC during chopping (temperature could vary depending on the species used). Additional ingredients are then added. Chopping continues at high speed for 10 min. The paste is stuffed into casings. After standing at room temperature for 1 h, the links are heat processed by immersion in a 90ëC water bath for 20 min. They are then cooled by immersion in a low temperature cooler (below 5ëC) until reaching an internal temperature of below 30ëC and stored in a 2ëC cooler.

9.9 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

References 1997. Chilling and freezing of fish. In Fish Processing Technology (Ed. Hall, G.M.), pp. 93±118: Chapman & Hall, New York. SLOAN, A.E. 1994. Top ten trends to watch and work on. Food Technol., 48(7): 89± 98. SKIPNES, D., OINES, S., ROSNES, J.T. and SKARA, T. 2002. Heat transfer in vacuum packed mussel (Mytilus edulis) during thermal processing. J. Aquatic Food Products Technol., 11(3/4): 5±19. PIGOTT, G.M. 1986. Surimi: the `high tech' raw materials from minced fish flesh. Food Rev. Int., 2: 213±246. PIGOTT, G.M. and TUCKER, B.W. 1990. Seafood: Effects of Technology on Nutrition, pp. 206±257, Marcel Dekker, Inc., New York. OHSHIMA, T., SUZUKI, T. and KOIZUMI, C. 1993. New development in surimi technology. Trends Food Sci. Technol., 4: 157±163. MAGNUSDOTTIR, E. 1995. Physical and chemical changes in stabilized mince from Pacific whiting during frozen storage. MS Thesis. Oregon State University, Corvallis, OR. LANIER, T.C. 1994. Functional food protein ingredients from fish. In Seafood Proteins: Minced Fish as a Food Ingredient (Eds Sikorski, Z.E., Pan B.S. and Shahidi, F.), pp. 127±159, Chapman & Hall, New York. BABBITT, J.K. 1986. Suitability of seafood species as raw materials. Food Technol., 40(3): 97±100, 134. JAHNCKE, M., BAKER, R.C. and REGENSTEIN, J.M. 1992. Frozen storage of unwashed cod (Gadus morhua) frame mince with and without kidney tissue. J. Food Sci., 57: 575±580. DINGLE, J.R. and HINES, J.A. 1975. Protein instability in minced flesh from fillets and frames of several commercial Atlantic fishes during storage at ±5ëC. J. Fish. Res. Board Can., 32: 775±783. SUZUKI, T. 1981. Fish and Krill Protein: Processing Technology, pp. 115±145, Applied Science Publishers Ltd., London. ABDEL-AAL, H.A. 2001. Using antioxidants for extending the shelf-life of frozen Nile karmout (Claries lazera) fish mince. J. Aquatic Food Product Technol., 10(4): 87±99. CHEN, H.H. 2002. Decoloration and gel-forming ability of horse mackerel mince by air-flotation washing. J. Food Sci., 67: 2970±2975. GARTHWAITE, G.M.

224 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

Maximising the value of marine by-products and JAHNCKE, M.L. 2000. Microbiological and color quality changes of channel catfish frame mince during chilled and frozen storage. J. Food Sci., 65: 151±154. MOLEDINA, K.H., REGENSTEIN, J.M., BAKER, R.C. and STEINKRAUS, K.H. 1977. Effects of antioxidants and chelators on the stability of frozen storage mechanically deboned flounder meat from racks after filleting. J. Food Sci., 42: 759±764. CHAPMAN, K.W., SAGI, I., HWANG, K.T. and REGENSTEIN, J.M. 1993. Extra-cold storage of hake and mackerel fillets and mince. J. Food Sci., 58: 1208±1211. SIMPSON, R., KOLBE, E., MACDONALD, G.A., LANIER, T.C. and MORRISSEY, M.T. 1993. The feasibility of producing surimi from partially processed and frozen Pacific whiting (Merluccius productus). J. Food Sci., 59: 272±276. LIAN, P.Z., LEE, C.M. and HUFNAGEL, L. 2000. Physical properties of frozen red hake (Urophycis chuss) mince as affected by cryopropective ingredients. J. Food Sci., 65: 1117±1123. PACHECO-AGUILAR, R., CRAWFORD, D.L. and LAMPILA, L.E. 1989. Procedures for the efficient washing of minced whiting (Merluccius productus) flesh for surimi production. J. Food Sci., 54: 248±252. YOON, K.S., LEE, C.M. and HUFNAGEL, L.A. 1991. Effect of washing on the texture and microstructure of frozen fish mince. J. Food Sci., 56: 294±298. HASTINGS, R.J. 1989. Comparison of the properties of gels derived from cod surimi and from unwashed and once-washed cod mince. Internat. J. Food Sci. Technol., 24: 93±102. PARK, J.W. and LIN, T.M. 2005. Surimi: manufacturing and evaluation. In Surimi and Surimi Seafood, Second edition, Revised/Expanded, (Ed. Park, J.W.), pp. 33±106, CRC Press, Boca Raton, FL. LANIER, T.C. 1986. Functional properties of surimi. Food Technol., 40(3): 107±114, 124. LEE, C.M. 1984. Surimi process technology. Food Technol., 38(11): 69±80. BERTAK, J.A. and KARAHADIAN, C. 1995. Surimi-based imitation crab characteristics affected by heating method and end point temperature. J. Food Sci., 60: 292±296. LEE, C.M. 1986. Surimi manufacturing and fabrication of surimi-based products. Food Technol., 40(3): 115±124. NIELSEN, R.G. and PIGOTT, G.M. 1996. Differences in textural properties in minced pink salmon (Oncorhynchus gorbuscha) processed with phosphate-treated proteins and gums. J. Aquatic Food Product Technol., 5(3): 81±104. HSIEH, Y.L. and REGENSTEIN, J.M. 1989. Texture changes of frozen stored cod and ocean perch minces. J. Food Sci., 54: 824±826, 834. CHENG, C.S., HAMANN, D.D., WEBB, N.B. and SIDWELL, V. 1979. Effects of species and storage time on minced fish gel texture. J. Food Sci., 44: 1087±1092. HOLMQUIST, J.F., BUCK, E.M. and HULTIN, H.O. 1984. Properties of Kamaboko made from red hake (Urophycis chuss) fillets, mince, or surimi. J. Food Sci., 49: 192±196. VERREZ, V., BENYAMIN, Y. and ROUSTAN, C. 1992. Detection of marine invertebrates in surimi-based products. Dev. Food Sci., 30: 441±448. BABBITT, J.K. and REPPOND, K.D. 1987. Manufacturing of a crab analogue to determine the quality of US shore-based produced surimi. Marine Fisheries Review, 49(4): 34±36. MOSKOWITZ, H.R. and PORRETTA, S. 2002. Contrasting customer and operator concept and product requirements: the case of surimi. Food Serv. Tech., 2(3): 115±130. PRZYBYLA, A.E. 1988. Supply and quality critical to growth of seafood. Food SUVANICH, V., MARSHALL, D.L.

Mince from seafood processing by-product and surimi as food ingredients 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

225

Engineering, July: 34±38. BUCK, E.M. and FAFARD, R.D. 1985. Development of a frankfurter analog from red hake surimi. J Food Sci., 50: 321±324, 329. ROCKWOER, R.K., DENG, J.C., OTWELL, W.S. and CORNELL, J.A. 1983. Effect of soy flour, soy protein concentrate and sodium alginate on the textural attributes of minced fish patties. J. Food Sci., 48: 1048±1052. DESTURA, F.I. and HAARD, N. 1999. Development of intermediate moisture fish patties from minced rockfish meat (Sebastes sp.). J. Aquatic Food Product Technol., 8(2): 77±94. VENUGOPAL, V. and SHAHIDI, F. 1998. Traditional methods to process underutilized fish species for human consumption. Food Rev. Int., 14: 35±97. PARK, J.W., LIN, T.M. and YONGSAWATDIGUL, J. 1997. New development in manufacturing of surimi and surimi seafood. Food Rev. Int., 13: 577±610. WENDEL, A., PARK, J.W. and KRISTBERGSSON, K. 2002. Recovered meat from Pacific whiting frame. J. Aquatic Food Product Technol., 11(1): 5±18. MARTIN, R.E. 1992. Seafood waste issues in the 1990s. J. Aquatic Food Product Technol., 1(1): 9±16. CRAPO, C. and HIMELBLOOM, B. 1994. Quality of mince from Alaska pollock (Theragra chalcogramma) frames. J. Aquatic Food Product Technol., 3(1): 7±17. SUVANICH, V., JAHNCKE, M.L. and MARSHALL, D.L. 2000. Changes in selected chemical quality characteristics of channel catfish frame mince during chill and frozen storage. J. Food Sci., 65: 24±29. MORRISSEY, M.T., LIN, T.M. and ISMOND, A. 2005. Waste management and byproduct utilization. In Surimi and Surimi Seafood, Second edition, Revised/Expanded, (Ed. Park, J.W.), pp. 279±434, CRC Press, Boca Raton, FL. HOKE, M.E., JAHNCKE, M.L., SILVA, J.L., HEARNSBERGER, J.O., CHAMUL, R.S. and SURIYAPHAN, O. 2000. Stability of washed frozen mince from channel catfish frames. J. Food Sci., 65: 1083±1086. LIN, T.M. 1996. Solubility and structure of fish myofibrillar proteins as affected by processing parameters. PhD Thesis. Oregon State University, Corvallis, OR. LIN, T.M. and PARK, J.W. 1997. Effective washing conditions reduce water usage for surimi processing. J. Aquatic Food Product Technol., 6(2): 65±79. HULTIN, H.O. and KELLEHER, S.D. 1999. Advanced protein technologies. Dec. 21, 1999. Process for isolating a protein composition from a muscle source and protein composition. US patent 6005073. CHOI, Y.J. and PARK, J.W. 2002. Acid-aided protein recovery from enzyme-rich Pacific whiting. J. Food Sci., 67: 2962±2967. KIM, Y.S. 2002. Physicochemical characteristics of fish myofibrillar and sarcoplasmic proteins treated at various pH conditions. MS Thesis. Oregon State University, Corvallis, OR. THAWORNCHINSOMBUT, S. 2004. Biochemical and gelation properties of fish protein isolate prepared under various pH and ionic strength conditions. PhD Thesis. Oregon State University, Corvallis, OR. HULTIN H.O., KRISTINSSON H.G., LANIER T.C. and PARK, J.W. 2005. Process for recovery of functional proteins by pH shifts. In Surimi and Surimi Seafood, Second edition, Revised/Expanded (Ed. Park, J.W.), pp. 107±139, CRC Press, Boca Raton, FL. REGENSTEIN, J.M. and REGENSTEIN, C.E. 1991. Introduction to Fish Technology, pp. 139±156. Van Nostrand Reinhold, New York. KOLBE, E. 1999. Freezing technology. In Surimi and Surimi Seafood. (Ed. Park,

226 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

66. 67. 68. 69. 70. 71. 72. 73. 74. 75.

Maximising the value of marine by-products J.W.), pp. 167±200, Marcel Dekker, Inc., New York.

and REGENSTEIN, J.M. 1992. Minced fish. In Encyclopedia of Food Science and Technology. (Ed. Hui, Y.H.), pp. 944±955, John Wiley & Sons, Inc., New York. TAKEDA, F. 1971. Technological history of frozen surimi industry. New Food Ind., 13: 27±31. KIM, J.S. and PARK, J.W. 2004. Characterization of acid-soluble collagen from Pacific whiting surimi processing byproducts. J. Food Sci., 69: C637±642. PARK, J.W. and MORRISSEY, M.T. 2000. Manufacturing of surimi from light muscle fish. In Surimi and Surimi Seafood. (Ed. Park, J.W.), pp. 23±58, Marcel Dekker, Inc., New York. SWAFFORD, T.C. 1987. Separation-recovery of soluble/insoluble proteins from surimi processing washwaters. Presented at the Pacific Fisheries Technologist Meeting, 1±13. NIKI, H., KATO, T., DEYA, E. and IGARASHI, S. 1985. Recovery of protein from effluent of fish meat in producing surimi and utilization of recovered protein. Bull. Japan. Soc. Sci. Fish., 51: 959±964. LIN, T.M., PARK, J.W. and MORRISSEY, M.T. 1995. Recovered protein and reconditioned water from surimi processing waste. J. Food Sci., 60: 4±9. NISHIOKA, F. and SHIMIZU, Y. 1983. Recovery of proteins from washings of minced fish by pH shifting method. Nippon Suisan Gakkaishi, 49: 795±800. HUANG, L. 1997. Application of membrane filtration to recover solids from protein solutions. PhD Thesis. Oregon State University, Corvallis, OR. SWAFFORD, T.C., BABBITT, J., REPPOND, K., HARDY, A., RILEY, C.C. and ZETTERLING, T.K.A. 1990. Surimi process yield improvement and quality contribution by centrifuging. In Proceedings of the International Symposium on Engineered Seafood including Surimi. (Eds. Martin, R.E. and Collette, R.L.), pp. 483±496, National Fisheries Institute, Seattle, WA. WENDEL, A.P. 1999. The recovery and utilization of Pacific whiting frame meat for surimi production. MS Thesis. Oregon State University, Corvallis, OR. PEDERSEN, L.D. 1990. Product recovery from surimi wash water, pp. 173±176. In International By-Products Conference: Anchorage, AL. MARSILI, R. 1993. Protein power: Functionality and versatility. Food Product Design. September: 67±80. CHANG, C.C. and REGENSTEIN, J.M. 1997. Textural changes and functional properties of cod mince proteins as affected by kidney tissue and cryoprotectants. J. Food Sci., 62: 299±304. PIPATSATTAYANUWONG, S. 1995. Alternative products from Pacific whiting: Fresh surimi and texturized mince. MS Thesis. Oregon State University, Corvallis, OR. HUIDOBRO, A. and TEJADA, M. 1993. Emulsifying capacity of fish mince from several species during frozen storage. J. Sci. Food Agric., 61: 333±338. INGOLFSDOTTIR, S., STEFANSSON, G. and KRISTBERGSSON, K. 1998. Seasonal variations in physicochemical and textural properties of North Atlantic cod (Gadus morhua) mince. J. Aquatic Food Product Technol., 7(3): 39±61. SRIKAR, L.N. and REDDY, G.V.S. 1991. Protein solubility and emulsifying capacity in frozen stored fish mince. J. Sci. Food Agric., 55: 447±453. CARPO, C. PAUST, B. and BABBIT, J. 1988. Recoveries and yields from Pacific fish and shellfish. National Marine Fishery Service, Marine Advisory Bulletin, No. 37, 10±11. MACDONALD, G.A., LELIEVRE, J. and WILSON, N.D.C. 1990. Strength of gels prepared HSIEH, Y.T.L., HSIEH, Y.L., HWANG, K.T., GOMEZ-BASURI, J.

Mince from seafood processing by-product and surimi as food ingredients

76. 77. 78.

79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93.

227

from washed and unwashed minces of hoki (Macruronus novaezelandiae) stored in ice. J. Food Sci., 55: 976±978, 982. ADU, G.A., BABBITT, J.K. and CRAWFORD, D.L. 1983. Effect of washing on the nutritional and quality characteristics of dried minced rockfish flesh. J. Food Sci., 48: 1053±1055, 1060. CHANG, C.C. and REGENSTEIN, J.M. 1997. Water uptake, protein solubility, and protein changes of cod mince stored on ice as affected by polyphosphates. J. Food Sci., 62: 305±309. SIMPSON, R., MORRISSEY M.T., KOLBE, E., LANIER, T.C. and MACDONALD, G.A. 1994. Effects of varying sucrose concentrations in Pacific whiting (Merluccius productus) stabilized mince used for surimi production. J. Aquatic Food Product Technol., 3(4): 41±52. NIELSEN, R.G. and PIGOTT, G.M. 1996. Differences in textural properties in minced pink salmon (Oncorhynchus gorbuscha) processed with phosphate-treated proteins and gums. J. Aquatic Food Product Technol., 5(2): 21±45. CHANG-LEE, M.V., LAMPILA, L.E. and CRAWFORD, D.L. 1990. Yield composition of surimi from Pacific whiting (Meriuccius products) and the effect of various protein additives on gel strength. J. Food Sci., 55: 83±86. PARK, J.W. 1994. Functional protein additives in surimi gels. J. Food Sci., 59: 525± 527. AN, H., WEERASINGHE, V.W., SEYMOUR, T.A. and MORRISSEY, M.T. 1994. Cathepsin degradation of Pacific whiting surimi proteins. J. Food Sci., 59: 1013±1017, 1033. YONGSAWATDIGUL, J., PARK, J.W., KOLBE, E., ABU DAGGA, Y. and MORRISSEY, M.T. 1995. Ohmic heating maximizes gel functionality of Pacific whiting surimi. J. Food Sci., 60: 10±14. TOYOHARA, H. and SHIMIDZU, Y. 1988. Relation between the modori phenomenon and myosin heavy chain breakdown in the threadfin-bream gel. Agric. Biol. Chem., 52: 255±257. BOYE, S.M. and LANIER, T.C. 1988. Effect of heat stable alkaline protease activity of Atlantic menhaden (Brevoorti tyrannus) on surimi gels. J. Food Sci., 53: 1340±1342. TOYOHARA, H., SAKATA, T., YAMASHITA, K., KINISHITA, M. and SHIMIDZU, Y. 1990. Degradation of oval-filefish meat gel caused by myofibrillar proteinase(s). J. Food Sci., 55: 364±368. PUTRO, S. 1989. Surimi prospects in developing countries. Infofish International, 5: 29±32. BANDARRA, N.M., BATISTA, I., NUNES, M.L., EMPIS, J.M. and CHRISTIE, W.W. 1997. Seasonal changes in lipid composition of sardine (Sardine pilcharardus). J. Food Sci., 62: 40±42. LEU, S-S., JHAVERI, S.N., KARAKOLTSIDIS, P.A. and CONSTANTINIDES, S.I. 1981. Atlantic mackerel (Scomber scombrus, L): Seasonal variation in proximate composition and distribution of chemical nutrients. J. Food Sci., 46: 1635±1638. PARK, J.W., KORHONEN, R.W. and LANIER, T.C. 1990. Effects of rigor mortis on gelforming properties of surimi and unwashed mince prepared from tilapia. J. Food Sci., 55: 353±355. LIN, T.M. and PARK, J.W. 1996. Extraction of proteins from Pacific whiting mince at various washing conditions. J. Food Sci., 61: 432±438. REDDY, G.V.S. and SRIKAR, L.N. 1991. Preprocessing ice storage effects on functional properties of fish mince protein. J. Food Sci., 58: 965±968. KELLEHER, S. 2005. Personal communication.

228 94. 95. 96. 97. 98. 99. 100. 101. 102.

Maximising the value of marine by-products NOGUCHI, S., OOSAWA, K. and MATSUMOTO, J.J. 1976. Studies on the control of denaturation of fish muscle proteins during frozen storage. VI. Preventive effect of carbohydrates. Bull. Japan. Soc. Sci. Fish., 42: 77±83. BABBITT, J.K., LAW, D.K. and CRAWFORD, D.L. 1976. Improved acceptance and shelf life of frozen minced fish with shrimp. J. Food Sci., 41: 35±37. GOELLER, L.M., AMATO, P.M., FARKAS, B.E., GREEN, D.P., LANIER, T.C. and KONG, C.S. 2004. Optimization of incorporation of low-molecular-weight cryoprotectants into intact fish muscle. J. Food Sci., 69: FEP164±171. PARK, J.W. 1994. Cryoprotection of muscle proteins and its mechanism during frozen storage ± a review. J. Aquatic. Food Product Technol., 3(3): 23±41. PARK, E.Y., BREKKE, C.J. and BRANEN, A.L. 1978. Use of Pacific hake (Merluccius products) in a frankfurter formulation. J. Food Sci., 43: 1637±1640, 1645. CRAWFORD, D.L., LAW, D.K., BABBITT, J.K. and MCGILL, L.S. 1979. The stability of frozen Pacific hake mince blocks. J. Food Sci., 44: 363±368. REGENSTEIN, J.M. 1986. The potential for minced fish. Food Technol., 40(3): 101±106. PARK, J.W. 2005. Surimi seafood: products, market, and manufacturing. In Surimi and Surimi Seafood, Second edition, Revised/Expanded, (Ed.) Park, J.W. pp. 375± 433, CRC Press, Boca Raton, FL., HOLLINGWORTH, T.A., KAYSNER, C.A., COLBURN, K,G., SULLIVAN, J.J., ABETY, C.,WALKER,

and WEKELL, M.M. 1991. Chemical and microbiological analysis of vacuum-packed, pasteurized flaked imitation crabmeat. J. Food Sci., 56: 164±167.

K.D., TORKELSON J.D., THROM, H.R.

10 Aquatic food protein hydrolysates H. G. Kristinsson, University of Florida, USA

10.1

Introduction

The proper utilization of limited aquatic resources has been a topic of great interest for many decades. As discussed in more detail in other chapters in this book, a major problem facing the seafood industry is that a substantial amount of material is left behind after processing. Segments of the industry are also faced with a tremendous amount of by-catch, which is not properly utilized. These materials, which are often discarded or used for animal feed or fertilizer, are rich in valuable and functional ingredients that can be recovered. Many years back it was found that adding enzymes to this `waste' material, with the aid of other processes such as filtration and centrifugation was an effective way to extract and recover proteins from the material. The application of proteases to fish processing waste leads to the hydrolysis of the proteins in the material, which can then be separated from other constituents of the muscle. This process produces what is known as fish protein hydrolysates (FPH). A number of research publications have demonstrated that using controlled enzymatic hydrolysis a broad range of protein ingredients of good quality can be produced from undesirable raw materials (e.g., Baek and Cadwallader, 1995; Shahidi et al., 1995; Vieira et al., 1995; Onodenalore and Shahidi, 1996; Kristinsson and Rasco, 2000a,b,c; 2002). On the commercial level, this promising technology has, however, been faced with economic obstacles, lack of usability and often low acceptance of the final products (Kristinsson and Rasco, 2000a). This is in part related to the very complex nature of the raw material, which can often lead to unstable, poorly functional and unacceptable final products. A few countries and companies have, however, been successful in producing FPH on a commercial level, and still are. The key to FPH success is their unique properties

230

Maximising the value of marine by-products

and the ability to produce FPH with very different properties from the same raw material, as elaborated in this chapter.

10.2

The enzymatic hydrolysis process

Many different processes have been reported for recovering and hydrolyzing proteins from aquatic food material with enzymes. The general principle of the process is simple (Fig. 10.1) but there are many factors that need to be carefully considered if the aim is to have good control over the process and produce a final product of consistent and good quality. For most processes the raw material is thoroughly minced and then added to water. A well homogenized raw material in water is important to allow for good mixing and good enzyme access. Too little water can significantly reduce the production/recovery of FPH (Slizyte et al., 2003), likely in large part due to high viscosity which limits enzyme access and would also hinder recovery attempts using filtration or centrifugation, particularly if the aim is to produce FPH of limited hydrolysis. Generally the raw materials for hydrolysis are a complex mixture which can present processing challenges, as well as having a major influence on the quality of the final product. Raw materials high in lipids and pro-oxidants (e.g., blood and heme proteins) have a tendency to oxidize during processing and may lead to significant lipid oxidation and color problems in the final material. To minimize this problem, antioxidants can be added before hydrolysis, or raw material can be washed to remove some of the heme proteins and lipids. Washing may, however, lead to lower protein recovery as proteins can be washed out.

Fig. 10.1 Outline of the main steps in the production of fish protein hydrolysates.

Aquatic food protein hydrolysates

231

Oxidation may also be enhanced at lower pH values and higher temperatures, which can be avoided with proper enzyme selection. Many raw materials have high levels of spoilage bacteria, which can be minimized by maintaining low temperatures, low pH (which unfortunately will accelerate oxidation) and applying proper enzyme inactivation temperatures, which also reduce the level of microorganisms. Furthermore, components such as skin, bones and scales may interfere with enzymatic hydrolysis, and ideally should be separated from the material prior to hydrolysis, e.g. via mechanical deboning. Recent advances, such as the advent of the acid- and alkali-aided solubilization/precipitation process to recover proteins from byproducts (Hultin et al., 2005) (see Chapter 4) now provide an economic way to start with a relatively pure protein material as the starting material for fish proteins hydrolysis. The next step in the process is to allow the homogenized mixture to reach the temperature of interest. This is important, as enzymes may have markedly different activities at different temperatures. The pH of the slurry is then adjusted to the desired value and the enzyme of interest added to the mixture. The choice of enzyme is a very important consideration when making FPH, since different enzymes have different specificities and reaction rates, and thus can yield very different products. The least expensive method is to utilize proteolytic enzymes present in the fish itself, e.g. mixing visceral material with the by-products. This autolytic process is still employed, but primarily for animal feed or fertilizer applications. There are, however, products produced with endogenous enzymes for human consumption, most notably fish sauce which consists of extensively hydrolyzed fish proteins. The major problem with using endogenous enzymes is that very little control is possible as visceral material can vary substantially in activity and enzyme levels. This may afford FPH that may be acceptable for animal or fertilizer applications, but typically not for food use (with the exception of fish sauce) where consistency of the final FPH is of great importance. Another problem is that endogenous enzymes have been reported to give significantly lower recoveries than commercial preparations (Hale, 1972; Shahidi et al., 1995). Kristinsson and Rasco (2000b) did, however, report similarly good protein recovery for an enzyme preparation made from fish pyloric ceca compared to commercial enzymes. The advantage of using visceral material as a source of enzymes is, however, lower processing cost. Another advantage is that endogenous fish enzymes often have high activities at low temperatures, in contrast to most commercial preparations (Kristinsson and Rasco, 2000d). This allows the reaction to operate at very low temperatures, which minimizes microbial growth as well as quality and functionality problems with the final product. Where controlled hydrolysis is required, commercial enzymes are added. A range of different commercial enzymes are available. Enzymes have either endo- or exopeptidase activities. The former pertains to enzymes cleaving peptide bonds within the protein, resulting in many peptides but relatively few free amino acids. Exo activity refers to an attack on either end of the protein polypeptide chain, thus giving many free amino acids and few large peptides.

232

Maximising the value of marine by-products

The two different enzyme activity forms can produce FPH with markedly different properties. If limited hydrolysis and large peptides are the desired end product, more specific enzymes have certain advantages. On the other hand, extensive hydrolysis requires the use of less specific enzymes, and typically a range of different enzymes. Normally a combination of enzymes with endo- and exopeptidase activity is used in commercial hydrolysis. Many different commercial enzyme preparations have been reported in the production of FPH. Acid proteases, such as pepsin, were early on believed to be the enzymes of choice, in large part since the low pH stabilized the reaction mixture with respect to microbial growth (Hale, 1969). The acid pH can, however, have a detrimental effect on the quality (mainly color and oxidative rancidity) and functionality of the FPH. In recent years, the use of enzymes working under pH conditions close to neutrality has been more common. The milder pH values give a more functional and higher quality FPH. Several successes have been reported with the enzyme preparations Alcalase, Flavourzyme and Protamex from Novo Nordisk (e.g., Quaglia and Orban, 1987; Sugiyama et al., 1991; Diniz and Martin, 1996; Benjakul and Morrissey, 1997; Kristinsson and Rasco, 2000b; Theodore and Kristinsson, 2005). The neutral proteases Corolase PN-L and 7089 have also been found to produce FPH of good recoveries and qualities (Kristinsson and Rasco, 2000b). After adding the enzyme, it acts very fast initially on the proteins in the mixture, significantly reducing the viscosity of the slurry. As more peptide bonds are broken, the slower the hydrolysis gets and eventually the reaction reaches its maximum level of hydrolysis (Fig. 10.2). The extent of the hydrolysis can be followed using different methods (Kristinsson and Rasco, 2000a). One of the most convenient methods is to follow the degree of hydrolysis (%DH) using the pH-stat procedure (Adler-Nissen, 1986). With this method the %DH is calculated from the volume and molarity of base or acid used to maintain constant pH. The method defines the degree of hydrolysis as percent ratio of the numbers of peptide bonds broken (h) to the total numbers of bonds per unit weight (htot; meq/kg protein, calculated from the amino acid composition of the substrate): %DH ˆ (h/htot)  100

(10.1)

%DH can also be expanded to: %DH ˆ (C  NB  100)/(  htot  MP)

(10.2)

where C is the acid or base consumption in ml (depending on the pH the hydrolysis is operated at; base if operating at neutral to alkaline pH; acid if operating at low pH), NB is the normality of the base (or acid),  is the average degree of dissociation of the -NH groups (if operating at neutral to alkaline pH) or COOH groups (if operating at low pH), and MP = mass of protein in grams (%N  6.25). This equation works well at neutral to alkaline pH values, but is difficult to use at acidic pH values, where other more appropriate methods are more useful (e.g., Nielsen et al., 2001). Essentially, the more the protein is being

Aquatic food protein hydrolysates

233

Fig. 10.2 An enzymatic hydrolysis curve for salmon muscle hydrolyzed with Flavourzyme 1000L at two different enzymatic activities (AzU = Azocoll Units).

broken down by the enzyme, the more the %DH increases (Fig. 10.2). Typically, the higher the %DH the higher the protein recovery (Kristinsson and Rasco, 2000b). The choice of pH and temperature for the reaction is highly dependent on what the operator wants to accomplish. Working at the enzyme optimal conditions will lead to very rapid and often extensive hydrolysis. Additionally, working close to the enzyme optima may in some cases compromise the stability of certain enzymes if the reaction is long. If the goal is limited hydrolysis, it is recommended to operate away from the enzyme optima, or at significantly lower enzyme concentrations, to be able to arrest the reaction more effectively at lower degrees of hydrolysis. The termination of the enzyme reaction is accomplished by irreversible denaturation of the enzyme. This can be done by heating the slurry typically to temperatures above 85ëC for at least 10 minutes (depending on enzyme). Another effective method is to use a combination of pH and temperature induced denaturation, e.g. reducing temperature to a very low pH in which case a milder heat treatment can be used. The proteins are then recovered by either filtration or centrifugation where they are separated from other components of the raw material (e.g., unhydrolyzed proteins, fat, bones, skin, scales, etc.) after which they are concentrated or dried. In some cases the FPH is concentrated or dried directly, if the raw material was a relatively clean source of protein. In many cases it may be necessary to add stabilizing agents, e.g. antioxidants or antimicrobials, or even introduce a deodorizing treatment.

234

10.3

Maximising the value of marine by-products

Properties of fish protein hydrolysates

Fish proteins have not only good functionality but also have a high nutritional value. One way to modify and improve the properties of fish proteins is to use proteases to produce a range of FPH. Most of the research in the past 50 years or so with FPH has concentrated on investigating and optimizing conditions to produce FPH of high protein yields as well as unraveling food functionality of FPH (Kristinsson and Rasco, 2002). These studies have demonstrated that the size and chemical properties of the peptides in FPH have a dramatic impact on their function. Therefore, good control of enzyme specificity and degree of hydrolysis (%DH) is critical if the goal is to produce hydrolysates with properties suitable for a wide array of products (Mullally et al., 1994; Kristinsson and Rasco, 2000b). Recently interest in FPH has shifted more to their potential bioactive properties rather than food functionality. Recent data on protein hydrolysates (including FPH) has demonstrated that they may provide significant physiological benefits to living systems. Some of the identified bioactive properties may also find applications in stabilizing food products, e.g. against lipid oxidation. However, very limited knowledge exists on the molecular mechanisms behind these activities.

10.4

Role in food systems

Fish protein hydrolysates have been extensively investigated for their food functionality and may be used in numerous applications in food products, as summarized by Kristinsson and Rasco (2002). Food systems can be used as vehicles for the consumption of bioactive FPH, but FPH can also be used to improve function and quality of foods. Fish protein hydrolysates differ from intact myofibrillar proteins in being readily soluble at a range of ionic strength and pH (Shahidi et al., 1995; Vieira et al., 1995; Kristinsson and Rasco, 2000b), while intact myofibrillar proteins have only high solubility at high and low pH values (Fig. 10.3). The solubility reported for FPH has typically been between 90 and 100%. This is because upon hydrolysis the proteins are broken down into many smaller units with effectively more exposed amino and carboxyl groups than the parent protein, thus enabling more protein-water interactions (Mahmoud et al., 1992). This is an advantage, as they can effectively be added to food systems under very different conditions. The soluble nature of FPH has sparked interest in its use as an injectable or mixable protein material to improve water-binding in seafood products. Kristinsson and Rasco (2000b) found that adding FPH from salmon to minced salmon muscle led to less drip loss on thawing compared to adding no FPH. Studies by Varetzis et al. (1990) and Shahidi et al. (1995) demonstrated a higher cook yield when FPH was added to hamburgers and minced pork, respectively. Work in the author's laboratory has shown that fish fillets can be tumbled or injected with solutions containing FPH to increase product yield significantly on cooking, equal or better than injection/ tumbling fillets in phosphates (Fig. 10.4). The mechanism behind this functional

Aquatic food protein hydrolysates

Fig. 10.3

235

Protein solubility of fish protein hydrolysate (salmon) compared to fish muscle proteins (ground catfish muscle) as a function of pH.

Fig. 10.4 Weight increase and cook loss of catfish fillets injected with different solutions; NaCl, catfish FPH and salt, salt and phosphate, catfish FPH, salt and phosphate. The grey bars show the weight increase after injection. The white bars show weight loss after cooking, calculated based on the original pre-injected weight of the fillets. For example, a positive value refers to fillets that have a higher weight than the fillets had before injection, while a negative value refers to fillets that has a weight below the preinjected weight of the fillets after cooking.

236

Maximising the value of marine by-products

property of FPH is not well understood, but FPH could be a valuable natural substitute for chemicals such as phosphates. Work with FPH has demonstrated that they may possess cryoprotective properties. Khan and coworkers (2003) reported that surimi containing FPH had better gel-forming ability than surimi with no FPH added. Surimi with FPH also demonstrated higher residual ATPase activity, which is a sign that it had a protective effect on myosin during freezing and thawing. Several studies have also demonstrated that FPH may protect proteins on drying (Zhang et al., 2002; Hossain et al., 2003; Khan et al., 2003). One problem during the production of FPH is that bitterness can develop. This may greatly limit their food use. Furthermore, if FPH are produced from a raw material that is susceptible to oxidation, rancid flavors may be associated with the final product. Liu et al. (2000) demonstrated a good correlation between the level of lipid oxidation and development of bitterness during autolytic hydrolysis of frigate mackerel. In addition, Hoyle and Merritt (1994) showed that defatted raw materials lead to less bitterness in the final isolate. Preventing bitterness in FPH is a difficult task which requires extensive experimentation, since certain functional properties may be compromised when the goal is to produce a peptide profile of minimal bitterness. The cause of bitterness is complicated and not well known. A high proportion of hydrophobic peptides in hydrolysates may in part account for increased bitterness (Tamura et al., 1990). There seems to be a sensitive balance between %DH and the extent of bitterness, and results vary in the literature. In general, it appears that limited and extensive hydrolysis leads to less bitterness rather than intermediate hydrolysis (Sugiyama et al., 1991; Yu and Fazidah, 1994; Vieira et al., 1995; Kristinsson and Rasco, 2000a). A number of studies have also revealed that not only the degree of hydrolysis but also the type of enzyme used can have a dramatic influence on the level of bitterness (Sugiyama et al., 1991; Hoyle and Merritt, 1994). Several enzyme preparations are now commercially available with the goal to greatly limit the development of bitterness and at the same time obtain good functional properties, e.g. Flavourzyme and Protamex from Novo Nordisk (Bagsvaerd, Denmark). Certain chemical treatments can also reduce bitterness, such as passing the FPH through activated charcoal (Shahidi et al., 1995), extraction with chemical solvents (Lalasidis et al., 1978; Chakrabarti, 1983), or applying a second hydrolysis step in the FPH with exopeptidases (Lalasidis et al., 1978; Sugiyama et al., 1991). Fish protein hydrolysates which have been extensively hydrolyzed or have a high proportion of small peptides (primarily di or tripeptides) have been reported to have flavor enhancement effects similar to MSG (Fujimaki et al., 1973; Noguchi et al., 1975; In, 1990; Imm and Lee, 1999). In fact several commercial seafood flavors made of or with FPH are on the market and are used for a variety of different products, such as soups, sauces, value-added seafood products, imitation seafood products, snacks and seasonings. Fish protein hydrolysates have been found to have excellent interfacial/ surface properties (Liceaga-Gesualdo and Li-Chan, 1999; Kristinsson and

Aquatic food protein hydrolysates

237

Rasco, 2000b; Jeon et al., 1999) and thus may have potential use as emulsifying and emulsion stabilizing ingredients in a variety of products (e.g., dressing, margarine and meat batters) as well as aid in the formation and stability of foambased products (e.g., whipped cream, meringues and mousse). There are, however, also studies reporting relatively poor interfacial/surface properties of FPH (Miller and Groninger, 1976; Shahidi et al., 1995; Vieira et al., 1995; Onodenalore and Shahidi, 1996; Sathivel et al., 2003). Many of those studies, however, have not specified the level of hydrolysis which makes it very difficult to interpret the data. The level of hydrolysis, i.e. peptide size, is very important for interfacial/surface activity of the FPH (Jeon et al., 1999). Several studies have shown that hydrolyzed fish muscle has an increased emulsification formation and stability compared to unhydrolyzed muscle (Spinelli et al., 1972; Liceaga-Gesualdo and Li-Chan, 1999). Limited hydrolysis (larger peptides) generally leads to improved emulsification and foaming properties of fish proteins, while extensive hydrolysis (small peptides) reduces these properties (Quaglia and Orban, 1990; Liceaga-Gesualdo and Li-Chan, 1999; Kristinsson and Rasco, 2000b; Jeon et al., 1999). Very small peptides don't have the ability to form a good stable cohesive protein network around oil droplets or air pockets. There is also evidence that as %DH increases (i.e. higher level of small peptides), FPH exhibits less oil binding (Kristinsson and Rasco, 2000b). Enzyme specificity is very important for interfacial/surface activity of FPH, since different peptides are produced from different enzymes. Kristinsson and Rasco (2000b) reported that different enzymes used to produce salmon FPH gave different emulsification capacity and stability even at the same %DH. Studies on a variety of different food proteins have shown that protein hydrolysates have antioxidative properties, and as such may play a role in both food and physiological systems in controlling peroxidative damage. Certain peptides and amino acids, which can be found in muscle foods, have been found to have excellent antioxidative properties (Karel et al., 1966; Chan and Decker, 1994). The action of these peptides and amino acids has been a subject of much investigation lately. Some postulated mechanisms are that they can scavenge radicals formed during peroxidation and chelate transition metals, which are potent prooxidants. Fish protein hydrolysates, like other hydrolysates, are rich in peptides and several studies have demonstrated their potential as a food antioxidant. Shahidi and coworkers have performed a number of studies on the antioxidative properties of FPH. Shahidi et al. (1995) reported that FPH made from capelin effectively reduced lipid oxidation when added to ground pork by up to 60.4%, as assessed by measuring thiobarbituric reactive substances (TBARS). Amarowicz and Shahidi (1997) fractionated capelin FPH into four fractions with gel filtration column chromatography, and found that different fractions had different activities, some being prooxidative while others were antioxidative. The fraction with the highest molecular weight was found to be most effective. Other work has also demonstrated that larger peptides rather than very small peptides are more effective. Work presented by Wu and coworkers (2003) showed that intermediate peptides (1±1.5 kDa) more effectively

238

Maximising the value of marine by-products

increased the lag time before oxidation when added to a linoleic acid emulsion peroxidation system, compared to small molecular weight peptides. It was proposed that this was due to the reducing ability of FPH as well as its ability to chelate metals. Jeon et al. (1999) also demonstrated that different FPH fractions representing different molecular weight ranges have different antioxidative activities. It was reported that fractions below 5 kDa most effectively reduced peroxidation of linoleic acid, and were as good as tocopherol. An extensive study by Theodore and Kristinsson (Theodore, 2005; Theodore and Kristinsson, 2005) on the potential antioxidative mechanisms of FPH made from isolated catfish muscle proteins at different %DH, demonstrated that the mechanism is far from being a simple one. Both the whole FPH and the soluble fractions of FPH were investigated for various different antioxidative tests. Both systems were found to have high radical scavenging activity (as tested by two methods), which increased as %DH increased (Fig. 10.5). Both systems were also found to have good metal chelation ability, again higher as the %DH increased. In addition, both systems exhibited reducing power, but in contrast to the other results, the reducing power decreased as %DH increased. Interestingly, however, when hydrolysates were tested in a linoleic acid peroxidation system they did not display any antioxidative effect. The soluble fractions of the hydrolysates (i.e. having smaller peptides), however, did display a strong antioxidant activity. Many of the above studies employ systems where metal ions are the prooxidants. Other prooxidants, such as heme proteins and lipoxygenases, are present in muscle foods and work via different mechanisms than transition metals. Chuang and coworkers (2000) demonstrated that FPH (from mackerel)

Fig. 10.5 The antioxidative activity (Trolox equivalence) of catfish protein hydrolysates at different degrees of hydrolysis as assessed by the oxygen radical absorbance capacity (ORAC) method (adapted from Theodore and Kristinsson, 2005). Control refers to unhydrolyzed catfish proteins.

Aquatic food protein hydrolysates

239

had the ability to decrease hemoglobin-mediated lipid oxidation in a linoleic acid system. Although no direct evidence was provided, the authors believed that this was possibly due to dipeptides containing histidine, such as anserine and carnosine, which are known to scavenge free radicals. Work done in the authors' laboratory shows that FPH (channel catfish) and fractions of FPH may have an antioxidative effect in a model fish muscle matrix system with hemoglobin as the added prooxidant. There is also a possibility that the antioxidative activity displayed by the FPH in the various studies presented above, is not necessarily due to peptides formed during the hydrolysis process. There are a number of compounds in fish muscle which display good antioxidative effects which would be present in the FPH preparations. Undeland and coworkers (2003) have demonstrated that the `press juice' of fish muscle (i.e. the aqueous fraction of muscle after extensive ultracentrifugation) has strong antioxidative activity on hemoglobin-mediated oxidation in a washed fish muscle matrix. Work in the authors' laboratory has demonstrated that the soluble fraction of catfish protein isolates has in some cases equal or better antioxidative activities that FPH from the isolate, suggesting the action of small molecular weight compounds, yet to be identified.

10.5

Physiological role in humans and animals

Much research in the past 50 years or so has shown that protein hydrolysates have an excellent amino acid balance, good digestibility, rapid uptake and the presence of certain bioactive peptide components. Hydrolyzed fish material is widely used as feed for a variety of farmed animals as well as cultured food fish (Dong et al., 1993; Gildberg, 1993; Kristinsson and Rasco, 2000a). Many studies have demonstrated that FPH have excellent protein quality and are readily utilized (Atia and Shekib, 1992; Sugiyama et al., 1991; Diniz and Martin, 1996; Liaset et al., 2000; Abdul-Hamid et al., 2002; Bechtel et al., 2003). Studies with laboratory rats show that adding FPH to rat diets leads to more rapid growth and higher body weight compared to rats fed casein (Kienkas, 1974; Ballester et al., 1977; Atia and Shekib, 1992). Fish protein hydrolysates present great promise for the aquaculture sector. Barrias and Olivia-Teles (2000) pointed out that FPH containing fish diets lead to a better nitrogen retention and have better feed conversion ratio than most commercial fishmeal feeds. Several studies have also demonstrated that FPH may be particularly good in the early stages of fish growth, as they increase the survival rate of young trout and salmon (Lian and Lee, 2003; Gildberg, 2003). Refstie and coworkers (2004) conducted an extensive study on post-smolt Atlantic salmon, by comparing the inclusion of 0, 5, 10 and 15% FPH in fishmeal fed to salmon. It was found that the higher the FPH inclusion the higher the feed consumption (FPH appeared to act as a feeding stimulant) and the higher the growth. Fish protein hydrolysates were also found to lead to increased protein retention and digestibility. Studies by Gildberg and coworkers (Gildberg et al., 1996; Bogwald et al., 1996;

240

Maximising the value of marine by-products

Gildberg, 2003) have shown that FPH may make fish more disease resistant and may stimulate the immune system of fish. It should, however, also be pointed out that several studies have shown no or a negative effect of feeding aquatic species with FPH. In one study fishmeal was replaced by FPH which did not lead to an increased growth or feed utilization of juvenile turbot (Olivia-Teles et al., 2000). Fish protein hydrolysates from krill led to lower growth when fed to shrimp, possibly due to an excessive amount of low-molecular-weight peptides which may lead to an imbalance of amino acid absorption (Cordova-Murueta and Garcia-Carreno, 2002). The positive effects FPH may have on animals suggest that they may have positive effects on humans as well. Just as FPH stimulates the immune system in fish, peptides in fish sauce (essentially a version of liquid FPH) also stimulated the proliferation of white blood cells in human subjects (Thongthai and Gildberg, 2004). Fish protein hydrolysates have also been found to have a blood thinning effect, i.e. increase flow of red blood cells (Chuang et al., 2000). A small protein or protein fragment with strong anticoagulant and antiplatelet properties has recently been isolated from yellowfin sole FPH (Rajapakse et al., 2005). A recent study demonstrated that a commercial preparation of FPH from Pacific whiting shows promise in improving the health and function of the digestive system (Fitzgerald et al., 2005). When cultured rat epithelia intestinal and colonic cells were given FPH, cell growth was increased and injury was reduced significantly. It was also found that most of the active components, glutamine containing di- and tripeptides, were soluble in ethanol and not water. One of the most interesting effects FPH have on humans is their effect on the neurological system. Studies in France have shown that FPH from cod and mackerel may reduce anxiety as well as improve memory and learning in humans (Dorman et al., 1995; Le Poncin, 1996a,b). Bernet et al. (2000) reported that FPH administered to rats reduced their level of stress, in a similar fashion as valium does. Just as FPH have been found to have antioxidative effects in vitro and in food systems, they have been found to reduce peroxidation in vivo. Boukortt et al. (2004) reported that overall antioxidant status was increased by 35% in hypertensive rats when they were fed fish protein hydrolysates, compared to feeding them casein. Chuang et al. (2000) demonstrated that FPH inhibited the activity of lipoxygenase, which is an enzyme implicated in low density lipoprotein oxidation. Recently significant interest has developed in the potential use of FPH to reduce blood pressure. A number of studies show that FPH made from different species have the ability to inactivate the angiotensin I converting enzyme (ACE) (Kohama et al., 1991; Ukeda et al., 1992; Matsumura et al., 1993; Wako et al., 1996). The compound angiotensin I is converted by ACE to angiotensin II which has been connected to hypertension. Bordenave et al. (2002) found that FPH from sardines and cod were able to inhibit almost 30% of ACE activity and shrimp FPH inhibited 57% of ACE activity. Theodore and Kristinsson (2005) found that FPH made from isolated catfish muscle proteins was able to inhibit ACE activity in vitro by close to 90%. Interestingly, the intact fish proteins (i.e.

Aquatic food protein hydrolysates

241

unhydrolyzed) also exhibited high ACE inactivation activity. Intact proteins would, however, not play a role directly in ACE regulation, as they would be hydrolyzed in the digestive system. Some studies have shown that the ACE inhibition is sensitive to peptide size. The ability of cod FPH fractions to reduce ACE activity was in the following order 3 kDa > 5 kDa > 10 kDa > 30 kDa, suggesting small molecular weight peptides are more effective. Jung et al. (2004) also found that lower molecular weight fractions (<5 kDa) of yellowfin sole FPH more effectively inhibited ACE than high-molecular-weight fractions. There is also evidence that FPH may play a role in regulating blood pressure in vivo. A 9% drop in blood pressure was recorded when rats were fed feed containing FPH compared to casein. This was believed to be due to the high concentrations of cysteine, methionine and arginine in FPH, all known to have an effect on hypertension. When hypertensive rats were fed a 300 mg dose of purified peptides from sea bream scale hydrolysates (i.e. collagen hydrolysates), their blood pressure dropped significantly (Fahmi et al., 2004). These peptides were found to have higher ACE inhibitory activity in vitro than the commercial hypertension drug enalapril maleate. Related to cardiac disease, it has also been demonstrated that FPH from salmon frames administered to rats reduced total cholesterol and increased HDL cholesterol (Wergedahl et al., 2004). In light of the many potential benefits FPH may provide to humans, it would be useful if commercial products were available with functional and active FPH. However, very few commercial products contain added FPH, for many different reasons. Significant strides have been made to incorporate FPH into the human diet, with mixed results. As has been mentioned before, seafood flavors, many of which are essentially FPH, are available. It has been suggested that to improve the protein quality of cereal products, they could be mixed with FPH (Yanez et al., 1976; Morales de Leon et al., 1990). Enriching legume products with FPH has also been reported, and it was found that the majority of sensory panelists responded favorably to FPH supplemented products (Morales de Leon et al., 1990). Some reports have shown that FPH can be successfully incorporated into bakery products. Chevalier and Noel (1982) patented a biscuit (a type of `energy bar') which was made with dried skim milk (15%) and fish protein hydrolysate (14%). Yu and Tan (1990) researched the incorporation of tilapia FPH into crackers, which were later fried. The crackers were found to be `highly acceptable' by panelists even at a 10% FPH addition level. It must be kept in mind, however, that the acceptance of products containing FPH will likely be greatly influenced by the culture and ethnic backgrounds the panelists represent.

10.6

Role in plant growth and propagation

Humans have for a long time recognized the benefits of fish and fish waste as a fertilizer for crops. The amino acid and peptide profile of FPH makes it an excellent source of nitrogen for plants, and it is readily adsorbed and utilized.

242

Maximising the value of marine by-products

Some commercial applications include fertilizer for cranberry and cherry trees (George Pigott, personal communication, 1997), golf courses (Stephen D. Kelleher, personal communication, 1999) and house plants. Shetty and coworkers (Eguchi et al., 1997; Milazzo et al., 1999) have demonstrated that FPH can have an effect beyond just basic nutrition for plants. It was reported that FPH is able to stimulate somatic embryogenesis in anise (Pimpinella anisum) better than proline, and has the potential to become a proline and amino acid substitute for propagating plants. It has also been demonstrated that FPH can stimulate plants to express high levels of bioactive compounds. Andarwulan and Shetty (1999, 2000) found that FPH from mackerel stimulated the production of phenolic compounds and rosemaric acid in oregano and phenolic compounds in anise root cultures. Vattem and Shetty (2002) later reported that FPH increased the level of phenolics in cranberry pomace. The presence of high levels of glutamic acid and proline in the FPH is thought to be a possible reason for its ability to stimulate the production of phenolic compounds (Andarwulan and Shetty, 1999). Fish protein hydrolysates can therefore find a niche market where they are used to express the production of potentially very valuable plant compounds.

10.7

Role as growth media for microorganisms

An often overlooked application of FPH is as a specialty growth media for microorganisms. Extensively hydrolyzed FPH are an excellent growth media for a variety of microorganisms (Gildberg et al., 1989; de la Broise et al., 1998; Gildberg 2003). It was reported that FPH was able to stimulate the growth of lactic acid bacteria in skim milk (Yugushi, 1984). Fish protein hydrolysates have reportedly performed better than commercial peptones, and have been a successful media in cultivating fish pathogens (Gildberg, 2003). A very recent study compared culture media with FPH from hake by-product to conventional culture media and found that the FPH media was as good as a substrate to support the growth of a variety of microorganisms (Martone et al., 2005).

10.8

Future trends

For many decades a number of devoted researchers and developers have worked tirelessly to find ways to better utilize our limited aquatic resources. The production and utilization of fish protein hydrolysates has come a long way, and has received a surge of interest in recent years after a period of relatively slow development. Future applications of FPH are now more likely to lie in specialty products and markets, and not so much in the food functionality ingredient market. Recent evidence shows that FPH have some unique properties such as antioxidative activity, and as such may find use as a natural ingredient in several food systems (likely seafood based) to improve product quality and shelf life.

Aquatic food protein hydrolysates

243

The physiological benefits demonstrated in various studies suggest FPH may find applications as a nutritional supplement, and products containing dried fish proteins and FPH are now found on the market with various health claims which require further investigation. Recent developments in the extraction and isolation of proteins from by-products or underutilized species further increase the possibility that very high grade and consistent FPH can be produced, since the starting material would consist mainly of protein. The future of FPH is bright and as more information is generated on its unique properties and how to engineer FPH with specific unique properties it is likely that FPH will find a strong market as an ingredient and supplement in the future.

10.9

References

and H B GAN. Nutritional quality of spray dried protein hydrolysate from black tilapia (Oreochrombis mossambicus). Food Chem 78: 69±74, 2002. ADLER-NISSEN J. Enzymic Hydrolysis of Food Proteins, Elsevier Applied Science Publishers, Barking, UK, 1986. AMAROWICZ R and F SHAHIDI. Antioxidant activity of peptide fractions of capelin protein hydrolysates. Food Chem 58: 355±359, 1997. ANDRAWULAN N and K SHETTY. Influence of acetyl salicylic acid in combination with fish protein hydrolysates on hyperhydricity reduction and phenolic synthesis in oregano (Origanum vulgare) tissue cultures. J Food Biochem 23: 619±635, 1999. ANDRAWULAN N and K SHETTY. Stimulation of novel phenolic metabolite, epoxy-pseudoisoeugenol-(2-methylbutyrate) (EPB), in transformed anise (Pimpinella anisum L.) root cultures by fish protein hydrolysates. Food Biotechnol 14(1±2): 1±20, 2000. ATIA M and L SHEKIB. Preparation of fish protein hydrolysate from bolti frame (Tilapia nilotica) and evaluation of its chemical composition. Assiut J Agric Sci 23: 75±87, 1992. BAEK H H and K R CADWALLADER. Enzymatic hydrolysis of crayfish processing byproducts. J Food Sci 60: 929±934, 1995. BALLESTER D, E YANEZ, O BRUNSER, A STEKEL, P CHADUD, G CASTANO and F MOCKENBERG. Safety evaluation of an enzymatic fish protein hydrolysate: 10-month feeding study and reproduction performance in rats. J Food Sci 42: 407±409, 1977. BARRIAS C and A OLIVA-TELES. The use of locally produced fish meal and other dietary manipulations in practical diets for rainbow trout Oncorhynchus mykiss (Walbaum). Aquaculture Res 31: 213±218, 2000. BECHTEL P J, S SATHIVEL, A C M OLIVEIRA, S SMILEY and J BABBITT. Properties of hydrolysates from pink salmon heads and viscera. In: Proceedings of the First Joint Trans-Atlantic Fisheries Technology Conference ± TAFT 2003. pp. 284±285. June 10±14, Reykjavik, Iceland. Published by The Icelandic Fisheries Laboratories. BENJAKUL B and M T MORRISSEY. Protein hydrolysates from Pacific whiting solid wastes. J Agric Food Chem 45: 3423±3430, 1997. BERNET F, V MONTEL, B NOEL and J P DUPOUY. Diazepam-like effects of a fish protein hydrolysate (Gabolysat PC60) on stress responsiveness of the rat pituitary-adrenal system and sympathoadrenal activity. Psychopharmacol 149: 34±40, 2000. ABDUL-HAMID A, JAMILAH-BAKAR, GAN-HOCKBEE

244

Maximising the value of marine by-products

and A GILDBERG. The stimulatory effect of a muscle protein hydrolysate from Atlantic cod, Gadus morhua L., on Atlantic salmon, Salmo salar l., head kidney leucocytes. Fish Shellfish Immunol 6: 3±16, 1996. BORDENAVE S, I FRUITIER, I BALLANDER, F SANNIER, A GILDBERG, I BATISTA and J M PIOT. HPLC preparation of fish waste hydrolysate fractions. Effec on guinea pig ileum and ACE activity. Prep Biochem Biotechnol 32: 65±77, 2002. BOUKORTT F O, A GIRARD, J PROST, D AIT-YAHIA, M BOUCHENAK and J BELLEVILLE. Fish protein improves the total antioxidant status of streptozotocin-induced diabetes in spontaneously hypertensive rat. Med Sci Monit 10: BR397±404, 2004. DE LA BROISE D, G DAUER, A GILDBERG and F GUERARD. Evidence of positive effect of peptone hydrolysis rate on Escherichia coli culture kinetics. J Mar Biotechnol 6(2): 111±115, 1998. CHAKRABARTI R. A method of debittering fish protein hydrolysate, J Food Sci Technol 20(4): 154±158, 1983. CHAN K M and E M DECKER. Endogenous skeletal muscle antioxidants. Crit Rev Food Sci Nutr 34: 403±426, 1994. CHEVALIER J and B NOEL. Food product of high nutritional efficiency. French Patent Application no. FR 2,495,442 A1, 1982. CHUANG W-L, B SUN PAN and J-S TSAI. Inhibition of lipoxygenase and blood thinning effects of mackerel protein hydrolysate. J Food Biochem 24: 333±343, 2000. CORDOVA-MURUETA J H and F L GARCIA-CARRENO. Nutritive value of squid and hydrolyzed protein supplement in shrimp feed. Aquaculture 210: 371±384, 2002. DINIZ F M and D M MARTIN. Use of response surface methodology to describe the combined effects of pH, temperature and E/S ratio on the hydrolysis of dogfish (Squalus acanthias) muscle. Int J Food Sci Tech 31: 419±426, 1996. DONG F M, W T FAIRGRIEVE, D I SKONBERG and B A RASCO. Preparation and nutrient analyses of lactic acid bacteria ensiled salmon viscera. Aquaculture 109: 351±366, 1993. DORMAN T, L BERNARD, P GLAZE, J HOGAN, R SKINNER, D NELSON, L BOWKER and D HEAD. The effectiveness of Garum armoricum (stabilium) in reducing anxiety in college students. J Adv Med 8: 193±200, 1995. EGUCHI Y, J S BELA and K SHETTY. Simulation of somatic embryogenesis in Anise (Pimpinella anisium) using fish protein hydrolysates and proline. J Herbs and Spices 5(3): 61±68, 1997. FAHMI A, S MORIMURA, H C GUO, T SHIGEMATSU, A KIDA and Y UEMURA. Production of Angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochem 39: 1195±2000, 2004. FITZGERALD A J, P S RAJ, T MARCHBANK, G W TAYLOR, S GHOSH, B W RITZ and R J PLAYFORD. Reparative properties of a commercial fish protein hydrolysate preparation. Gut 54(6): 775±781, 2005. FUJIMAKI M, S ARAI, M YAMASHITA, H KATO and M NOGUSHI. Taste peptide fractionation from a fish protein hydrolysate. Agric Biol Chem 37: 2891±2895, 1973. GILDBERG A. Enzymic processing of marine raw materials. Process Biochem 28: 1±15, 1993. GILDBERG A J. Enzymes and bioactive peptides from fish waste related to fish silage: Fish feed and fish sauce production. In: Proceedings of the First Joint Trans-Atlantic Fisheries Technology Conference ± TAFT 2003. pp. 328±331. June 10±14, Reykjavik, Iceland. Published by The Icelandic Fisheries Laboratories. GILDBERG A, I BATISTA and E STROM. Preparation and characterization of peptone obtained BOGWALD J, R A DALMO, R LEIFSON-MCQUEEN, E STENBERG

Aquatic food protein hydrolysates

245

by a two-step enzymatic hydrolysis of whole fish. Biotech Appl Biochem 11: 413± 423, 1989. GILDBERG A, J BOGWALD, A JOHANSEN and E STENBERG. Isolation of acid peptide fractions from a fish protein hydrolysate with strong stimulatory effect on Atlantic salmon (Salmo salar) head kidney leucocytes. Comp Biochem Physiol B 114(1): 97±101, 1996. HALE M B. Relative activities of commercially-available enzymes in the hydrolysis of fish proteins. Food Technol 23: 107±110, 1969. HALE M B. Making fish protein concentrate by enzymatic hydrolysis, NOAA Technical Report NMFS SSRF-675, US Department of Commerce, Seattle, WA, USA, 1±31, 1972. HOSSAIN M A, T ISHIHARA, K HARA, K OSATOMI, M A A KHAN and Y NOZAKI. Effect of proteolytic squid protein hydrolysate on the state of water and dehydration-induced denaturation of lizard fish myofibrillar proteins. J Agric Food Chem 51: 4769± 4774, 2003. HOYLE N and J H MERRITT. Quality of fish protein hydrolysates from herring (Clupea harengus). J Food Sci 59: 76±79, 1994. HULTIN H O, H G KRISTINSSON and T C LANIER. Process for Recovery of Functional Proteins by pH Shifts. In J W Park, ed. Surimi and Surimi Seafood, 2nd edn. New York: Marcel Dekker Inc. 2005. IMM J Y and C M LEE. Production of seafood flavor from red hake (Urophycis chuss) by enzymatic hydrolysis. J Agric Food Chem 47: 2360±2366, 1999. IN T. Seafood flavourants produced by enzymatic hydrolysis. In: M N Voigt and J R Botta, eds. Advances in Fisheries Technology and Biotechnology for Increased Profitability. Lancaster, PA: Technomic Publishing Co. Inc, 1990, pp. 425±436. JEON Y-J, H-G BYUN and S-E KIM. Improvement of functional properties of cod frame protein hydrolysate using ultrafiltration membranes. Process Biochem 35: 471±478, 1999. JUNG W K, E MENDIS, J Y JE, P J PARK, B W SON, H C KIM, Y K CHOI and S K KIM. Angiotensin Iconverting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneous hypertensive rats. J Food Biochem 29: 108±116, 2005. KAREL M, S R TANNENBAUM, D H WALLANCE and H MALONEY. Autoxidation of methyl linoleate in freeze-dried model systems. III. Effects of added amino acids. J Food Sci 31: 892±896, 1966. KHAN, M A A, M A HOSSAIN, K HARA, K OSATOMI, T ISHIHARA and Y NOZAKI. Effect of enzymatic fish protein hydrolysate from fish scrap on the state of water and denaturation of lizard fish (Saurida wanieso) myofibrils during dehydration. Food Sci Technol Res 9: 257±263, 2003. KIENKAS I. Biological value of protein hydrolysate of cod muscle obtained by enzymic hydrolysis and dialysis. Izvestiya Akademii Nauk Latviiskoi-SSR 7: 92±96, 1974. KOHAMA Y, H OKA, Y KAYAMORI, K TSUJIKAWA, T MIMURA, Y NAGASE and M SATAKE. Potent synthetic analogues of angiotensin-converting enzyme inhibitor derived from tuna muscle. Agric Biol Chem 55(8): 2169±2170, 1991. KRISTINSSON H G and B A RASCO. Fish protein hydrolysates: production, biochemical and functional properties. CRC Crit Rev Food Sci Nutr 32: 1±39, 2000a. KRISTINSSON H G and B A RASCO. Biochemical and functional properties of Atlantic salmon (Salmo salar) muscle proteins hydrolyzed with various alkaline proteases. J Agric Food Chem 48: 657±666, 2000b. KRISTINSSON H G and B A RASCO. Hydrolysis of salmon muscle proteins by an enzyme

246

Maximising the value of marine by-products

mixture extracted from Atlantic salmon (Salmo salar) pyloric caeca. J Food Biochem 24: 177±187, 2000c. KRISTINSSON H G and B A RASCO. Kinetics of the hydrolysis of Atlantic salmon (Salmo salar) muscle proteins by alkaline proteases and a visceral serine protease mixture. Proc Biochem 36: 131±139, 2000d. KRISTINSSON H G and B A RASCO. Fish Protein Hydrolysates and Their Potential use in the Food Industry. In M Fingerman and R Nagabhushanam, eds. Recent Advances in Marine Biotechnology Vol 7; Enfield, NH: Science Publishers, Inc., 2002, pp. 157±181. LALASIDIS G, S BOSTROM and L-B SJOBERG. Low molecular weight enzymatic fish protein hydrolysates: Chemical composition and nutritive value. J Agric Food Chem 26(3): 751±756, 1978. LE PONCIN M. Experimental study: stress and memory. Eur Neuropsychopharmacol 6: 110-P10-2, 1996a. LE PONCIN M. Nutrient presentation of cognitive and memory performances. Eur Neuropsychopharmacol 6: 187-P19-4, 1996b. LIAN P and C M LEE. Characterization of squid hydrolysates for its potential as aquaculture feed ingredient. In: Proceedings of the First Joint Trans-Atlantic Fisheries Technology Conference ± TAFT 2003, pp. 379±380. June 10±14, Reykjavik, Iceland. Published by The Icelandic Fisheries Laboratories. LIASET B, E LIED and M ESPE. Enzymatic hydrolysis of by-products from the fish-filleting industry; chemical characterisation and nutritional evaluation. J Sci Food Agric 80(5): 581±589, 2000. LICEAGA-GESUALDO A M and E C Y LI-CHAN. Functional properties of fish protein hydrolysate from herring (Clupea harengus). J Food Sci 64(6): 1000±1004, 1999. LIU C, K MORIOKA, Y ITOH and A OBATAKE. Contributions of lipid oxidation to bitterness and loss of free amino acids in the autolytic extract from fish wastes: Effective utilization of fish wastes. Fisheries Sci 66: 343±348, 2000. MAHMOUD M I, W T MALONE and C T CORDLE. Enzymatic hydrolysis of casein: effect of degree of hydrolysis on antigenicity and physical properties. J Food Sci 57(5): 1223±1229, 1992. MARTONE C B, O P BORLA and J J SANCHES. Fishery by-product as a nutrient source for bacteria and archaea growth media. Biores Technol 96(3): 383±387, 2005. MATSUMURA N, M FUJII, Y TAKEDA and T SHIMIZU. Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from bonito bowels. Biosci Biotechnol Biochem 57(10): 1743±1744, 1993. MILAZZO M C, Z ZHENG, G KELLETT, K HAYNESWORTH and K SHETTY. Stimulation of benzyladenine-induced in vitro shoot organogenesis and endogenous proline in melon (Cucumis melo L.) by fish protein hydrolysates in combination with proline analogues. J Agric Food Chem 47(4): 1771±1775, 1999. MILLER R and H S GRONINGER. Functional properties of enzyme-modified acylated fish protein derivatives. J Food Sci 41: 268±272, 1976. MORALES-DE-LEON J, A GALVEZ-MARISCAL and V TELLEZ-STILL. Preparation of fish protein isolate and hydrolyzate (Mugil cephalus) and their incorporation into Mexican foods. Arch Latinoamericanos Nutr 40: 55±68, 1990. MULLALLY M M, D M O'CALLAGHAN, R J FITZGERALD, W J DONNELLY and J P DALTON. Proteolytic and peptidolytic activities in commercial pancreatin protease preparations and their relationship to some whey protein hydrolysate characteristics. J Agric Food Chem 42: 2973±2981, 1994.

Aquatic food protein hydrolysates

247

and C DAMBMANN. Improved method for determining food protein degree of hydrolysis. J Food Sci 66(5): 642±646, 2001. NOGUCHI M, S ARAI, M YAMASHITA, H KATO and M FUJIMAKI. Isolation and identification of acidic oligopeptides in a flavor potentiating fraction from a fish protein hydrolysate. J Agric Food Chem 23(1): 49±53, 1975. OLIVA-TELES A, A L CERQUEIRA and P GONCALVES. The utilization of diets containing high levels of fish protein hydrolysate by turbot (Scophthalmus maximus) juveniles. Aquaculture 179: 195±201, 2000. ONODENALORE A C and F. SHAHIDI. Protein dispersions and hydrolysates from shark (Isurus oxyrinchus). J Aquat Food Prod Technol 5: 43±59, 1996. QUAGLIA G B and E ORBAN. Influence of the degree of hydrolysis on the solubility of the protein hydrolsyates from sardine (Sardina pilchardus). J Sci Food Agric 38: 271± 276, 1987. QUAGLIA G B and E ORBAN. Influence of enzymatic hydrolysis on structure and emulsifying properties of sardine (Sardina pilchardus) protein hydrolysates. J Food Sci 55(6): 1571±1573, 1990. RAJAPAKSE N, W K JUNG, E MENDIS, S H MOON and S K KIM. A novel anticoagulant purified from fish protein hydrolysate inhibits factor XIIa and platelet aggregation. Life Sci 76(22): 2607±2619, 2005. REFSTIE S, J J OLLI and H STANDAL. Feed intake, growth and protein utilization by postsmolt Atlantic salmon (Salmo salar) in response to graded levels of fish. Aquaculture 239(1±4): 331±349, 2004. SATHIVEL S, P J BECHTEL, J BABBITT, S SMILEY, C CRAPO, K D REPPOND and W PRINYAWIWATKUL. Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates. J Food Sci 68: 2196±2200, 2003. SHAHIDI F, X-Q HAN and J SYNOWIECKI. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem 53: 285±293, 1995. SLIZYTE R, E DAUKSAS, E FALCH and T RUSTAD. Functional properties of different fractions generated from hydrolysed cod (Gadus morhua) by-products. In: Proceedings of the First Joint Trans-Atlantic Fisheries Technology Conference ± TAFT 2003, pp. 301±303. June 10±14, Reykjavik, Iceland. Published by The Icelandic Fisheries Laboratories. SPINELLI J, B KOURY and R MILLER. Approaches to the utilization of fish for the preparation of protein isolates; enzymic modifications of myofibrillar fish proteins. J Food Sci 37: 604±608, 1972. SUGIYAMA K, M EGAWA, H ONZUKA and K OBA. Characteristics of sardine muscle hydrolysates prepared by various enzymic treatments. Nippon Suisan Gakkaishi. 57(3): 475±479, 1991. TAMURA M, N MORI, T MIYOSHI, S KOYAMA, H KOHRI and H OKAI. Practical debittering using model peptides and related compounds. Agric Biol Chem 54: 41±50, 1990. THEODORE A E. Bioactive and functional properties of catfish protein hydrolysates and catfish protein isolates. MS thesis. University of Florida, Gainesville, FL, 2005. THEODORE A E and H G KRISTINSSON. 2005. Bioactive properties of fish protein hydrolysates at varying degrees of hydrolysis made from catfish protein isolates. Annual IFT Meeting Book of Abstracts. July 16±20, New Orleans, LA. Abstract 50±5. THONGTHAI C and A GILDBERG. 2005. Asian fish sauce as a source of nutrition. In: Asian Functional Foods, New York, Marcel Dekker Inc., 215±265. UKEDA H, H MATSUDA, K OSJIMA, H MATUFUJI, T MATSUI and Y OSJIMA. Peptides from peptic hyrolysate of heated sardine meat that inhibit angiotensin I converting enzyme. NIELSEN P M, D PETERSEN

248

Maximising the value of marine by-products

Nippon Nogeikagaku Kaishi 65(8): 1223±1228, 1992. and M P RICHARDS. Aqueous extracts from some muscles inhibit hemoglobin-mediated oxidation of cod muscle membrane lipids. J Agric Food Chem 51(10): 3111±3119, 2003. VARELTZIS K, N SOULTOS, F ZETOU and F TSIARAS. Proximate composition and quality of a hamburger type product made from minced beef and fish protein concentrate. Lebensm Wiss u Technol 23(2): 112±115, 1990. VATTEM D A and K SHETTY. Solid-state production of phenolic antioxidants from cranberry pomace by Rhizopus oligosporus. Food Biotechnol 16: 189±210, 2002. VIERA G H F, A M MARTIN, S SAKER-SAMPAIAO, S OMAR and R C F GONCALVES. Studies on the enzymatic hydrolysis of Brazilian lobster (Panulirus spp.) processing wastes. J Sci Food Agric 69: 61±65, 1995. WAKO Y, S ISHIKAWA and K MURAMOTO. Angiotensin I-converting enzyme inhibitors in autolysates of squid liver and mantle muscle. Biosci Biotechnol Biochem 60(8), 1353±1355, 1996. WERGEDAHL H, B LIASET, O A GUDBRANDSEN, E LIED, M ESPE, Z MUNA, S MORK and R K BERGE. Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA: Cholesterol acyltransferase activity in liver of Zucker rats. J Nutr 134(66): 1320±1327, 2004. WU H, H CHEN and C SHIAU. Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Res Int 36: 949±957, 2003. YANEZ E, D BALLESTER and F MONCKEBERG. Enzymatic fish protein hydrolysate: chemical composition, nutritive value and use as a supplement to cereal protein. J Food Sci 41: 1289±1292, 1976. YU S Y and L K TAN. Acceptability of crackers (`Keropok') with fish protein hydrolsyates. Int J Food Sci Technol 25(2): 204±210, 1990. YU S Y and S FAZIDAH. Enzymic hydrolysis of proteins from Aristichthys noblis by protease P`Amano'3. Trop Sci 34: 381±391, 1994. YUGUSHI H. Studies on utilization of the fish meat hydrolysate for fermented milk products. VII. Correlation of the chemical composition of the fish meat hydrolyate with the stimulatory activity for the growth of lactic acid bacteria and for the decrease of curd tension of fermented milk products. Japanese J Dairy Food Sci 33: A81±A91, 1984. ZHANG H, Y YAMASHITA and Y NOZAKI. Effect of protein hydrolysate from Antarctic krill meat on the state of water and denaturation by dehydration of lizard fish myofibrils. Fish Sci 68: 672±679, 2002. UNDELAND I, H O HULTIN

11 Engineering and functional properties of powders from underutilized marine fish and seafood products S. Sathivel and P. J. Bechtel, University of Alaska Fairbanks, USA

11.1

Introduction

In Japan and many other Asian countries there is much interest in marinederived ingredients; however, there is some demand for marine-derived functional ingredients in North America. The projected functional food market for North American is anticipated to grow by more than 20% per year for the next several years (Barrow, 2005). In recent years, by-products from fishing industries have been used as raw materials to produce some common bioactive supplies for supplements or functional ingredients. The functional food market in North American is currently valued at more than US$30 billion; however, marine-derived ingredients account for only a small portion of this total. Marine by-products that have potential as functional food ingredients include protein hydrolysates from fish processing waste and underutilized fish species (Barrow, 2005). Large amounts of protein-rich by-products from the seafood industry are discarded or processed into fish meal. Novel processing methods are needed to convert seafood by-commodities into marketable products. Many of these protein-rich seafood by-products have a range of dynamic properties (Phillips et al., 1994) and can potentially be used in foods as binders and emulsifiers. Soy and milk proteins are widely used in many segments of the food industry, while amino acids and peptides are gaining popularity for use in energy drinks and other applications (O'Donnell and Dornblaser, 2002). Proteins from fish processing by-products can be modified to improve their quality and functional characteristics using enzymatic hydrolysis (Shahidi,

250

Maximising the value of marine by-products

1994). Utilizing proteolytic enzymes, fish protein hydrolysates (FPHs) can be prepared with peptides having new and/or improved properties. Functional properties can be defined as physical and chemical properties, which affect the behavior of proteins in food systems during processing, storage, preparation, and consumption (Kinsella, 1976). Functional properties of fish proteins are related to their physical, chemical, and conformational properties. This chapter will discuss engineering and functional properties of proteins and hydrolysates made from marine fish and their by-products.

11.2

Fish protein powder as bio-active ingredients

A wide range of food products including protein supplements, infant formula, formulas for the elderly and beverages contain protein hydrolysates as stabilizers (Frokjaer, 1994; Kristinsson and Rasco, 2000). Some fish protein and protein hydrolysates in addition to having good functional properties may also have specific health benefits, including the ability to lower blood pressure, reduce the risk of type-II diabetes (McCarty, 2003), and improve glucose tolerance and insulin sensitivity (Lavigne et al., 2000). In Japan, sardine protein hydrolysate is widely available in supplements. Sardine protein hydrolysate incorporated at a dosage of 0.5 g into a vegetable drink was shown to lower systolic blood pressure (Kawasaki et al., 2002). Protein hydrolysates derived from non-marine food products such as soy and whey reduce blood pressure; however, marinederived hydrolysates may be effective at lower doses. Hydrolysates from fish can be used in a number of food applications such as enhancing water binding, and hydrolysates made from caplin and peptide were shown by Shahidi et al. (1995) to inhibit oxidation, and peptides derived from fish can also been shown to have antioxidant properties (Amarowicz and Shahidi, 1997; Shahidi and Amarowicz, 1996). Antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been commonly used by the food industry to improve product quality during storage, resulting in increased product shelf life. To meet consumer demands for safer foods, numerous studies are currently focused on using natural ingredients to enhance food quality and shelf life in order to avoid the use of synthetic preservatives. Sathivel et al. (2003) reported that fish protein hydrolysates had antioxidant properties. Arrowtooth flounder protein that was partially hydrolyzed by endogenous enzymes was used to coat salmon fillets and resulted in reduced lipid oxidation when compared to non-coated controls (Sathivel, 2005).

11.3

Functional properties of fish protein powders

Amino acids and peptides are finding more uses in energy drinks and other applications (O'Donnell and Dornblaser, 2002), and soy and milk protein, unlike

Engineering and functional properties of powders from seafood products

251

fish protein, are widely used in many segments of the food industry. Fish byproducts from coldwater marine species are good sources of high quality proteins and there is an opportunity to use more fish processing by-products as protein sources for food and feed ingredients and industrial applications. In 1993, the Association of Danish Fish Processing Industries and Exporters commercially produced fish-based protein powders for use in frozen products to enhance water binding and stability properties when frozen (Urch, 2001). Phillips et al. (1994) reported that protein-rich seafood by-products had a range of dynamic properties and could potentially be used in foods as binders and emulsifiers. Sathivel et al. (2004) reported that protein powders from herring and arrowtooth flounder were good sources of high quality fish protein with many desirable functional properties. Solubility is one of the most important properties of proteins (Kinsella, 1976; Mahmoud et al., 1992). The nitrogen solubility values of hydrolysates from red salmon ranged from 17.2% to 54.4% (Sathivel et al., 2005b) and solubility of the soluble fraction of herring hydrolysates was 84.9% (Sathivel et al., 2003). High solubility of fish protein hydrolysates is often due to cleavage of proteins into smaller peptide units that usually have increased solubility (Shahidi, 1994), which is mainly due to a reduction in the molecular weight and an increase in the number of polar groups. Increased solubility is not only due to smaller peptide size but also to the balance of hydrophilic and hydrophobic elements in the peptides. An emulsion is defined as a heterogeneous system consisting of at least two immiscible liquid phases, one of which is dispersed in the other in the form of droplets (Das and Kinsella, 1990). Gauthier et al. (1993) and Jost et al. (1977) examined the role of peptide characteristics on emulsification properties. They reported that hydrophobicity and peptide lengths influenced the emulsifying properties. Peptides often have reduced emulsifying properties (Chobert et al., 1988). A positive correlation between surface activity and peptide length was reported by Jost et al. (1977), and Lee et al. (1987) reported that peptides should have a minimum length of 20 residues to possess good emulsifying and interfacial properties. Sathivel et al. (2003) reported that emulsion stability of protein powders was similar to a soy protein concentrate. Emulsifying stability of red salmon head hydrolysates ranged from 66.9±100% (Sathivel et al., 2005b). Emulsifying stability values of 52 to 61.0% and 48.5 to 54.2% were reported for Atlantic salmon protein hydrolysates (Kristinsson and Rasco, 2000) and for herring protein hydrolysates (Sathivel et al., 2003), respectively.

11.4 Flow properties analysis of emulsion containing fish protein powders Proteins extracted from fish are good sources of high quality proteins, have a range of dynamic properties, and can potentially be used in foods as emulsifiers (Sathivel et al., 2004). Sathivel et al. (2005a) reported that proteins extracted

252

Maximising the value of marine by-products

from arrowtooth flounder provide desirable emulsifying properties in the emulsion system that exhibits pseudoplastic and viscoelastic characteristics. It may be possible to substitute egg yolk with fish protein powders in an oil-inwater emulsion system. The power law (Eq. 11.1) and the Casson equation (Eq. 11.2) can be used to analyze the flow properties of the emulsion containing fish protein.  ˆ K n

…11:1†

where  ˆ shear stress (Pa.s), ˆ shear rate (sÿ1), K ˆ consistency index (Pa.sn), and n ˆ flow behavior index. The logarithms are taken on both sides of Equation 11.1, and a plot of log  versus log is constructed, and the magnitude of K and n are determined from the resulting straight line intercepts for log K and the slope for n values. 0:5 ˆ o ‡ K1 0:5

…11:2†

where K = constant and o ˆ yield stress (Pa). The square root of shear stress is plotted against the square root of shear rate and o and K1 are obtained from the square of the intercept and the slope of the straight line, respectively. The flow parameters of the flow index (n), consistency index (K), and the Casson yield stress values of emulsion containing arrowtooth protein powders are shown in Table 11.1. The flow index values for all mayonnaise samples were less than 1.0, which indicated that they were pseudoplastic fluids (Paredes et al., 1989). Values for n of 0.13 to 0.91 have been reported for some commercial mayonnaises and model mayonnaise systems (Dickie and Kokini, 1983; Steffe, 1992). Higher K values of emulsion samples indicate a more viscous consistency (Paredes et al., 1989). Values obtained for emulsions made with arrowtooth flounder protein powder were within the range of yield stress values (9 to 91 Pa) that Steffe (1992) reported for commercial mayonnaises. The wide range of n, K, and o values reported by Dickie and Kokini (1983) and Steffe (1992) was due in part to different methods or use of different shear rate ranges. Table 11.1 Flow parameters of mayonnaises containing fish protein powders Samples AFSPE1 AFSPE2 AFISPE3 AFISPE4

n 0.5 0.6 0.9 0.8

   

K (Pa.s) 0.0 0.02 0.02 0.01

5.6 4.2 0.2 0.3

   

0.8 0.4 0.01 0.4

o (Pa) 40.5 30.5 0.7 1.6

   

1.0 1.8 0.3 0.1

n ˆ flow index; K ˆ consistency index; o ˆ yield stress. AFSPE1 contained 5% AFSP, 1% lemon juice, and 60.35% soybean oil. AFSPE2 contained 5% AFSP, 3% lemon juice, and 58.35% soybean oil. AFISPE3 contained 5% AFISP, 1% lemon juice, and 60.35% soybean oil. AFISPE4 contained 5% AFISP, 3% lemon juice, and 58.35% soybean oil. AFSP ˆ arrowtooth flounder soluble protein powder: AFISP ˆ arrowtooth flounder insoluble protein power. Data from Sathivel et al. (2005a).

Engineering and functional properties of powders from seafood products

253

11.5 Viscoelastic properties of emulsions containing fish protein powders Dynamic rheological tests can be used to characterize viscoelastic properties of emulsions. Equations (11.3) and (11.4) can be used to define viscoelastic behavior:   o 0 cos  …11:3† G ˆ

o   o sin  …11:4† G00 ˆ

o where G0 is the storage modulus, G00 (Pa) is the loss modulus, and tan  is the loss tangent. The storage modulus, G0 , characterizes the rigidity of the sample and can be viewed as the magnitude of the energy that is stored in the material per cycle of deformation. The loss modulus, G00 , characterizes the resistance of the sample to flow, and is a measure of the energy that is lost through viscous dissipation per cycle of deformation. For a perfectly elastic solid, all the energy is stored; that is, G00 is zero and the stress and the strain will be in phase. In contrast, for a liquid with no elastic properties, all the energy is dissipated as heat; that is G0 is zero and the stress and strain will be out of phase by 90ë (Rao, 1999). Dynamic rheological tests can be used to characterize viscoelastic properties of emulsions. The G0 and G00 of the emulsion samples containing arrowtooth flounder soluble protein powder and arrowtooth flounder insoluble protein powder were determined as a function of frequency (!) at a fixed temperature of 25ëC (Fig. 11.1).

Fig. 11.1 Rheology properties of emulsions containing arrowtooth flounder protein powers. G0 (Pa) and G00 (Pa) indicate storage and loss modulus, respectively. Panel a contained arrowtooth flounder soluble protein powder (AFSPE1); panel b contained insoluble arrowtooth protein powder (AFISPE4). Data from Sathivel et al. (2005b).

254

Maximising the value of marine by-products

Emulsion containing arrowtooth flounder soluble protein powder showed a gradual increase in both the loss modulus and the storage modulus and with increasing frequency. For emulsion made with arrowtooth flounder insoluble protein powder G0 crosses G00 as frequency increased. This behavior is typical of macromolecular solution where the polymer molecules are mutually entangled. At low frequency increases there is sufficient time for the entanglements to make and break. A high viscosity occurred in the emulsion system due to entanglement but there are no intermolecular cross-linkages (Oakenfull et al., 1997). Emulsions made with arrowtooth flounder soluble protein powder had higher G0 than G00 , which indicate a viscoelastic material with both G0 and G00 being independent of frequency. The higher G0 and G00 values of emulsion made with arrowtooth flounder soluble protein when compared to those made with arrowtooth flounder insoluble protein may be due to the soluble protein content that increases interactions between the neighboring droplets.

11.6

Thermal properties of fish protein powders

During extraction and preparation process, fish protein powders are subjected to temperature changes, which may alter their physical state. The most commonly occurring phase transition in protein is denaturation, which can alter the properties of protein and thus the quality of final products. DSC thermograms of the fish protein powder samples are shown in Fig. 11.2. The DSC thermogram for (herring body protein powder) HBP and WHP (whole herring protein powder) show a single small endothermic transition with total enthalpy,
Ht

Fig. 11.2 DSC thermograms of fish protein powders. Data from Sathivel et al. (2004).

Engineering and functional properties of powders from seafood products

255

values of 1.58 and 0.76 j/g. HGP (herring gonad protein powder), HHP (herring head protein powder), and APP (ˆ arrowtooth flounder protein powder) did not show the sharp peaks. Although the results indicate a small transition in HBP and WHP, all protein powders were subjected to a high degree of denaturation that may have occurred during fish protein powder preparation. Knowledge of thermal decomposition of the fish protein samples can be used to improve their stability and functional properties. A thermal gravimetric (TG) analyzer is a balance, which measures changes in weight as a function of changing temperatures. A series of the TG thermogram of the fish protein powder samples is shown in Fig. 11.3. Weight loss of FPP samples increased with increasing heating temperatures between 0 and 600ëC and the mass losses were slightly different among the protein powders. Four weight-loss temperature regions were identified for a series of protein powders made from herring and herring by-products. The TG curves indicated the thermal stability in the following order: HGP > HBP > WHP > HHP > AFP. Differences in thermal stability may be due to the presence of components that interact with protein in powders such as phospholipids, complexed metals (notably iron, calcium, and magnesium minerals), free fatty acids, and peroxides and their breakdown products. The presence of those components reduces the effectiveness of heat transfer to protein powders, and thus the mass losses of protein powders.

Fig. 11.3 TG thermograms showing weight loss curve for fish protein powders. WHP = whole herring protein powder; HBP = herring body protein powder; HHP = herring head protein powder; HGP = herring gonad protein powder; AFP = arrowtooth flounder protein powder. Data from Sathivel et al. (2004).

256

11.7

Maximising the value of marine by-products

Future trends

The functional food market for North America has been projected to grow by more than 20% per year over the next several years. Protein powders and hydrolysates made from fillets of underutilized species such as arrowtooth flounder, or protein derived from fish processing by-products have good functional properties that can be used as ingredients in both animal and human foods. Protein hydrolysates and protein powder derived from marine fish can expect to get into the North American functional food market in the next decade. In addition to fish protein the small amounts of marine oils found in most of these products are high in omega-3 fatty acids. The public is continuing to gain a positive nutritional image of products containing the long chain omega-3 fatty acids. However, to enhance utilization of fish protein hydrolysates and protein powders, progress in processing and formulation will be needed to enhance sensory, functional and nutritional properties and consumer education and awareness will also be addressed.

11.8

References

and SHAHIDI F (1997), `Antioxidant activity of peptide fractions of capelin protein hydrolysates', Food Chem, 58, 355±359. BARROW C (2005), `Marine by-products as functional food ingredients', Food Tech International, http://www.foodtech-international.com/index.htm CHOBERT J M, BERTRAND H C and NICOLAS M G (1988), `Solubility and emulsifying properties of caseins and whey proteins modified enzymatically by trypsin', J Agric Food Chem, 36, 883±889. DAS K P and KINSELLA J E (1990), `Stability of food emulsions: physicochemical role of protein and nonprotein emulsifiers', Res Adv Food Nutr, 43, 81. DICKIE A M and KOKINI J L (1983), `An improvement method for food thickness from nonNewtonian fluid mechanics in the mouth', J Food Sci, 48, 57±61, 65. FROKJAER S (1994), `Use of hydrolysates for protein supplementation', Food Technol, 48, 86±88. GAUTHIER S F, PAQUIN P, POULIOT Y and TURGEON S (1993), `Surface activity and related functional properties of peptides obtained from whey protein', J Dairy Sci, 76, 321±328. JOST R, MONTI J C and PAHUD J J (1977), `Partial enzymatic hydrolysis of whey protein by trypsin', J Dairy Sci, 60, 1387±1393. KAWASAKI T, JUN C J, FUKUSHIMA Y, KEGAI K, SEKI E, OSAJIMA K, ITOH K, MATSUI T and MATSUMOTO K (2002), `Antihypertensive effect and safety evaluation of vegetable drink with peptides derived from sardine protein hydroysates on mild hypertensive, high-normal and normal blood pressure subjects', Zasshi Fukuoka Igaku, 93(10), 208±218. KINSELLA J E (1976), `Functional properties of proteins in foods: a survey', CRC Crit Rev Food Sci Nutr, 8, 219±280. KRISTINSSON H G and RASCO B A (2000), `Biochemical and functional properties of Atlantic salmon (Salmo salar) muscle proteins hydrolyzed with various alkaline AMAROWICZ R

Engineering and functional properties of powders from seafood products

257

proteases', J Agric Food Chem, 48, 657±666. and JACQUES H (2000), `Cod and soy proteins compared with casein improve glucose tolerance and insulin sensitivity in rats', Am J Physiol Endocrinol Metab, 278: E491±E500. LEE S W, SHIMIZU M, KAMINOGAWA S and YAMAGUCHI K (1987), `Emulsifying properties of a mixture of peptides derived from the enzymatic hydrolysates of -casein', Agric Biol Chem, 51: 161±165. MAHMOUD M I, MALONE W T and CORDLE C T (1992), `Enzymatic hydrolysis of casein: effect of degree of hydrolysis on antigenicity and physical properties', J Food Sci, 57, 223±229. MCCARTY M F (2003), `ACE inhibition may decrease diabetes risk by boosting the impact of bradykinin on adipocytes', Medical Hypothesis, 60(6), 779±783. OAKENFULL D, PEARCE J and BURLEY R W (1997), `Protein Gelation' in Damodaran S and Paraf A, Food proteins and their applications, Marcel Dekker, Inc, New York. O'DONNELL C D and DORNBLASER (2002), `Amino acids/peptides', Prepared Foods, 117, 72±73. PAREDES M D C, RAO M A and BOURNE M (1989), `Rheological characterization of salad dressing. 2. Effect of storage', J Text Studies, 20, 235±250. PHILLIPS L G, WHITEHEAD D M and KINSELLA J (1994), Structure function properties of food proteins; Academic Press, San Diego, CA, USA. RAO M A (1999), Rheological of fluids and semisolids. Principal and applications, Aspen Publishers Inc, Gaitherburg, MD, USA. SATHIVEL S (2005), `Chitosan and protein coatings affect yield, moisture loss and lipid oxidation of pink salmon (Oncorhynchus gorbuscha) fillets during frozen storage', J Food Sci, 70, 455±459. SATHIVEL S, BECHTEL P J, BABBITT J, SMILEY S, CRAPO C, REPPOND K D and PRINYAWIWATKUL W (2003), `Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates', J Food Sci, 68, 2196±2200. SATHIVEL S, BECHTEL P J, BABBITT J, PRINYAWIWATKUL W, NEGULESCU I I and REPPOND K D (2004), `Properties of protein powders from arrowtooth flounder (Atheresthes stomias) and herring (Clupea harengus) byproduct', J Agric Food Chem, 52, 5040±5046. SATHIVEL S, BECHTEL P J, BABBITT J, PRINYAWIWATKUL W and PATTERSON M (2005a), `Functional, nutritional, and rheological properties of protein powders from arrowtooth flounder and their application in mayonnaise', J Food Sci, 70, 57±63. SATHIVEL S, SMILEY S, PRINYAWIWATKUL W and BECHTEL P J (2005b), `Functional and nutritional properties of red salmon (Oncorhynchus nerka) enzymatic hydrolysates', J Food Sci, 70, C401±C406. SHAHIDI F (1994), `Seafood processing byproducts', in Shahidi F and Botta J R, Seafoods: Chemistry, Processing Technology, and Quality, Blackie Academic & Professional, London, 321±334. SHAHIDI F and AMAROWICZ R (1996), `Antioxidant activity of protein hydrolysates from aquatic species', J Am Oil Chem Soc, 73, 1197±1199. SHAHIDI F, HAN X and SYNOWIECKI J (1995), `Production and characteristics of protein hydrolysates from caplin (Mallotus villosus)', Food Chem, 53, 285±293. STEFFE J F (1992), `Yield stress: Phenomena and measurement', in Singh R B and Wirakaratakusumah M A, Advances in Food Engineering, CRC Press, London. URCH S (2001), `Danish fish protein', Denmark in Depth Seafood International, 12, 35. LAVIGNE C, MARETTE A

12 Marine oils from seafood waste F. Shahidi, Memorial University of Newfoundland, Canada

12.1

Introduction

Marine oils originate primarily from the body of fatty fish, the liver of white lean fish, and the blubber of marine mammals such as seal. The main sources of fish oils are pelagic species caught in large quantities, particularly those with oily flesh, such as salmon, tuna, mackerel and herring or small fish such as anchovies and capelin. The oily flesh is often used for the purpose of fish meal and oil production, but fish oil can also be produced from offal from the fish processing industry since there is a sizable and growing world market demand for highquality fish oils. Thus, commercial fish oil production can be quite profitable if suitable raw materials are available (Bonnet et al., 1974). Marine oils provide for excellent sources of long-chain omega-3 fatty acids (Table 12.1). Both omega-3 and omega-6 fatty acids are essential polyunsaturated fatty acids (PUFA) that cannot be made in the human body (Din et al., 2004). The Western diet is abundant in omega-6 fatty acids, mainly from vegetable oils rich in linolenic acid (C18:2n-6). However, humans lack the necessary enzymes to convert omega-6 fatty acids to their omega-3 counterparts, and the latter must be obtained from separate dietary sources (Hulshof et al., 1999). The n-3 PUFA mainly include the essential fatty acid linolenic acid (ALA, C18:3n-3) and its long-chain metabolites eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) (Fig. 12.1). ALA is available from certain plants such as the seeds and oils of flax or linseed, and to a lesser extent perilla, soybean and canola (Newton and Snyder, 1997; Kamal-Eldin and Yanishlievab, 2002). EPA and DHA, however, are derived marine products (i.e.

Marine oils from seafood waste

259

Table 12.1 Dietary sources of various omega-3 polyunsaturated fatty acids (% of total fatty acids) Omega-3 Alpha-linolenic acid (18:3) Eicosapentaenoic acid (20:5) Docosahexaenoic acid (22:6) Freshwater fish (1±6%) Marine fish (~1%) Linseed (45±60%) Green leaves (56%) Rapeseed (10±11%)

Freshwater fish (5±13%) Pacific anchovy (18%) Capelin (codfish) (9%) Mackerel (8%) Herring (3±5%) Sardine (3%)

Freshwater fish (1±5%) Pacific anchovy (11%) Capelin (codfish) (3%) Mackerel (8%) Herring (2±3%) Sardine (9±13%)

Source: Newton and Snyder (1997).

fish and shellfish and algal species) (Newton and Snyder, 1997; (NDA) Scientific Panel on Dietetic Products, Nutrition and Allergies, 2005; Watanabe and Ackman, 1974; Ackman 1982; Myher et al., 1996; Mayzaud et al., 1999; Tanabe et al., 1999; Arts et al., 2001; Oliveira and Bechtel, 2005), but DPA is less abundant and found in less than 1% in most fish oils. Humans can synthesize, up to approximately 5%, EPA and DHA, through desaturation and elongation, from dietary ALA (Aliam, 2003). This pathway is an important source of these long-chain n-3 PUFA in strict vegetarians, who do not consume fish. Non-vegetarians can also obtain PUFA from a variety of food products (Gebhardt and Thomas, 2002). Table 12.2 shows fatty acid composition of lipid from several marine organisms. The main components of marine lipids are monounsaturated fatty

Fig. 12.1 The omega-3 fatty acid family.

260

Maximising the value of marine by-products

Table 12.2 Distribution of selected FA (% wet weight) in various marine organisms Component

14:0 C16:0 C18:0 SFA C16:1 C18:1 C20:1 C22:1 MUFA C18:2n-6 C18:3n-3 C18:4n-3 C20:4n-6 C20:5n-3 C22:5n-3 C22:6n-3 PUFA Other Component

14:0 C16:0 C18:0 SFA C16:1 C18:1 C20:1 C22:1 MUFA C18:3n-3 C18:4n-3 C20:5n-3 C22:5n-3 C22:6n-3 C18:2n-6 C20:4n-6 PUFA Other 1

Atlantic menhaden oil1

Common oyster (Crassostrea virginica)2

Northern krill (Meganyctiphanes norvegica)3

Atlantic salmon (Salmo gairdneri)4

7.3 19.0 4.2 30.5 9.1 13.2 2.0 0.6 24.9 1.3 1.3 2.8 0.2 11.0 1.9 9.1 27.6 17.0

3.5 25.9 3.6 36.0 4.2 8.2 5.0 0.3 17.7 2.0 3.3 2.6 2.3 11.2 Ð 9.7 31.1 15.2

4.6 18.4 2.2 25.2 3.5 14.7 2.3 0.9 21.4 1.8 1.3 3.1 1.0 10.8 1.0 28.6 45.8 7.6

5.5 10.2 2.7 18.4 8.1 16.9 15.1 14.4 54.5 4.5 0.9 1.7 0.6 6.2 1.8 9.1 24.8 2.3

Sardine (Sardine pilchardus)5

Anchovy (Engraulis encrasicholus)6

Cod (Gadus morhua)7

Skipjack tuna8

5.7 17.8 3.1 27.2 6.8 7.8 Ð 5.1 19.7 1.9 Ð 8.4 Ð 15.5 2.6 Ð 28.4 25.0

10.2 23.5 3.4 40.4 10.6 10.3 5.0 0.7 28.4 0.8 Ð 8.2 0.9 15.2 1.6 0.7 29.4 1.8

0.8 17.2 4.0 22.2 3.0 13.4 0.4 0.0 16.9 0.4 0.6 16.5 0.9 36.9 1.3 3.0 61.0 Ð

8.2 26.5 11.9 38.4 8.2 6.8 0.5 Ð 7.3 0.0 1.3 11.1 Ð 29.1 1.1 3.1 45.7 Ð

Ackman (1982); 2 Watanabe and Ackman (1974); 3 Mayzaud et al. (1999); 4 Oliveira and Bechtel (2005); 5 Newton and Snyder (1997); 6 Kalogeropoulos et al. (2004); 7 Hyvonen and Koivistoinen (1994); 8 Tanabe et al. (1999).

Marine oils from seafood waste

261

acids along with PUFA and some saturated fatty acids that are present in different proportions (Watanabe and Ackman, 1974; Ackman 1982; Myher et al., 1996; Mayzaud et al., 1999; Tanabe et al., 1999; Arts et al., 2001; Oliveira and Bechtel, 2005). Among these, the ratio of DHA to EPA and possible presence of DPA in modest amounts are most important (Shahidi, 2002). Although EPA and DHA are found abundantly in different marine oils, DPA is present in significant amounts only in seal blubber oil (Wanasundara et al., 1998; Aidos et al., 2002; Sathivel et al., 2003; Jayasinghe et al., 2003). The spatial distribution of fatty acids in triacylglycerols in fish and marine mammal oils differ in that fish oils contain long-chain PUFA mainly in the sn-2 position of triacylglycerols whereas marine mammal lipids have them predominantly in the sn-1 and sn-3 positions. These factors greatly influence the metabolism and potential health benefits of marine lipids (Shahidi, 1998).

12.2

Oil from fish processing by-products

The fish industry produces a considerable amount of by-catch as well as fish species that are primarily harvested for the fish meal industry with oil being a by-product. One example is menhaden oil. Menhaden (Brevoortin tyrannus) is a filter feeder fish that is available near the shores of Atlantic coast of the United States and Gulf of Mexico. Menhaden is used for production of meal for aquaculture and land-based animal feed. Menhaden oil, a by-product of menhaden fish meal industry, contains 18% EPA and nearly 10% DHA. The major fatty acids of menhaden oil are given in Table 12.3. Capelin (Mytilus edulis) is a small marine fish that is also widely processed for fish meal as well as whole body oil. It is a major prey species that is often used as a bait in some fisheries. Capelin contains 7±10% oil, mainly triacylglycerols and about 20% PUFA as shown in Table 12.3. The oil from capelin is used in aquaculture feed formulation as well as other applications. By-products from gutting, filleting, and other processing operations are good raw materials for fish meal and oil production. One can obtain oil from different parts of fish with diverse nutritional composition. Composition, lipid content and fatty acid profile of individual by-products is of increasing importance, as different by-products are being segregated and used for different end products. Shark liver is the principal site of lipid storage. The oil content and fatty acid composition in shark liver are influenced by the gender, season and species of shark. Table 12.4 presents the fatty acid profile of liver oil from male and female blue shark. In addition to fatty acids, shark liver oil contains high amounts of squalene, low-density lipids (diacylglyceryl ethers) and vitamin A (Jayasinghe et al., 2003). In recent years, scientific evidence has emerged in support of the therapeutic value of shark products, particularly shark fins, cartilage, and liver oil as a good source of n-3 PUFA. At present, in

262

Maximising the value of marine by-products Table 12.3 Major fatty acids of menhaden oil1 and capelin oil2 Fatty acid

Menhaden (%)

Capelin (%)

14:0 15:0 16:0 16:1 16:2 n-7 16:2 n-4 16:3 n-4 16:4 n-1 17:0 18:0 18:1 18:2 n-6 18:2 n-4 18-3 n-3 18:4 n-3 20:1 20:2 n-6 20:3 n-3 20:4 n-3 20:5 n-3 21:5 n-3 22:1 22:4 n-3 22:5 n-3 22:6 n-3

7.30 0.65 19.45 9.05 0.50 1.55 1.70 2.60 1.05 4.45 10.40 1.30 0.50 0.65 2.65 1.45 0.30 1.00 0.80 18.30 0.90 1.55 0.60 1.80 9.60

5.9 0.2 8.7 10.5 Ð Ð 0.8 2.0 Ð 0.6 6.0 0.5 0.1 0.2 1.2 17.6 Ð Ð Ð 9.3 Ð 27.8 Ð 0.7 4.1

1

Ackman (2005);

2

Copeman and Parrish (2004).

the market cod liver oil is dominant and it contains high levels of vitamins A and D (Engelhardt and Walker, 1974). Here it should be noted that cod as well as halibut liver oils, although rich in n-3 PUFA, are used primarily as a source of vitamins A and D. Salmon deposits oil mainly in its head, which contains approximately 15± 18% lipids. However, salmon oils can also be produced from viscera, whole fish (down-graded) and filleting by-products (heads, trimmings such as belly-flaps, as well as skin, and frame bones). Significant differences were found in the lipid and fatty acid content and composition of salmon by-products. Pink salmon heads had the highest lipid content and viscera the lowest. The n-3/n-6 ratios for pink salmon samples ranged from 7.7 to 10.5 and only viscera values were statistically different (Wanasundara et al., 1998; Aidos et al., 2002; Sathivel et al., 2003; Jayasinghe et al., 2003). The fatty acid composition of salmon oil depends on the composition of the raw material used (Table 12.5). The average content of n-3 PUFA in salmon oil is in the medium range compared with other

Marine oils from seafood waste

263

Table 12.4 Fatty acid composition in total lipids from Blue shark (P. glauca) liver (w/w %)1 Fatty acid

14:0 16:0 18:0 Total SFA 16:1 18:1 20:1 22:1 24:1 Total MUFA 18:2n-6 18:3 18:4n-3 20:5n-3 22:5n-3 22:6n-3 Total PUFA 1

Blue shark liver Male

Female

2.3 22.0 4.9 36.0 3.9 16.0 3.0 0.0 0.8 23.6 0.8 0.2 0.4 4.5 2.1 23.2 39.2

2.8 16.8 3.9 30.3 5.3 27.6 4.9 0.0 0.9 38.7 0.7 0.2 0.4 2.7 2.5 18.4 30.2

Jayasinghe et al. (2003).

fish oils. Even though the by-products from most other fish-processing industries tend to vary with season, both in quality and quantity, salmon slaughterhouses generate high-quality offal at a relatively constant rate (Park et al., 2004; Rora et al., 2005). The by-products of catfish processing consist of heads, frames, skin, and viscera, which often end up in landfills or rendering plants. Producing edible oil from viscera may add value to catfish viscera. The total unsaturated fatty acids in the purified oil from catfish viscera was 67.7% (Table 12.5). The combined n3 fatty acids of the purified catfish viscera oil was only 4.6 mg/g of oil (Sathivel et al., 2003). Herring oil is produced from three different types of by-products, only heads, mixed, and headless by-products are of interest (Sathivel et al., 2003). Even though by-products from heads and their oil have the highest oxidation levels and the lowest -tocopherol content, heads contain the lowest PUFA and the highest amount of saturated fatty acids (Table 12.6). No significant differences were found between the fatty acid composition of the mixed and the headless by-products or their oil (Aidos et al., 2002).

Table 12.5 Fatty acid profile of different by-product oil from Alaska pink salmon (Oncorhynchus gorbuscha), catfish (Ictalurus punctatus), cod (Gadus morhua) and of their respective whole fish oil (w/w %) Alaska pink salmon1

Fatty acid

14:0 16:0 18:0 Total SFA 16:1 18:1 20:1 22:1 24:1 Total MUFA 18:2n-6 18:3n-3 18:4n-3 20:5n-3 22:5n-3 22:6n-3 Total PUFA

Catfish2

Cod3

Whole fish

Head

Viscera

Whole wild fish

Viscera

Whole fish

Liver (crude)

Liver (RBD)

3.6 12.04 3.25 22.49 3.66 16.04 9.66 9.05 0.77 39.18 1.73 1.34 2.71 7.70 2.75 15.78 32.53

4.12 12.39 2.87 19.38 3.96 14.75 10.68 11.43 0.91 41.73 1.67 1.27 2.95 7.56 2.33 11.77 31.38

3.44 12.8 4.45 20.69 3.82 16.83 6.36 6.82 0.00 33.83 1.38 1.16 1.32 10.93 2.83 17.32 40.35

3.04 20.77 4.30 33.68 7.38 22.45 1.14 Ð Ð 32.68 0.27 0.51 4.02 0.73 3.69 4.65 26.00

3.45 12.91 15.75 32.90 10.56 21.62 6.26 0.00 0.00 38.44 20.30 3.30 0.00 0.00 Ð 1.18 28.62

2.60 16.85 7.31 28.75 2.99 14.18 3.13 0.09 Ð 20.97 0.24 Ð 0.17 8.32 1.57 28.59 46.93

2.01 9.42 3.33 15.28 6.19 22.13 11.3 7.81 0.00 48.06 0.81 0.57 0.65 13.9 1.43 16.9 34.66

3.33 11.1 3.89 19.17 7.85 21.16 10.4 9.07 0.00 49.07 0.74 0.46 0.61 11.2 1.14 14.8 29.29

RBD refers to crude and refined-bleached and deodorized (RBD) oils. 1 Oliveira and Bechtel (2005); 2 Bonnet et al. (1974); Sathivel et al. (2003);

3

Bonnet et al. (1974); Wanasundara et al. (1998).

Marine oils from seafood waste

265

Table 12.6 Fatty acid profile in extracted oils from different by-products of herring (Clupea harengus) and of whole body herring oil (w/w %)1 Fatty acid

Herring heads

Herring headless

Herring mixed

Whole fish oil

9.4 14.5 2.3 26.2 5.6 7.1 9.9 17.8 0.0 40.4 1.4 1.4 3.0 8.7 Ð 8.0 22.5

8.8 13.8 2.0 24.6 5.4 7.2 10.3 17.7 0.0 40.6 1.3 1.4 3.2 8.8 Ð 8.1 22.8

9.0 14.2 2.2 25.4 5.5 7.5 10.3 17.9 0.0 41.2 1.2 1.2 3.2 9.0 Ð 8.5 23.1

5.04 19.92 1.52 28.19 6.36 26.15 1.29 0.71 1.39 36.39 5.10 2.52 1.49 7.33 0.55 15.56 35.42

14:0 16:0 18:0 Total SFA 16:1 18:1 20:1 22:1 24:1 Total MUFA 18:2n-6 18:3n-3 18:4n-3 20:5n-3 22:5n-3 22:6n-3 Total PUFA 1

Aidos et al. (2002).

12.3

Marine mammal oils

Marine mammals are unique in that they possess a layer of insulating fat under their skin, known as blubber, which allows their survival in the cold waters of the Arctic and Antarctic (Holmer, 1989). Blubber also helps the animals with their movement and buoyancy. The blubber may vary in thickness, depending on a number of variables, but is on average about 5 cm thick for seals. Seal blubber oil is a by-product of seal meat and seal industries. The oils from marine mammals contain various lipid classes, including triacylglycerols, diacylglycerols, monoacylglycerols, free fatty acids, wax esters, cholesterol, cholesterol esters, hydrocarbons, vitamins, and ether lipids. Triacylglycerols (TAG) of seal blubber oils are the main component of neutral lipids which contain a variety of lipid classes. Neutral lipids account for 98.9% of blubber in contrast to intramuscular lipids (78.8% neutral and 21.1% polar lipids) (Shahidi et al., 1994). In addition to TAG, wax esters (long-chain alcohols esterified to fatty acids) are another important group of neutral lipids found in marine mammals. Most species of marine mammals have C32, C34, C36, and C38 (total of alcohol plus acid) as major components (Lee and Patton, 1989). Whale oils are especially interesting because some contain fatty acids that are largely in the form of wax esters (Gruger, 1967). The oils from the blubber of the Physeteridae may consist mainly of wax esters. The sperm whale blubber oil consists of a mixture of about 79% wax esters and 21% TAG (Hansen and Cheah, 1969). The dwarf sperm whale (K. simus) blubber oils consist of 42% wax esters and 58% TAG

266

Maximising the value of marine by-products

(Litchfield et al., 1975). The blubber fat of beaked whales (Berardius, Hyperoodon, and Ziphius) is composed almost entirely of wax esters (94±99%) along with low levels of TAG (2±6%) (Litchfield et al., 1976). A number of possible functions for wax esters in marine mammals has been proposed; these include their role as a reserve energy store, buoyancy, metabolic water, thermal insulation, and biosonar (Nevenzel, 1970; Sargent et al., 1976; Sargent, 1978). Among unsaponifiable matters, hydrocarbons, especially long-chain hydrocarbons, are found in detectable amounts in marine mammal oils. Some marine oils contain less than 0.1% hydrocarbons, while others contain as much as 90% (Heller et al., 1957). In the liver of the seal, Arctocephalus (Pinnipedia), liver squalene was 0.50% of the oil (Karnovsky and Rapson, 1947). High squalene contents (90, 91 and 92.8%) occur in shark liver oils (Karnovsky and Rapson, 1947; Heller et al., 1957). Total hydrocarbons were present at 0.3% of dry matter weight of the blubber, 1.6% in liver, and 1.3% in the muscle (Bottino, 1978). Among cetaceans, limited data for two dolphins have been published: in Delphinus longirostris liver very long-chain hydrocarbons (C44) were detected; and zamene was present in Langenorynchus acutus (Blumer and Thomas, 1965). The fatty acid composition of marine lipids varies significantly, but all contain a large proportion of long-chain highly unsaturated fatty acids, similar to fish oils. However, the proportion of fatty acids in fish and marine mammals varies considerably (Shahidi, 1998). A marine oil typically contains some 40 different fatty acids with carbon numbers varying from 10 to 24, resulting in a large number of different TAG with the same carbon number, but with different levels of unsaturation (Shahidi et al., 1994; Borch-Jensen and Mollerup, 1996). Even-numbered carbon fatty acids make up about 97% of the total fatty acids, with a few notable exceptions (Hansen and Cheah, 1969). Some fatty acids with odd-numbered carbon chain such as C15:0 and C17:0, along with traces of C13:0 and C19:0 have also been found in marine oils (Ackman, 1989). Besides, monomethyl branched fatty acids have been isolated from marine oils, such as 3-methyldodecanoic acid from blubber of the sperm whale physeter catodon (Ackman, 1989). In contrast to relatively small amounts of saturated fatty acids, marine mammal oils have been characterized by high amounts of monounsaturated fatty acids (MUFA) and n-3 PUFA (Bang et al., 1976, 1980). For instance, the content of MUFA in neutral and polar lipids in seal blubber are more than 60 and 46%, respectively (Shahidi et al., 1996). Most of these fatty acids are long chain with 20 to 22 carbon atoms and have !3 configurations. Ackman et al. (1965) have pointed out that the total C20 and C22 MUFA and PUFA in each layer of whale blubber is nearly constant, but the ratios of the monounsaturated to polyunsaturated fatty acids change very significantly. The most common long chain PUFA in marine lipids are EPA and DHA as well as a smaller amount of DPA, all of which belong to the !3 family (Wanasundara, 1997). The high content of !3 fatty acids in marine lipids is suggested to be a consequence of cold temperature adaptation, because at lower habitat temperatures, !3 PUFA remain liquid and oppose any tendency to crystallize (Ackman, 1989). Most of

Marine oils from seafood waste

267

the long chain PUFA are formed in unicellular phytoplankton and multicellular sea algae and eventually pass through the food web and become incorporated into the body of fish and other higher marine species, including marine mammals which often eat fish (Yongmanichai and Ward, 1989). The fatty acid composition of oils from most species of marine mammals has been summarized (Ackman and Lamothe, 1989). Seal oils, due to the increasing interest in seal fishery and product development, have been in focus and frequently studied by researchers. The fatty acid composition of oils from different species of seal has been reviewed (Shahidi, 1998). Table 12.7 shows the fatty acids and their contents in blubber lipid from six species of seals. The fatty acid composition of blubber of marine mammals such as seals is regulated by their diet (Grahl-Nielsen and Mjaavatten, 1991), location (West et al., 1979a,b), season as well as physiological conditions such as age (Engelhardt and Walker, 1974) and sex (West et al., 1979a,b) of the animal. In some marine mammals, the depot fats are largely dietary fatty acids laid down with a minimum change, but the fatty acids of the lipids of the essential organs have terrestrial characteristics (Ackman and Lamothe, 1989). Fatty acid composition also depends on tissue and species of the animal. However, differences are most apparent among tissues. Seal blubber, for example, had a high content of monounsaturated fatty acids, but was low in arachidonic acid, dimethyl acetals and DHA. Lung tissue lipids were high in palmitic acid and heart tissue lipids had a higher content of linolenic acid. The proportions and fatty acid Table 12.7

Fatty acid composition (g/100g) of blubber of various species of seal1

Fatty acid 14:0 16:0 16:0 16:1 18:0 18:1 18:1 18:0 18:1 18:1 18:2 20:1 20:4 20:5 22:0 22:1 22:5 22:6

DMA !7 DMA !9 DMA !7 DMA !9 !7 !6 !9 !6 !3 !11 !3 !3

Bearded

Gray

Harbor

Harp

Hooded

Ringed

3.05 3.830.03 4.520.13 4.660.49 4.400.38 3.360.66 ND ND ND ND ND ND 10.14 6.610.08 8.030.38 6.240.44 9.811.57 4.822.07 17.77 12.770.09 19.260.53 14.930.46 10.090.35 23.120.18 ND ND ND ND ND ND ND ND ND ND ND ND ND 0.450.01 ND 0.460.00 ND ND 2.15 0.940.02 0.850.02 0.950.03 1.830.31 0.420.19 16.76 24.500.44 18.610.55 18.591.01 22.772.66 19.721.33 9.49 4.950.09 5.160.44 3.570.36 3.750.47 5.030.46 2.30 1.280.00 1.270.04 1.360.20 1.630.20 2.580.02 5.08 12.500.43 9.060.33 12.562.92 13.001.86 6.712.17 0.94 0.510.00 0.440.00 0.360.96 0.310.03 0.300.02 8.28 4.850.13 9.310.21 6.820.69 5.211.65 8.721.06 0.63 <0.3 1.190.02 <0.3 <0.3 0.750.67 0.27 0.620.03 0.310.01 0.770.61 0.860.33 0.340.01 4.26 5.060.05 4.220.14 4.780.25 2.290.08 5.460.47 7.22 8.910.29 7.760.98 10.481.98 9.562.36 9.451.74

DMA: dimethyl acetal. ND: not detected. 1 Durnford and Shahidi (2002); Durnford et al. (2003).

268

Maximising the value of marine by-products

Table 12.8 Fatty acid distribution in different positions of triacylglycerols of harp seal blubber oil1 Fatty acid

sn-1

sn-2

sn-3

Total saturates Total unsaturates Total monounsaturates Total polyunsaturates Eicosapentaenoic acid (EPA) Docosapentaenoic acid (DPA) Docosahexaenoic acid (DHA) Total omega-3 Total omega-6

6.34 90.51 62.91 27.60 8.36 3.99 10.52 25.65 0.75

25.56 73.25 65.98 7.27 1.60 0.79 2.27 5.56 1.58

4.32 94.32 51.09 43.23 11.21 8.21 17.91 38.87 3.34

1

Shahidi (1998).

constituents in different tissues are different, most probably due to their varying functional requirements (Durnford and Shahidi, 2002). The lipids of vital organs of seals and whales contain high proportions of fatty acids of the !6 family, similar to those of terrestrial animals. The distinction between the fatty acids of functional organs such as liver, heart, and other organs with depot fat has been discussed (Ackman et al., 1972; Durnford and Shahidi, 2002). As explained earlier, the fatty acid distribution in the TAG molecules in blubber oil are different from fish oil and the omega-3 fatty acids are located primarily in the sn-1 and sn-3 positions of TAG (Table 12.8), while in fish oils they are located abundantly in the sn-2 position of TAG (Wanasundara and Shahidi, 1997). Mag (2000) has reported that the different distribution of fatty acids may influence the metabolism and potential health benefits of marine lipids, and moreover, may account for the higher oxidative stability of marine mammal oils compared to fish oils.

12.4

Algal oil

As an alternative to usual marine oils, PUFA can be obtained from microorganisms. Microorganisms, in particular the marine algae, are widely used in the aquaculture industry, mostly to feed fish, crustaceans, and bivalves directly. They are also used indirectly, although to a much lesser extent, via feeding of rotifers, copepods, and brine shrimps, which are in turn used as live feed. Therefore, they are thought to be the primary producers of n-3 PUFAs in the marine food chain (Coutteau et al., 1997; Wikfors et al., 2001). Although marine fish and mammals appear to have some capacity for in vivo biosynthesis of n-3 PUFAs, the majority of the PUFAs in their body originate from their diet (Ackman et al., 1964). Depending on the microbial species and environmental conditions, the lipid content of microorganisms may vary between a few percent to over 80% of the

Marine oils from seafood waste

269

biomass on a dry weight basis (Ratledge, 1993; Leman, 1997). To make some kind of a distinction, the term oleaginous has been introduced and these microorganisms accumulate over 20±25% lipid on a dry biomass basis (Ratledge and Evans, 1989). Oleaginous microorganisms store lipids mainly in the form of triacylglycerols. Various eukaryotes can accumulate large amounts of triacylglycerols (Ratledge, 1993). Microbial oil or single cell oil (SCO) production via fermentation is a relatively new concept, first proposed in the twentieth century (Ratwan, 1991; Ratledge, 2001). In SCO processes, microorganisms that are able to produce the desired oil are cultivated in a bioreactor (Sijtsma and de Swaaf, 2004). In the industrial-scale fermentation, cells are harvested at maximum volumetric productivity followed by drying and further processing of the oil (Kyle, 1997). Since heterotrophic cultivation is independent of light, this production system possesses many advantages such as axenical operation, optimal controlled conditions, increased reproducibility, higher biomass concentrations, and straightforward scale-up of the fermentation process (Chen, 1996). High levels of DHA are found in heterotrophic marine algae including Traustochitrium, Schizochytrium and Crypthecodinium cohnii species, except for Amphidinium sp. Phototrophic alga contains a relatively higher level of EPA (Table 12.9) (Myher et al., 1996; Sijtsma and de Swaaf, 2004; Molina et al., 1993; Viso and Marty, 1993; Servel et al., 1994; Singh and Ward, 1996; Vazhappilly and Chen, 1998; Shields et al., 1999; Meireles et al., 2002). A major drawback of Schizochytrium sp. for producing DHA is the presence of DPA (n-6) in the microbial oils, in addition to DHA (Sijtsma and de Swaaf, 2004). Algal oils have many benefits in functional foods due to their high n-3 fatty acids and the lack of environmental toxins. Furthermore, algae usually contain one specific PUFA rather than a mixture of various PUFA. This gives the algal oils an added advantage compared to fish oils, which contain mixtures of PUFA. In addition, PUFA can be purified more easily (and thus more economically) from oils, which contain one PUFA instead of a mixture of PUFA (Kendrick and Ratledge, 1992).

12.5

Marine oil manufacturing process

Crude oils contain varying amounts of substances that may impart undesirable flavor and color, including free fatty acids, phospholipids, proteins, water, pigments, and fat oxidation products. Therefore, crude oils are subjected to a number of commercial refining processes designed to remove undesirable materials (Bell et al., 2003). Fish oil refining steps include extraction of the crude oil, degumming, neutralizing, bleaching, and deodorizing. Both insoluble and soluble impurities are removed through degumming, if necessary; neutralization of crude oil with caustic soda removes free fatty acids. Bleaching removes soap, trace metals, sulphurous compounds, and part of the more stable pigments and pigment-breakdown products. This process also degrades

Table 12.9 Fatty acid profile of selected algal oils1 Organism Thraustochytrium aureum(H,76) Schizochytrium sp. (H,77) Crypthecodinium cohnii (H,33) Amphidinium carterae (H,78) Isochrysis galbana (P,79) Skeletonema costatum (P,80) Amphidinium sp. (P,81) Pavlova lutheri (P,82)

14:0 3 18 15 8 12 17 5 14

H: heterotrophic growth, P: phototrophic growth. 1 Sijtsma and de Swaaf (2004).

14:1

30

16:0 8 38 15 15 15 10 17 27

16:1

18:0

6 2 5 11 11

1 2 3 1

10

18 1

18:1

18:2

18:3

16 5 15 5 3 2 17 3

2

2 1

1 6 2 1 2

17 2

18:4

20:4 n-6 20:5 n-3 22:5 22:6 n-3 3 1 6

11 6 4

1

9

4 25 41 8 12

4

52 18 37 2 11 7 17 7

Marine oils from seafood waste

271

hydroperoxides to their respective aldehydes, ketones and other products. Finally, deodorization is carried out in order to remove residual free fatty acids, aldehydes, and ketones, which are responsible for unacceptable odor and flavor of the oil (Aidos et al., 2003; Dauksas et al., 2005). Even though fish and fish oils are the main sources of PUFA, the quality of fish oil, however, is variable and depends on fish species, season and location of catch (Moffat, 1995; He and Daviglus, 2005). Marine fish oils may contain environmental pollutants and problems associated with the typical fishy smell and unpleasant taste may exist (Sijtsma and de Swaaf, 2004). Fish oils also contain EPA which is undesirable for use in infant formulas because it leads to reduced arachidonic acid levels and hence reduced rates of infant weight gain (Carlson, 1996; Heird and Lapillonne, 2005). In order to meet the rapidly growing demand for PUFA in human nutrition, fish feeds for aquaculture operations and pharmaceutical applications, and to circumvent the drawbacks of fish oils, alternative production processes for PUFA have been developed. These include the development of novel refining techniques of fish oils (Shahidi and Wanasundara, 1998; Carvalho and Malcata, 2005) as well as production of concentrates with varying proportions of EPA and DHA. In addition, exploitation of microbial PUFA has taken place (Ishihara et al., 2000; Meireles et al., 2002).

12.6

Health effects of PUFA

Recognition of the health benefits associated with consumption of seafoods (n-3 fatty acids) is one of the most promising developments in human nutrition and disease prevention research in the past three decades. According to the current knowledge, long-chain n-3 PUFA play an important role in the prevention and treatment of coronary artery disease (Alexander, 1998), hypertension (Howe, 1997), diabetes (Krishna Mohan and Das, 2001), arthritis and other inflammatory (Babcock et al., 2000), and autoimmune disorders (Kelly, 2001), as well as cancer (Rose and Connolly, 1999; Akihisa et al., 2004) and are essential for normal growth and development, especially for the brain and retina (Anderson et al., 1990). The most direct and complete source of n-3 oils is found in fish oils and the blubber of certain marine mammals, especially harp seal. Among its advantages is that the body's absorption of n-3 fatty acids from marine mammal blubber may be faster and more thorough than is the case with flaxseed and fish oils (Mag, 2000). Since marine mammal oils contain a high concentration of monounsaturated fatty acids (MUFA), it is possible that some of their beneficial effects may be ascribed to their MUFA or to the combined effect of MUFA and n-3 PUFA (Hansen et al., 1994). A pilot study indicated that a low dose of seal oil supplementation can reduce atherogenic risk indices in young healthy individuals, and the effects are strongly dependent on the integrated n-3 fatty acids dose (Deutch et al., 2000; Bonefeld Jorgensen et al., 2001). The essential fatty acids found in seal oil include a high level of DPA (up to ten times that of

272

Maximising the value of marine by-products

fish oils). There is growing evidence that DPA is the most important of fatty acids that keep artery walls soft and plaque-free (Mag, 2000). Marine oils are also attractive from a nutritional point of view because they are thought to provide specific physiological functions against thrombosis, cholesterol build-up and allergies (Kimoto et al., 1994). Oils from the blubbers of seal and whale have beneficial effects on selected parameters that play a role in cardiovascular disease; it has been hypothesized that the effect of whale oil is not mediated by its n-3 fatty acids alone (Osterud et al., 1995). The difference in the beneficial effects of whale and seal oils on cardiovascular disease may argue against the distribution of n-3 fatty acids in TAG as being relevant to the superiority of whale oil, since the n-3 fatty acids are mainly in the sn-1 and sn-3 positions of both of these oils. The effect of whale oil is probably not mediated by n-3 fatty acids alone as the content of these fatty acids is relatively low in whale oil. Thus, in addition to !3 fatty acids, other dietary factors may play a role in the protective effects against atherosclerosis and thrombosis in Greenland Eskimos (Osterud et al., 1995). The beneficial effects of PUFA have also been ascribed to their ability to lower serum TAG, to increase membrane fluidity and to reduce thrombosis by conversion to eicosanoids (Kinsella, 1986). Both EPA and DHA, induced increases in the serum concentrations of the corresponding fatty acids as well as their relative contents in platelets (Vognild et al., 1998). However, distribution of n-3 PUFA in TAG molecules influences glycerolipid metabolism and arachidonic acid contents of serum and liver phospholipids, as well as thromboxane (TX) A2 production. In rats that were fed marine oils, for instance, plasma and liver TAG concentrations were more effectively reduced by dietary seal oil than by fish oil. Furthermore, dietary seal oil reduced arachidonic acid content in liver phosphatidylcholine and phosphatidylethanolamine, and serum phosphatidylcholine more effectively than fish oil. Activities of fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH) and the malic enzyme were significantly lowered when hamsters were fed seal oil (Yoshida et al., 2001). The predominant effect of seal oil was due to the suppression of fatty acid synthesis in the liver (Yoshida et al., 1999). In addition, reduction of TX A2 production of platelets and whole blood platelet aggregation by seal oil has been observed (Ikeda et al., 1998; Brox et al., 2001). Benefits of DPA in health have been described (Rissanen et al., 2000; Yazawa, 2001).

12.7

References

(1982). Fatty acid composition of fish oils. In Barlow, S.M. and Stansby, M.E. eds. Nutritional evaluation of long-chain fatty acids in fish oils. Academic Press, London, pp. 25±88. ACKMAN, R.G. (1989). Fatty acids. in: Marine Biogenic Lipids, Fats, and Oils, Vol 1,. Ackman, R.G. ed., CRC Press, Inc., Boca Raton, FL, pp. 103±138. ACKMAN, R.G. (2005). Fish oils. in Bailey's Industrial Oil and Fat Products, Shahidi, F. ed., John Wiley, Hoboken, NJ, pp. 279±317. ACKMAN, R.G.

Marine oils from seafood waste

273

and LAMOTHE, F. (1989). Marine mammals. in: Marine Biogenic Lipids, Fats, and Oils, Vol 2, Ackman, R.G. ed., CRC Press, Inc., Boca Raton, FL, pp. 179±382. ACKMAN R.G., JANGAARD, P.M., HOYLE, R.J. and BROCKERHOFF, H. (1964). Origin of marine fatty acids. Analysis of the fatty acids produced by the diatom Skeletonema costatum. J. Fish Res. Board Can., 21, 747±756. ACKMAN, R.G., EATON, C.A. and JANGAARD, P.M. (1965). Lipids of the Finwhale (Balaenoptera physalus) from North Atlantic Waters. Can. J. Biochem., 43, 1513±1520. ACKMAN, R.G., HOOPER, S.N. and HINGLEY, J. (1972). The harbor seal Phoca vitulina concolor De Kay: comparative details of fatty acids in lung and heart phospholipids and triglycerides. Can. J. Biochem., 50, 833±838. AIDOS, I., MASBERNAT-MARTINEZ, S., LUTEN, J. B., BOOM, R.M. and PADT A.V. (2002). Composition and stability of Herring oil recovered from sorted byproducts as compared to oil from mixed byproducts. J. Agric. Food Chem., 50, 2818±2824. AIDOS, I., SCHELVIS-SMIT, R., VELDMAN, M., LUTEN, J.B., PART, A.V.D. and BOOM, R.M. (2003). Chemical and sensory evaluation of crude oil extracted from Herring byproducts from different processing operations. J. Agric. Food Chem., 51, 1897±1903. ACKMAN, R.G.

AKIHISA, T., TOKUDA, H., OGATA, M., UKIYA, M., IIZUKA, M., SUZUKI, T., METORI, K., SHIMIZU, N.

and NISHINO, H. (2004). Cancer chemopreventive effects of polyunsaturated fatty acids. Cancer Letters, 2±5, 9±13. ALEXANDER, J.W. (1998). Immunonutrition: the role of omega-3 fatty acids. Nutrition 14, 627±633. ALIAM, S.S.M. (2003). Long chain polyunsaturated fatty acids, nutritional and healthy aspects. Review article. Rivista Italiana delle sostanze grasse., 80, 85±92. ANDERSON, G.J., CONNOR, W.E. and CORLISS, J.D. (1990). Docosahexaenoic acid is the preferred dietary n-3 fatty acid for the development of the brain and retina. Pediatr. Res., 27, 89±97. ARTS M.T., ACKMAN R.G. and HOLUB, B.J. (2001). Essential fatty acids in aquatic ecosystems: a crucial link between diet and human health and evolution. Can. J. Fish Aquat. Sci., 58, 122±137. BABCOCK, T., HELTON, W.S. and ESPAT, N.J. (2000). Eicosapentaenoic acid (EPA): an antiinflammatory !-3 fat with potential clinical applications, Nutrition 16, 1116±1118. BANG, H.O., DYERBERG, J. and HJOORNE, N. (1976). Composition of food consumed by Greenland Eskimos. Acta Med. Scand., 200, 69±73. BANG, H.O., DYERBERG, J. and SINCLAIR, H.M. (1980). Composition of the Eskimo food in north-western Greenland. Am. J. Clin. Nutr., 33, 2657±2661. BELL, J G., DOUGLAS, R., TOCHER, R., HENDERSON, J., DICK, J.R. and CRAMPTON, V.O. (2003). Altered fatty acid compositions in Atlantic salmon (Salmo salar) fed diets containing linseed and rapeseed oils can be partially restored by a subsequent fish oil finishing diet. J. Nutr., 2793±1801. BLUMER, M. and THOMAS, D.W. (1965). `Zamane', isomeric C19 monoolefins from marine zooplankton, fishes and mammals. Science, 148, 370±371. BONEFELD JORGENSEN, E.C., MOLLER, S.M. and HANSEN, J.C. (2001). Modulation of atherosclerotic risk factors by seal oil: a preliminary assessment. Int. J. Circumpolar. Health, 60, 25±33. BONNET, J.C., SIDWELL, V.D. and FOOK, E.G. (1974). Chemical and nutritive values of several fresh and canned finfish, crustaceans, and mollusks. II. Fatty acid composition. Marine Fisheries Rev., 36, 8±14.

274

Maximising the value of marine by-products

and MOLLERUP, J. (1996). Supercritical fluid chromatography of fish, shark and seal oils. Chromatographia 42, 252±258. BOTTINO, N.R. (1978). Lipids of the antarctic sei whale, Balaenoptera borealis. Lipids, 13, 18. BORCH-JENSEN, C.

BROX, J., OLAUSSEN, K., OSTERUD, B., ELVEVOLL, E. O., BJORNSTAD, E., BRENN, T., BRATTEBO, G.

and IVERSEN, H. (2001). A long-term seal- and cod-liver-oil supplementation in hypercholesterolemic subjects. Lipids, 36, 7±14. CARLSON, S.E. (1996). Arachidonic acid status of human infants: Influence of gestational age at birth and diets with very long chain n-3 and n-6 fatty acids. J. Nutr., 126, S1092±S1098. CARVALHO, A.P. and MALCATA, F.X. (2005). Preparation of fatty acid methyl esters for gaschromatographic analysis of marine lipids: insight studies. J. Agric. Food Chem., 53, 5049±5059. CHEN, F. (1996). High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol., 14, 421±426. COPEMAN, L. and PARRISH, C.C. (2004). Lipid classes, fatty acids, and sterols in seafood from Gilbert Bay, Southern Labrador. J. Agric. Food Chem., 52, 4872±4881. COUTTEAU, P., GEURDEN, I., CAMARA, M.R., BERGOT, P. and SORGELOOS, P. (1997). Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture, 155, 149±164. DAUKSAS, E., FALCH, E., SLIZYTE, R. and RUSTAD, T. (2005). Composition of fatty acids and lipid classes in bulk products generated during enzymic hydrolysis of cod (Gadus morhua) by-products. Process-Biochem., 40, 2659±2670. DEUTCH, B., BONEFELD JORGENSEN, E.C. and HANSEN, J.C. (2000). N-3 PUFA from fish or seal oil reduce atherogenic risk indicators in Danish women. Nutr. Res., 20, 1065± 1077. DIN, J.N., NEWBY, D.E. and FLAPAN, A.D. (2004). Omega 3 fatty acids and cardiovascular disease ± fishing for a natural treatment. British Med. J., 328, 30±35. DURNFORD, E. and SHAHIDI, F. (2002). Analytical and physical chemistry ± comparison of FA compositions of selected tissues of phocid seals of Eastern Canada using oneway and multivariate techniques. J. Am. Oil Chem. Soc., 79, 1095±1102. DURNFORD, E., SHAHIDI, F. and ACKMAN, R.G. (2003). Processing and engineering technology ± Letters to the editor ± Phthalates and the overestimation of docosanoic acid in seal lipids. J. Am. Oil Chem. Soc., 80, 405. ENGELHARDT, F.R. and WALKER, B.L. (1974). Fatty acid composition of the harp seal, Pagophilus groenlandicus (Phoca groenlandica), Comp. Biochem. Physiol. B., 47, 169±179. GEBHARDT, S. E. and THOMAS, R.G. (2002). Nutritive value of foods. US Department of Agriculture, Home and Garden Bulletin, 72, 1±103. GRAHL-NIELSEN, O. and MJAAVATTEN, O. (1991). Dietary influence on fatty acid composition of blubber fat of seals as determined by biopsy: a multivariate approach. Marine Biology 110, 59±64. GRUGER, E.H. JR. (1967). Fatty acid composition, in: Fish Oils, Stansby, M.E. ed., The AVI Publishing Company Inc., Westport, CT, pp. 3±30. HANSEN, I.A. and CHEAH, C.C. (1969). Related dietary and tissue lipids of the sperm whale. Comp. Biochem. Physiol., 31, 757±761. HANSEN, J.C., SLOTH PEDERSEN, H. and MULVAD, G. (1994). Fatty acids and antioxidants in the Inuit diet. Their role in ischemic heart disease (IHD) and possible interactions with other dietary factors. A review. Arctic. Med. Res., 53, 4±17.

Marine oils from seafood waste

275

and DAVIGLUS, M. (2005). A few more thoughts about fish and fish oil. J. Amer. Diet. Assoc., 350±351. HEIRD, W.C. and LAPILLONNE, A. (2005). The role of essential fatty acids in development. Annu. Rev. Nutr., 25, 549±571. HELLER, J.H., HELLER, M.S., SPRINGER, S. and CLARK, E. (1957). Squalene content of various shark livers. Nature, 179, 919±920. HOLMER, G.K. (1989). Triglycerides. in: Marine Biogenic Lipids, Fats, and Oils, Vol 1, Ackman, R.G. ed. CRC Press, Inc., Boca Raton, FL, pp. 139±174. HOWE, P.R.C. (1997). Dietary fats and hypertension: Focus on fish oil, Ann. NY Acad. Sci., 827, 339±352. HE, K.

HULSHOF, K.F.A.M., VAN ERP-BAART, M.A., ANTTOLAINEN, M., BECKER, W., CHURCH, S.M., COUET, C., HERMANN-KUNZ, E., KESTELOOT, H., LETH, T., MARTINS, I., MOREIRAS, O., MOSCHANDREAS, J., PIZZOFERRATO, L., RIMESTAD, A.H., THORGEIRSDOTTIR, H., VAN

and VAN (1999). Intake of fatty acids in Western Europe with emphasis on trans fatty acids: the transfair study. Eur. J. Clin. Nutr., 53, 143±157. HYVONEN, L. and KOIVISTOINEN, P. (1994). Fatty acid analysis, TAG equivalents as net fat value, and nutritional attributes of fish and fish products. J. Food Comp. Anal., 7, 44±58. IKEDA, I., YOSHIDA, H., TOMOOKA, M., YOSET, A., IMAIZUMI, K., TSUJI, H. and SETO, A. (1998). Effects of long-term feeding of marine oils with different positional distribution of eicosapentaenoic and docosahexaenoic acids on lipid metabolism, eicosanoid production, and platelet aggregation in hypercholesterolemic rats. Lipids, 33, 897±904. ISHIHARA, K., MURATA, M., KANENIWA, M., SAITO, H., KOMATSU, W. and SHINOHARA, K. (2000). Purification of stearidonic acid 1(8:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) from algal fatty acid with lipase and medium pressure liquid chromatography. Biosci. Biotechnol. Biochem., 64, 2454±2457. JAYASINGHE, C., GOTOH, N., TOKAIRIN, S., EHARA, H. and WADA, S. (2003). Inter species changes of lipid compositions in liver of shallow-water sharks from the Indian Ocean. Fisheries Sci., 69, 644±653. KALOGEROPOULOS, N., ANDRIKOPOULOS, N.K. and HASSAPIDOU, M. (2004). Dietary evaluation of Mediterranean fish and mollusks pan-fried in virgin olive oil. J. Sci. Food Agric., 84, 1750±1758. KAMAL-ELDIN, A. and YANISHLIEVAB, N.V. (2002). N-3 fatty acids for human nutrition: stability considerations. Eur. J. Lipid Sci. Technol., 104, 825±836. KARNOVSKY, M.L. and RAPSON, W.S. (1947). Application of the Fuelson method of `Squalene' determination to some marine oils. J. Soc. Chem. Ind. London, 66, 124. KELLY, D.S. (2001). Modulation of human immune and inflammatory responses by dietary fatty acids. Nutrition, 17, 669±673. KENDRICK, A. and RATLEDGE, C. (1992). Lipids of selected molds grown for production of n-3 and n-6 polyunsaturated fatty acids. Lipids, 27, 15±20. KIMOTO, H., ENDO, Y. and FUJIMOTO, K. (1994). Influence of. interesterification on theoxidative stability of marine oil triacylglycerols, J. Am. Oil Chem. Soc., 71, 469±473. KINSELLA, J.E. (1986). Food components with potential therapeutic benefits: the n-3 polyunsaturated fatty acids of fish oils. Food Technol., 40, 89±97. KRISHNA MOHAN, I. and DAS, U.N. (2001). Prevention of chemically induced diabetes mellitus in experimental animals by polyunsaturated fatty acids. Nutrition, 17, 126±151. AMELSVOORT, J.M.M., ARO, A., KAFATOS, A.G., LANZMANN-PETITHORY, D.

POPPEL, G.

276

Maximising the value of marine by-products

(1997). Production and use of a single cell oil highly enriched in arachidonic acid. Lipid Technol., 9, 116±121. LEE, R.F. and PATTON, J.S. (1989). Alcohol and waxes. in: Marine Biogenic Lipids, Fats, and Oils, Vol 1, Ackman, R.G. ed., CRC Press, Inc., Boca Raton, FL, pp. 73±102. LEMAN, J. (1997). Oleaginous microorganisms: an assessment of the potential. Adv. Appl. Microbiol,, 43, 195±243. LITCHFIELD, C., GREENBERG, A.J., CALDWELL, D.K., CALDWELL, M.C., SIPOS, J. and ACKMAN, R.G. (1975). Comparative lipid patterns in acoustical and nonacoustical fatty tissues of dolphins, porpoises and toothed whales. Conp. Biochem. Physiol. B, 50, 591±597. LITCHFIELD, C., GREENBERG, A. J. and MEAD, J.G. (1976). Distinctive character of zyphiidae head and blubber fats. Cetology, 23, 1±10. MAG, T. (2000). Patent WO00/44862A1, PCT International Patent Application. MAYZAUD, P., VIRTUE, P. and ALBESSARD, E. (1999). Seasonal variations in the lipid and fatty acid composition of the euphausiid Meganyctiphanes norvegica from the Ligurian Sea. Mar. Ecol. Prog. Ser., 186, 199±210. MEIRELES, L.A., GUEDES, A.C. and MALCATA, F.X. (2002). Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the microalgae Pavlova lutheri folllowing random mutagenesis. Biotechnol. Bioeng., 81, 50±55. MOFFAT, C.F. (1995). Fish oil triglycerides: a wealth of variation. Lipid Technol., 7, 125±129. MOLINA, G.E, SANCHEZ, P.J.A, GARCIA. C.F., GARCIA, S.J.L. and LOPEZ, A.D. (1993). N-3 PUFA productivity in chemostat cultures of microalgae. Appl. Microbiol. Biotechnol., 38, 599±605. MYHER, J.J., KUKSIS, A., GEHER, K., PARK, P.W. and DIERSEN-SCHADE, D.A. (1996). Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids. Lipids, 31, 207±215. NDA (SCIENTIFIC PANEL ON DIETETIC PRODUCTS, NUTRITION AND ALLERGIES). (2005). Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to nutrition claims concerning omega-3 fatty acids, monounsaturated fat, polyunsaturated fat and unsaturated fat. The EFSA J., 253, 1±29. NEVENZEL, J.C. (1970). Occurrence, function and biosynthesis of wax esters in marine organisms. Lipids 5, 308±319. NEWTON, I. and SNYDER, D. (1997). Nutritional aspects of long-chain omega-3 fatty acids and their use in bread enrichment. Cereal Foods World, 42, 126±131. OLIVEIRA, A.C.M. and BECHTEL, P.J. (2005). Lipid composition of Alaska pink salmon (Oncorhynchus gorbuscha) and Alaska Walleye pollock (Theragra chalcogramma) byproducts. J. Aquatic Food Product Technol., 14, 73±89. KYLE D.J.

OSTERUD, B., ELVEVOLL, E., BARSTAD, H., BROX, J., HALVORSEN, H., LIA, K., OLSEN, J.O., OLSEN,

and VOGNILD, E. (1995). Effect of marine oils supplementation on coagulation and cellular activation in whole blood. Lipids, 30, 1111± 1118. PARK, Y., KELLEHER, S.D., MCCLEMENTS, D.J. and DECKER, E.A. (2004). Incorporation and Stabilization of Omega-3 Fatty Acids in Surimi Made from Cod, Gadus morhua. J. Agric. Food Chem., 52, 597±601. RATLEDGE, C. (1993). Single cell oils-have they a biotechnological future? Tibtech., 11, 278±284. RATLEDGE, C. (2001). Microorganisms as sources of polyunsaturated fatty acids. In: Gunstone, F.D. eds. Structured and modified lipids. Marcel Dekker, New York, pp. 351±399. R. L., SISSENER, C., REKDAL, O.

Marine oils from seafood waste

277

and EVANS, C.T. (1989). Lipids and their metabolism. In: Rose, A.H. and Harrison, J.S. eds, The yeasts, 2nd edn. Academic Press, London, pp. 367±455. RATWAN, S.S. (1991). Sources of C20-polyunsaturated fatty acids for biotechnological use. Appl. Microbiol Biotechnol, 35, 421±430. RISSANEN, T., VOUTILAINEN, S., NYYSSONEN, K., LAKKA, T.A. and SALONEN, J.T. (2000). Fish oil derived fatty acids, docosahexaenoic acid and docosapentaenoic acid and the risk of acute coronary events: the Kuopio Ischaemic Risk Factor Study. Circulation, 102, 2677±2679. RORA, A.M.B., BIRKELANDB, S., HULTMANNC, L., RUSTADC, H., RAB, S.T. and BJERKENG, B. (2005). Quality characteristics of farmed Atlantic salmon (Salmo salar) fed diets high in soybean or fish oil as affected by cold-smoking temperature. L.W.T., 38, 201±211. ROSE, D.P. and CONNOLLY, J.M. (1999). Omega-3 fatty acids as cancer chemopreventive agents. Pharmcol. Ther., 83, 217±244. SARGENT, J.R. (1978). Marine wax esters. Sci. Prog. Oxford, 65, 437±458. SARGENT, J.R., LEE, R.F. and NEVENZEL, J.C. (1976). Marine waxes. in: Chemistry and Biochemistry of Natural Waxes, Kolattukudy, P. ed., Elsevier, Amsterdam, pp. 49±91. SATHIVEL, S., PRINYAWIWATKUL, W., KING, J.M., GRIMM, C.C. and LLOYD, S. (2003). Oil production from catfish viscera. J. Amer. Oil Chem. Soc., 80, 377±382. SERVEL, M.O., CLAIRE, C., DERRIEN, A., COIFFARD, L. and ROECK-HOLTZHAUER, Y. DE. (1994). Fatty acid composition of some marine microalgae. Phytochem., 36, 691±693. SHAHIDI, F. (1998). Seal blubber. in: Shahidi, F. ed., Seal Fishery and Product Development, Science Tech, NF, Canada, pp. 99±146. SHAHIDI, F. (2002). Marine nutraceuticals. Inform., 13, 57±62. SHAHIDI, F. and WANASUNDARA, U.N. (1998). Omega-3 fatty acid concentrates: nutritional aspects and production technologies. Trends in Food Sci. Technol., 9, 230±240. SHAHIDI, F., SYNOWIECKI, J., AMAROWICZ, R. and WANASUNDARA, U. (1994). Omega-3 fatty acid composition and stability of seal lipids, in: Ho, C.T. and Hartaman, T.G. eds., Lipids in Food Flavors, ACS Symposium Series 558, American Chemical Society, Washington, DC, pp. 233±243. SHAHIDI, F., WANASUNDARA, U.N. and AMAROWICZ, R. (1996). in: Chemistry and Novel Foods, Mills, O., Okai, H., Spanier, A.M. and Tamura, M. eds., American Chemical Society, Washington, DC. SHIELDS, R.J., BELL, J.G., LUIZI, F.S., GARA, B., BROMAGE, N.R. and SARGENT, J.R. (1999). Natural copepods are superior to enriched artemia nauplii as feed for Halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr., 129, 1186±1194. SIJTSMA, L. and DE SWAAF, M.E. (2004). Biotechnological production and applications of the !-3 polyunsaturated fatty acid docosahexaenoic acid. Appl. Microbiol. Biotechnol., 64, 146±153. SINGH, A. and WARD, O.P. (1996). Production of high yields of docosahexaenoic acid by Traustochytrium roseum ATCC 20810. J. Ind. Microbiol., 16, 370±373. TANABE, T., SUZUKI, T., OGURA, M. and WATANABE, Y. (1999). High proportion of docosahexaenoic acid in the lipid of juvenile and young skipjack tuna, Katsuwonus pelamis from the tropical western Pacific. Fish. Sci., 65, 806±807. VAZHAPPILLY, R. and CHEN F. (1998). Heterotrophic production of potential omega-3 polyunsaturated fatty acids by microalgae and algaelike microorganisms. Bot. Mar., 41, 553±558. VISO, A.C. and MARTY, J.C. (1993). Fatty acids from 28 marine microalgae. Phytochem., 34, RATLEDGE, C.

278

Maximising the value of marine by-products 1521±1533.

and OSTERUD, B. (1998). Effects of dietary marine oils and olive oil on fatty acid composition, platelet membrane fluidity, platelet responses, and serum lipids in healthy humans. Lipids, 33, 427±436. WANASUNDARA, U.N. (1997). Marine oil: stabilization, structural characterization and omega-3 fatty acid concentration. PhD Thesis, Memorial University of Newfoundland. WANASUNDARA, U.N. and SHAHIDI, F. (1997). Structural Characteristics of Marine Lipids and Preparation of omega 3 Concentrates, in: Flavor and Lipid Chemistry of Seafoods, Shahidi, F. and Cadwallader, K. K. eds., American Chemical Society, Washington, DC. WANASUNDARA, U.N., SHAHIDI, F. and AMAROWICZ, R. (1998). Effect of processing on constituents and oxidative stability of marine oils. J. Food Lipids, 5, 29±41. WATANABE, T. and ACKMAN, R.G. (1974). Lipids and fatty acids of the American (Crassostrea virginica) and European flat (Ostrea edulis) oysters from a common habitat and after one feeding with Dicrateria inornata or Isochrysis galbana. J. Fish. Res. Board Can., 31, 403±409. WEST, G.C., BURNS, J.J. and MODAFFERI, M. (1979a). Fatty acid composition of Pacific walrus skin and blubber fats. Can. J. Zool., 57, 1249±1255. WEST, G.C., BURNS, J.J. and MODAFFERI, M. (1979b). Fatty acid composition of blubber from the four species of bering sea phocid. Seals, Can. J. Zool., 57, 189±195. WIKFORS, G.H. (2001). Impact of algal research in aquaculture. J. Phycol., 37, 968±974. YAZAWA, K. (2001). Fatty acids and lipids ± new findings. World Rev. Nutr. Diet., 88, 249±252. YONGMANICHAI, W. and WARD, O.P. (1989). Omega-3 fatty acids: Alternative sources of production. Prog. Biochem., 24, 117±125. YOSHIDA, H., MAWATARI, M., IKEDA, I., IMAIZUMI, K., SETO, A. and TSUJI, H. (1999). Effect of dietary seal and fish oil on triacylglycerol metabolism in rats. J. Nutr. Sci. Vitaminol., 45, 411±421. YOSHIDA, H., IKEDA, I., TOMOOKA, M., MAWATARI, M., IMAIZUMI, K., SETO, A. and TSUJI, H. (2001). Effect of dietary seal and fish oils on lipid metabolism in hamsters. J. Nutr. Sci. Vitaminol., 47, 242±247. VOGNILD, E., ELVEVOLL, E., BROX, J., OLSEN, R. L., BARSTAD, H., AURSAND, M.

13 Collagen and gelatin from marine by-products J. M. Regenstein and P. Zhou, Cornell University, USA

13.1

Introduction

Collagen is one of the major structural components of both vertebrates and invertebrates, and is found widely in skin, bone and other connective tissues (Balian and Bowes, 1977). Gelatin, on the other hand, is a class of protein fractions that have no existence in nature, but are derived from the parent protein collagen, by procedures involving the destruction of cross-linkages between polypeptide chains of collagen along with some level of breakage of polypeptide bonds. Most commercial collagen and gelatin are obtained from mammals, mainly from bovine bone, bovine hide, and porcine skin. In recent years studies on collagen and gelatin obtained from seafood processing by-products have drawn extensive interest, in part due to the requirements for kosher and halal food product development and consumers' concern about bovine spongiform encephalopathy (BSE, `mad cow disease') in products from mammals. This chapter provides a brief review of the literature for the following three main topics, namely, manufacture of collagen and gelatin from seafood processing byproducts; chemical, physical and nutritional properties of marine collagen and gelatin; and food and non-food applications of collagen and gelatin.

13.2

Key drivers of marine collagen and gelatin

At present, most of the world's production of collagen and gelatin comes from porcine skin, bovine hide or bovine bone (Table 13.1, Gelatine Manufacturers of Europe, 2004). Unfortunately, these sources of collagen and gelatin present religious and safety-orientated concerns for various consumer communities.

280

Maximising the value of marine by-products

Table 13.1 World gelatin market data from 2001 to 2003 2001

2002

2003

Production Percentage Production Percentage Production Percentage (metric ton) (metric ton) (metric ton) Pork skin 110 400 Bovine hides 77 200 Bones 80 800 Fish and others 1 000 Total 269 400

41.0 28.6 30.0 0.4

113 600 77 500 79 600 1 800 272 500

41.7 28.4 29.2 0.7

117 950 81 650 76 750 1 950 278 300

42.4 29.3 27.6 0.7

Source: Research by Gelatin Manufacturers of Europe, 2004.

13.2.1 Requirement of kosher and halal product development Kosher and halal certifications are important components of the food business. Kosher food, in particular, has become a major part of modern food production. Many companies in the food industry would prefer to operate all of their processing lines as kosher because of the complications and cost involved in switching back and forth between non-kosher and kosher. On the other hand, the number of Muslims in the world is more than 1.3 billion people, and trade in halal products is currently about 150 billion US dollars (Regenstein et al., 2003). The absence of any widely accepted collagen and gelatin for both Jewish and Muslim groups is probably the most significant factor holding up further expansion of kosher and halal food products. Although some lenient rabbis permit some types of traditional collagen and gelatin as kosher, the mainstream Orthodox kosher supervision agencies, such as the OU, OK, Star-K or Kof-K, only certify collagen and gelatin from kosher fish (fish with fins and removable scales) and kosher slaughtered animals (Regenstein et al., 1996). Similarly, for most Muslims, pork gelatin is forbidden and gelatin from non-religiously slaughtered cattle is not desirable; but most fish collagen and gelatin can be certified as halal. 13.2.2 Consumers' safety concerns BSE, which has been spread in Europe but is also found in many other countries, raised a serious health concern with respect to collagen and gelatin. In many countries such as the United States, the government agencies issued specific rules to regulate the source and processing of collagen and gelatin to reduce the potential risk posed by BSE. Although the scientific evidence did not show that collagen and gelatin were BSE carrier materials, many consumers still have concerns about the safety of these products. 13.2.3 Economic drivers from the fishing industry's point of view The waste from fish processing after filleting can account for as much as 75% of the total catch weight (Shahidi, 1995). It includes the heads, skin and scales, guts/

Collagen and gelatin from marine by-products 281 Table 13.2

Marine collagen and gelatin sources Species

Tissues

Invertebrate

Cuttlefish, octopus and squid Jellyfish Starfish Sea urchin Sea cucumber

Outer skin and cartilaginous tissues Exumbrella and mesogela Body wall Test (or shell) Body wall

Sea mammal

Seal and whale

Skin

Fish

Jawless fish Cartilaginous fish Bony fish

Skin, notochord and cartilaginous tissues Skin and cartilage Skin, scale. bone, and swim bladder

internal organs, frames (bone rack with adhering meat), and trim (pieces cut from the fillets during processing) (Regenstein, 2004). About 30% of such waste consists of skin, bone and scale with high collagen content that could be used to produce collagen and gelatin (Young and Lorimer, 1960; GoÂmez-GuilleÂn et al., 2002; Sadowska et al., 2003). Thus, preparation of collagen and gelatin from marine byproducts can not only satisfy the kosher and halal requirement and consumers' concern for BSE, but can also increase the economic return for the fishing industry.

13.3

Sources of marine collagen and gelatin

Collagen and gelatin can be obtained from various marine sources. The marine species available for collagen and gelatin manufacture can be roughly divided into three categories: marine invertebrates, sea mammals and fishes (Table 13.2). Fish, based on their living environments, are usually subdivided into four groups: hot-water fish, warm-water fish, cold-water fish (Eastoe and Leach, 1977), and ice-water fish. Cold-water fishes, such as pollock, cod and salmon, account for a large part of commercial fish capture (FAO, 2002). They are often processed into the form of skinned and boned fillets, leaving large amounts of fish skin, scales and bones as waste. These by-products, especially the skin, usually contain a large amount of protein, most of which is collagens (Young and Lorimer, 1960). Warm-water fish account for most fresh-water fish aquaculture production (FAO, 2002), and currently, many commercial fish gelatins come from fish in this category. In the following sections the studies of collagen and gelatin from both marine sources and some fresh water fish species will be reviewed to examine the variation among different sources.

13.4

Manufacture of marine collagen and gelatin

The yield and properties of collagen and gelatin would be influenced by the source of the raw material, the nature and concentration of acid or alkali used

282

Maximising the value of marine by-products

during pretreatment, the temperature and time of pretreatment and extraction, and other process variables depending on the details of the process selected. 13.4.1 Manufacture of collagen Collagens, based on the different preparation processes, can be divided into salt soluble collagen, acid soluble collagen (ASC), and pepsin soluble collagen (PSC). Although a few studies applied cold neutral salt solutions for collagen extraction (Young and Lorimer, 1960, 1961), the soluble fraction obtained by this method has a very low collagen yield and contains a large portion of noncollagenous proteins. So, in this section only the acid process and the enzymatic method will be covered (Fig. 13.1). Pretreatment Since collagen is only one of the constituents of the raw materials, before collagen extraction, one or several pretreatments might be applied to remove the contaminants and increase the purity of the final extract. Non-collagenous proteins, pigments, and lipids are usually treated as contaminants during collagen extraction. Alkaline pretreatment can be used to remove non-collagenous proteins and pigments (Nagai et al., 2002), and a diluted NaOH solution is widely used. To remove lipids, the raw materials can be treated with 10% butyl alcohol, and washed with distilled water (Nagai and Suzuki, 2000); a diluted alkaline solution can also partly satisfy this function. Some raw materials, such as fish bones (Nagai and Suzuki, 2000), fish scales (Kimura et al., 1991; Nomura et al., 1996), the testes of sea urchin (Omura et al.,

Fig. 13.1 Manufacturing procedures for marine collagen.

Collagen and gelatin from marine by-products 283 1996) and the body wall of starfish (Kimura et al., 1993) are highly calcified. Two different solvents can be used for demineralization: 0.05 M ethylenediaminetetraacetic acid (EDTA) in neutral buffer (Nagai and Suzuki, 2000) or HCl solution (Nomura et al., 1996). Both solvents can remove most of the Ca in the raw materials; but loss of protein is higher in treatments using HCl solution compared to those using 0.05 M EDTA. In addition, collagen may undergo a partial hydrolytic deterioration during the demineralization under acid condition. Thus, the demineralization is better done with an EDTA treatment (Nomura et al., 1996). Acid extraction process The acid extraction process has been widely used for collagen preparations. The most common acid extraction condition uses a 0.5 M acetic acid solution (Kimura and Ohno, 1987; Kimura et al., 1991; Nomura et al., 1996; Sivakumar and Chandrakasan, 1998; Nagai and Suzuki, 2000; Sadowska et al., 2003; Muyonga et al., 2004a). In some other cases, acid processes with citric acid solutions are applied (0.1 M cold citrate buffer at pH 3.5, in Young and Lorimer, 1960; 0.5 M citric acid, in Sadowska et al., 2003). The collagen obtained by an acid extraction process is usually called acid soluble collagen. During the acid process, the collagen yield may be influenced by many factors, such as acid concentration, the ratio of raw material to acid solution, incubation temperature and incubation time. Increases in acetic acid concentration from 0.1 M to 0.5 M result in slight increases in cod skin collagen yield (from 52% to 59% for minced cod skin, Table 13.3). Increasing the acid solution portion in an extraction mixture could also increase the yield (Sadowska et al., 2003); however, because this change may require extra effort to concentrate in the subsequent preparation steps, it is generally not technically useful to do so. In addition, most of the acid extractions are done at 4ëC. Although an increase in incubation temperature can offer a higher collagen yield (Muyonga et al., 2004a), it may also cause the degradation of the peptide chains of collagen (especially for the cold-water fish), which is not desirable for the end product. The incubation time for acid extraction is usually 24 h or longer (Table 13.3). Compared to simply prolonging the extraction time, applying several consecutive extractions may get better ASC yield (Sadowska et al., 2003). The yield of ASC varies not only with process conditions, but also with raw materials based on species and tissues (Table 13.3). In addition, Sadowska and coworkers (2003) suggested that minced cod skin could give a higher yield of ASC compared to the whole skin (Table 13.3); and some other researchers also prepared smaller pieces of the raw materials to facilitate the collagen extraction (Kimura and Ohno, 1987). By using acid extraction, especially with the consecutive process, a relatively high collagen yield could be obtained from fish skin and bone. However, for some marine species, particularly marine invertebrates, the yield of ASC is low (Table 13.3). Thus, a further extraction process with a partial enzymatic digestion is often applied to improve collagen yield.

Table 13.3 Extraction conditions and resultant yields of manufactured marine collagens Raw materials Solution Octopus, outer skin Squid, cranial cartilage Cod, skin (minced) Cod, skin (whole) Carpa, bones Carpa, scales Japanese sea bass, bone Japanese sea bass, skin Nile percha (adult), skin Nile percha (young), skin Ocellate puffer fish, skin Sardine, scales Shark, cartilage #

Yield#

Extraction conditions

0.5 M acetic acid 0.5 M acetic acid with 0.5 M acetic acid 0.5 M acetic acid with 0.1 M acetic acidb 0.25 M acetic acidb 0.5 M acetic acidb 0.5 M acetic acidb 0.5 M citric acidc 0.5 M citric acidc 0.5 M acetic acid 0.5 M acetic acid 0.5 M acetic acid 0.5 M acetic acid 0.5 M acetic acid 0.5 M acetic acid 0.5 M acetic acid 0.5 M acetic acid with 0.5 M acetic acid 0.5 M acetic acid with 0.5 M acetic acid with 0.5 M acetic acid 0.5 M acetic acid with

pepsin pepsin

pepsin pepsin pepsin pepsin

Temperature

Time

4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 4ëC 15ëC 15ëC 4ëC 4ëC 4ëC 4ëC 15ëC 4ëC 4ëC

224 h 48 h ± ± 24 h 24 h 24 h 24 h 24 h 324 h 24 h 24 h 72 h+48 h 72 h+48 h 16 h 16 h 72 h 48 h ± 96 h 96 h ± ±

5% 50% 12% 60% 52% 54% 59% 20% 10±25% 70±90% 20% 7% 41% 51% 59% 63% 11% 45% 5% 14% 71% 20% 54%

Reference

Nagai and Suzuki (2002) Nagai and Suzuki (2002) Sivakumar and Chandrakasan Sivakumar and Chandrakasan Sadowska et al. (2003) Sadowska et al. (2003) Sadowska et al. (2003) Sadowska et al. (2003) Sadowska et al. (2003) Sadowska et al. (2003) Kimura et al. (1991) Kimura et al. (1991) Nagai and Suzuki (2000) Nagai and Suzuki (2000) Muyonga et al. (2004a) Muyonga et al. (2004a) Nagai et al. (2002) Nagai et al. (2002) Nomura et al. (1996) Nomura et al. (1996) Nomura et al. (1996) Sivakumar and Chandrakasan Sivakumar and Chandrakasan

, The yield was on the basis of the dry weight of skin. , Fresh water fish species; b, the ratio of skin : acid solution was 1:6; c, the ratio of skin : acid solution was in the range of 1:4 to 1:20.

a

(1998) (1998)

(1998) (1998)

Collagen and gelatin from marine by-products 285 Enzymatic method To increase the yield of collagen extracted, various enzymes, such as trypsin and -amylase, have been used to facilitate the solubilization of collagen (Johns and Courts, 1977). For marine collagen extraction, extraction with limited pepsin proteolysis is widely used, and it can be applied alone or right after the acid extraction process. Pepsin digestion of collagen is usually done in 0.5 M acetic acid at low temperature, and collagen obtained by this method is called pepsin soluble collagen. As mentioned before, this method is often applied to those raw materials where collagen is hard to extract with the acid process alone. By using this method, the collagen yield from cuttlefish outer skin (Nagai et al., 2001), octopus outer skin (Nagai and Suzuki, 2002), and fish skin and scales (Nomura et al., 1996; Nagai et al., 2002) has been significantly improved. Nomura and coworkers (1996) suggested that the yield and the properties of the resultant soluble collagen were highly influenced by the incubation temperature and time. Although increases in temperature and time can increase the yield significantly, they may also result in a significant amount of low molecular weight components. Therefore, most of the preparation steps are done at low temperature (usually 4ëC) and a reasonable time period (24 to 96 h).

13.4.2 Manufacture of gelatin Most manufacturers produce gelatin, instead of collagen, owing to the wider industrial uses of gelatin. The ultimate aim in gelatin production is the conversion of collagen into gelatin with a maximum yield and good physicochemical properties. The yield and qualities of gelatin are influenced not only by the species or tissue from which it is extracted, but also by the manufacturing process. In gelatin manufacture two methods are usually used: the acid process and the alkaline process. The gelatin prepared by the acid process is called type A gelatin, while that prepared by the alkaline process is called type B gelatin (Hinterwaldner, 1977). Although the properties of marine raw materials are different from those of mammals and avian species, the marine gelatin extraction processes may still be divided into these two categories: an acid process and an alkaline process (Table 13.4). Acid process For gelatin extraction, the acid process refers to a pretreatment of raw materials with an acid solution followed by an extraction that is carried out in an acid medium (Devictor et al., 1995; GoÂmez-GuilleÂn and Montero, 2001; Gilbert et al., 2002; Arnesen and Gildberg, 2002; Muyonga et al., 2004b). During the acid pretreatment, the breakage of some inter-chain cross-linkages occurs, which facilitates the following extraction process. On the other hand, the acid pretreatment can partly exclude the degradation of collagen by endogenous

Table 13.4 Pretreatment and extraction conditions and the resultant yields of manufactured marine gelatin Raw materials

Pretreatment Pretreatment steps

Nile perch* (adult), skin Nile perch* (adult), bones Nile perch* (young), skin Nile perch* (young), bones Megrim, skin Sole, skin Tilapia*, skin Cod, skin Cod, skin Hake, skin Lumpfish, skin Pollock, skin #

Yield# Reference

Water extraction

Temperature

0.01 M H2SO4, 16 h 20±25ëC 3% HCl, 9±12 d 20±25ëC 20±25ëC 0.01 M H2SO4, 16 h 3% HCl, 9±12 d 20±25ëC 0.2 M NaOH, 1.5 h; 5ëC; room 0.05 M acetic acid, 3 h 0.2 M NaOH, 1.5 h; 5ëC; room 0.05 M acetic acid, 3 h 0.2% NaOH, 2 h; All 15±27ëC 0.2% H2SO4, 2 h; then 1% citric acid, 2 h 0.1±0.4% NaOH, 2 h; All room 0.1±0.4% H2SO4, 2 h; then 0.4±1.4% citric acid, 2 h 0.2 M NaOH, 1.5 h; 5ëC; room 0.05 M acetic acid, 3 h 0.2 M NaOH, 1.5 h; 5ëC; room 0.05 M acetic acid, 3 h 0.1 M NaOH, 24 h; All 5ëC 0.1 M HCl, 20 h All 2ëC 0.12 M Ca(OH)2, 2 h; then 0.1 M acetic acid, 1 h

The yield was on the basis of the wet weight of skin; * Fresh water fish species.

50, 60, 70 and 95ëC, 50, 60, 70 and 95ëC, 50, 60, 70 and 95ëC, 50, 60, 70 and 95ëC, 45ëC, overnight

each each each each

5h 5h 5h 5h

16% 2.4% 12% 1.3% 7.4%

Muyonga et al. Muyonga et al. Muyonga et al. Muyonga et al. GoÂmez-GuilleÂn

(2004b) (2004b) (2004b) (2004b) et al. (2002)

45ëC, overnight

8.3% GoÂmez-GuilleÂn et al. (2002)

40±50ëC, overnight

15%

45ëC, overnight

Grossman and Bergman (1992)

11±14% Gudmundsson and Hafsteinsson (1997)

45ëC, overnight

7.2% GoÂmez-GuilleÂn et al. (2002)

45ëC, overnight

6.5% GoÂmez-GuilleÂn et al. (2002)

55ëC, 1 h

14%

Osborne et al. (1990)

50ëC, 3 h

18%

Zhou and Regenstein (2004)

Collagen and gelatin from marine by-products 287 proteases and minimize the enzymatic breakage of intra-chain peptide bonds of collagen during extraction (Zhou and Regenstein, 2005). The period of acid pretreatment varies with raw materials, but is usually within one day. The pretreatment temperature is critical for the acid process; increasing the temperature may facilitate the pretreatment, but it will cause the loss of collagen. For sea mammals and warm-water fish, the pretreatment can be done at room temperature, while for cold-water fish gelatin extractions, the optimal temperature is lower than 10ëC (Zhou and Regenstein, 2004). Alkaline process The alkaline process refers to a pretreatment of raw materials with an alkaline solution, in most cases followed by the neutralization with an acid solution, so the extraction may be carried out in an alkaline, neutral or acid medium. One advantage of this process is that the pretreatment with an alkaline solution can remove considerable amounts of non-collagenous materials (Johns and Courts, 1977; Zhou and Regenstein, 2005). The alkaline pretreatment also breaks some inter-chain cross-linkages and excludes the effects of proteases on collagen. The neutralization with an acid solution provides an optimal weak acid extraction medium, which guarantees a high extraction yield with good gel quality. During all the pretreatments, the temperature should be controlled within a certain optimal range. For cold-water fish, this temperature is very critical, and should be kept below 10ëC to avoid the extensive loss of collagen during pretreatment processes. During the studies on pollock skin gelatin, Zhou and Regenstein (2005) also suggested that during the alkaline pretreatment, the type of alkali does not make a significant difference, but the concentration of alkali is critical. The acid type and concentration during the acid neutralization process determines the final pH of the extraction medium, and affects the yield and gel quality of the gelatin extracts. 13.4.3 Stabilizing marine collagen and gelatin Marine collagens and gelatins, especially those from fish, have lower melting points than mammalian gelatins, and care is needed in their preparation and storage because they are extremely susceptible to microbiological attack and thermal hydrolysis (Jones, 1977). Fish collagens and gelatins are commercially available in both concentrated liquid or solid form. The liquid form usually requires a mixture of methyl and propyl hydroxybenzoates or other preservatives to prevent bacterial attack (Norland, 1990). The solid collagen and gelatin usually contain less than 15% moisture. In most situations, collagen and gelatin are dried and processed into a powder or sheet, and the quality of the final products is influenced by the methods applied. It has been reported that freezedried gelatins have better gel properties than air-dried gelatins (Gudmundsson and Hafsteinsson, 1997; FernaÂndez-DõÂaz et al., 2001).

288

13.5

Maximising the value of marine by-products

Properties of marine collagen and gelatin

13.5.1 Chemical properties The amino acid composition of gelatin is very close to its parent collagen, and no amino acid sequence rearrangements occur during the collagen-gelatin conversion. Glycine (Gly), alanine (Ala), proline (Pro) and hydroxyproline (Hyp) are four of the most abundant amino acids in collagen and gelatin; and the frequency of occurrence of Gly is about 1 out of every 3 amino acids (Eastoe and Leach, 1977). The amino acid sequence is characterized by the repeating sequence of Gly-X-Y triplets, where X is mostly Pro and Y is mostly Hyp. The presence of Gly at every three residues is a critical requirement for the collagen super-helix structure. Gly contains no side chain, which allows it to come into the center of the super helix without any steric problems to form a close packing structure (te Nijenhuis, 1997). The superhelix structure is further stabilized by the steric restrictions that are imposed by the pyrrolidine residues and the hydrogen bonds that are formed between amino acid residues. The polypeptide forming collagen is an  chain, which contains about 1000 amino acids and has a MW of around 100 kD depending on the source. The  chain forms a left-handed helix by itself, and three  chains together form a right-handed super triple helix. The conversion of collagen to gelatin yields molecules of varying mass: each is a fragment of the collagen chain from which it is cleaved. Therefore, gelatin is a mixture of fractions varying in MW from 15 to 400 kD (Gelatin Manufacturers Institute of America, 1993). By optimizing the pretreatment and extraction conditions, a gelatin extract with a higher molecular weight distribution can be obtained, where certain inter-chain crosslinkages present in the collagen are destroyed but with less breakage of peptide bonds. The amino acid composition of collagen and gelatin may vary, depending mainly on the source (Tables 13.5 to 13.10). Except for those from sea mammals, marine collagens and gelatins vary according to the living environments of their sources, particularly with respect to water temperature. In general, those collagens and gelatins from warm-water fish have a lower amino acid content than those from mammals, but a higher imino acid content than those from coldwater fish and ice fish; while those from marine invertebrates are much less consistent and differ even between similar species. In addition, the amino acid composition may vary among collagens and gelatins prepared from different tissues and/or with different methods. During the studies on a fresh water fish, Nile perch, Muyonga and coworkers (2004a,b) suggested that the maturation stage of the source could also influence the amino acid composition of the collagen and gelatin extracts. 13.5.2 Physical properties The formation of thermo-reversible gels in water is one of gelatin's characteristics. When an aqueous solution of gelatin with a concentration greater than a

Collagen and gelatin from marine by-products 289 Table 13.5

Ala Arg Asx Cys Glx Gly His Hyl Hyp Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Amino acids Total Reference

Amino acid composition of collagen from ice fish R. glacialis Skin ASC

T. eulepiodotus Skin ASC

T. leonnbergii Skin ASC

83 49 61 ± 77 332 7 3 47 15 45 23 11 16 104 70 30 ± 2 25 151 1000 Rigby (1968)

99 47 63 ± 73 339 8 5 45 13 30 32 12 15 98 67 28 ± 3 23 143 1000 Rigby (1968)

103 47 57 ± 70 350 8 5 47 11 28 32 10 15 100 65 26 ± 4 22 147 1000 Rigby (1968)

ASC, acid soluble collagen.

critical point is cooled down, it may form a gel. The critical gelation concentration and temperature depend mainly on the amino acid composition and the molecular weight distribution of the gelatin. Gel strength is the major physical property of gelatin gels, and the commercial value of gelatins is principally based on their gel strength. Besides the influence of amino acid composition and molecular weight distribution of the gelatin itself, the strength of a gelatin gel also varies with gelatin concentration, thermal history (gel maturation temperature and time), pH, and the presence of any additives (Choi and Regenstein, 2000). In addition, the size and shape of the container used to form the gelatin gel and the parameters of the instrument applied in the determination of gel strength will also affect the final value. Thus, bloom strength, the gel strength determined by the standard bloom method at 10ëC with certain well-defined requirements (Wainewright, 1977) was developed to standardize the test and it has become the most critical standard used to commercially assess the grade and quality of a gelatin. A detailed review of gel strength and its determination can be found in Wainewright (1977). The gel strength of most commercial gelatin varies from less than 100 bloom to more than 300 bloom. Although warm-water fish gelatin gels can

290

Maximising the value of marine by-products

Table 13.6 Amino acid composition of collagen and gelatin from cold-water fish Alaska pollock Cod Skin Swim Skin bladder ASC GB ASC ASC GB Ala Arg Asx Cys Glx Gly His Hyl Hyp Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Imino acids Total Aa Reference

108 51 51 ± 74 358 8 6 55 11 20 26 16 12 95 63 25 ± 3 18 150 1000 a

114 51 54 ± 75 365 6 0 59 9 19 27 12 10 96 65 24 ± 2 11 155 1000 b

114 51 52 ± 75 348 8 8 52 12 22 25 17 13 94 65 24 ± 4 16 146 1000 a

107 51 52 ± 75 345 8 6 53 11 23 25 13 13 102 69 25 ± 5 19 155 1002 c

96 56 52 ± 78 344 8 6 50 11 22 29 17 16 106 64 25 ± 3 18 156 1001 d

Hake Lumpfish Salmon Skin Skin Skin GB

GB

ASC

119 54 49 ± 74 331 10 5 59 9 23 28 15 15 114 49 22 ± 4 19 173 999 d

100 54 53 3 62 333 7 8 65 11 24 28 12 17 112 58 23 ± 3 27 177 1000 e

106 52 52 ± 77 361 10 8 58 10 19 25 16 12 104 51 22 ± 2 15 162 1000 f

ASC, acid soluble collagen; GB, alkaline processed gelatin. References: a, Kimura and Ohno, 1987; b, Zhou and Regenstein, 2005; c, Rigby, 1968; d, GoÂmezGuilleÂn et al., 2002; e, Osborne et al., 1990; f, Matsui et al., 1991.

reach a bloom strength of 300, some cold-water fish gelatin solutions remain liquid at 10ëC (Norland, 1990). As a thermo-reversible gel, a gelatin gel will start melting when the temperature increases above a certain point. This point is called the melting point and is usually lower than the human body temperature. This melt-in-themouth property has become one of the most important characteristics of gelatin gels, and is widely applied in the food and pharmaceutical industries. No other biopolymer has this unique property, although many efforts have been made to find a substitute for gelatin. The melting point of gelatin from cow and pig ranges from 30 to 33ëC, but fish gelatins show lower melting temperatures than mammalian gelatin, due to their lower imino acid content. The melting point for warm-water fish gelatin usually ranges from 23 to 29ëC, while cold-water fish gelatin may melt around 10ëC. Viscosity is the other commercially important property of gelatin samples. Low viscosity gives short, brittle gelatin gels; while high viscosity gives tougher

Collagen and gelatin from marine by-products 291 Table 13.7 Amino acid composition of collagen and gelatin from warm-water fish and hot-water fish Mackerel Skin ASC Ala Arg Asx Cys Glx Gly His Hyl Hyp Ile Leu Lys Met Phe Por Ser Thr Trp Tyr Val Imino acids Total Aa Reference

124 55 49 ± 72 334 6 6 66 9 23 26 14 14 108 44 27 ± 3 20 174 1000 a

Warm-water fish Megrim Puffer fish Sole Skin Skin Skin GB PSC GB 123 54 48 ± 72 350 8 5 60 8 21 27 13 14 115 41 20 ± 3 18 175 1000 b

106 54 50 2 87 351 8 67 12 23 19 14 10 103 48 25 ± 4 17 170 1000 c

122 55 48 ± 72 352 8 5 61 8 21 27 10 14 113 44 20 ± 3 17 174 1000 b

Tilapia* Skin GB

Hot-water fish Lungfish* Skin G

123 47 48 ± 69 347 6 8 79 8 23 25 9 13 119 35 24 ± 2 15 198 1000 d

126 54 44 ± 77 327 5 6 78 10 20 24 3 14 129 42 24 ± 1 18 207 1002 e

* Fresh water fish species. ASC, acid soluble collagen; PSC, pepsin soluble collagen; GB, alkaline processed gelatin; G, gelatin. References: a, Kimura, 1983; b, GoÂmez-GuilleÂn et al., 2002; c, Nagai et al., 2002; d, Sarabia et al., 2000; e, Eastoe and Leach, 1977.

and extensible gels. For many applications, gelatins of high viscosity are preferred and command higher prices, given that other properties are equal (Wainewright, 1977). Besides the chemical properties of the gelatin itself, the viscosity of gelatin depends on its concentration, temperature, pH and the presence of additives. Molecular weight distribution appears to play a more important role in the effect on viscosity than it does on gel strength and melting point. The viscosity of gelatin is normally measured at 60ëC and a 6.6% gelatin concentration, in both Europe and North America. Most commercial gelatins have a viscosity between 15 and 75 mPs (Gelatin Manufacturers Institute of America, 1993; Poppe, 1997). It is worth noting that no single physical property can adequately convey the texture and sensory appreciation of a gel by a consumer, and an overall evaluation of gelatin combining all the critical physical properties may be necessary.

292

Maximising the value of marine by-products

Table 13.8 Amino acid composition of collagen and gelatin from jawless fish and cartilaginous fish

Ala Arg Asx Cys Glx Gly His Hyl Hyp Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Imino acids Total Ala Reference

Jawless fish Hagfish Skin ASC

ASC

Cartilaginous fish Shark Skin PSC G

108 52 46 ± 58 367 8 5 59 13 16 19 16 10 98 67 31 ± 4 23 157 1000 a

123 54 41 0 75 321 9 9 67 19 25 25 16 14 111 43 22 ± 3 23 178 1000 b

124 54 41 0 74 323 9 9 69 19 24 24 16 13 111 42 23 ± 1 24 180 1000 b

119 50 43 ± 66 333 7 5 79 19 24 24 10 14 113 45 26 ± 1 22 192 1000 c

Dogfish Skin ASC 106 51 43 ± 68 338 13 6 60 15 25 27 18 13 106 61 23 ± 3 25 166 1001 d

ASC, acid soluble collagen; PSC, pepsin soluble collage; G, gelatin. References: a, Kimura and Matsui, 1990; b, Yoshimura et al., 2000; c, Eastoe and Leach, 1977; d, Piez et al., 1963.

13.5.3 Nutritional properties Since the nutritional quality of proteins can vary greatly and is affected by many factors, it is important to have standards for evaluating quality. Quick assessment of a protein's nutritive value can be obtained by determining its content of amino acids, and comparing it with the essential amino acid pattern of an ideal reference protein (Fennema, 1996). The ideal pattern of essential amino acids in proteins (reference protein) for preschool children (2±5 years) is used as the standard for all age groups except infants (Table 13.11). Collagen or gelatin completely lacks the essential amino acid Trp and is deficient in several others (Table 13.11), and it has a chemical score of 0 out of 100. Therefore, gelatin alone is of very low nutritive value. Many studies performed to evaluate the nutritional value of collagen/gelatin attest to the inadequacies of dietary collagen/gelatin as a nutritional protein source. It has been suggested that diets

Collagen and gelatin from marine by-products 293 Table 13.9

Ala Arg Asx Cys Glx Gly His Hyl Hyp Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Imino acids Total Aa Reference

Amino acid composition of gelatin from mammals Harp seal Skin GA

Minke whale Skin GA

Pork Skin GA

Cattle Skin GB

108 52 45 0 76 316 6 7 101 10 25 26 5 14 120 38 23 0 4 23 221 999 a

104 53 48 0 80 302 6 9 85 12 28 30 6 15 126 40 29 0 5 22 211 1000 a

112 49 46 ± 72 330 4 6 91 10 24 27 4 14 132 35 18 ± 3 26 223 1003 b

112 46 46 ± 71 333 5 6 98 12 23 28 6 12 129 37 17 ± 2 20 227 1003 b

GA, acid processed gelatin; GB, alkaline processed gelatin. References: a, Arnesen and Gildberg, 2002; b, Eastoe and Leach, 1977.

containing inadequate amounts of essential amino acids might threaten the health of adults and depress the normal growth of children (Fennema, 1996). In addition, over-consumption of any particular amino acid can lead to an `amino acid antagonism' or toxicity. Excessive intake of one amino acid often results in an increased requirement for other essential amino acids. This is due to competition among amino acids for absorption sites on the intestinal mucosa (Fennema, 1996). The nutritional quality of a protein that is deficient in an essential amino acid can be improved by mixing it with another protein that is rich in that essential amino acid, or by supplementing it with essential free amino acids that are under-represented (Fennema, 1996). However, in the case of collagen/gelatin as a major protein in the diet, it will be very difficult to achieve a satisfactory improvement, because collagen/gelatin not only contains no Trp but also is deficient in several other essential amino acids. Although gelatin alone cannot serve as a main dietary protein, as a supplementary protein, it may have some advantages. Gelatin hydrolysates are used

Table 13.10 Amino acid composition of collagen from marine invertebrates Cuttlefish S. lycidas

Ala Arg Asx Cys Glx Gly His Hyl Hyp Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Imino acids Total Aa Reference

Jellyfish

S. officinalis

Sea cucumber Sea urchin

S. meleagris

S. japonicus

A. ijimai

Starfish A. amurensis A. pectinifera

A. planci

Outer skin PSC

Cartilage PSC

Cornea PSC

Exumbrella PSC

Body wall PSC

Test PSC

Body wall PSC

Body wall PSC

Body wall PSC

83 56 64 2 92 318 18

65 47 105 ± 72 326 5 19 126 16 23 8 10 8 96 28 20 ± 2 18 222 994 b

65 46 102 ± 67 348 4 14 127 15 21 7 10 7 94 27 18 ± 2 17 221 991 b

82 52 79 ± 98 309 2 27 40 22 34 38 4 10 82 45 35 0 6 35 122 1000 c

99 52 74 ± 112 278 7 9 48 24 30 17 11 18 85 48 42 ± 14 32 133 1000 d

78 59 61 ± 94 325 5 9 84 11 28 7 12 4 97 56 41 ± 6 23 181 1000 e

113 55 59 0 85 342 2 5 60 17 17 14 13 3 94 69 25 ± 5 22 154 1000 f

123 59 57 0 86 346 2 4 67 11 13 14 14 3 90 48 33 ± 6 24 157 1000 f

128 55 66 1 75 342 2 6 76 15 13 13 10 2 100 39 28 ± 6 23 176 1000 f

90 21 29 13 1 10 98 48 29 ± 5 23 188 1000 a

PSC, pepsin soluble collagen. References: a, Nagai et al., 2001; b, Sivakumar et al., 2003; c, Nagai et al., 1999; d, Saito et al., 2002; e, Omura et al., 1996; f, Kimura et al., 1993.

Collagen and gelatin from marine by-products 295 Table 13.11 Recommended essential amino acid pattern for food proteins and the essential amino acid pattern of gelatin powder Essential Aa

His Ile Leu Lys Met + Cys Phe + Tyr Thr Trp Val Total

Recommended pattern1 (mg/g protein) Infant

Preschool child

26 46 93 66 42 72 43 17 55 434

19 28 66 58 25 63 34 11 35 320

Pork skin gelatin2

Cod skin gelatin2

Tilapia skin gelatin3

6 12 30 38 5 27 20 0 28 165

8 11 22 29 27 19 25 0 18 159

6 8 23 25 9 15 24 0 15 125

1

The ideal pattern of essential amino acids in proteins (reference protein) for preschool children is used as the standard for all age groups except infants, Fennema, 1996; 2 Sarabia et al., 2000; 3 GoÂmez-GuilleÂn et al., 2002.

widely as nutritional supplements due to their high protein content, availability, ease of preparation, ease of addition, and low cost.

13.6

Food applications

Collagen and gelatin are usually used as functional ingredients rather than nutritional ingredients in food applications. 13.6.1 Food applications of collagen Collagen can be used to produce edible casing for sausages. Sausage casings were originally derived from the gastrointestinal tract of cattle, sheep and pig. However, with the rapid growth in demand for sausage products, the collagen casings were developed, and they had some advantages over the natural gut casings, due to the convenience, economic efficiency and uniformity of the regenerated collagen casings (Hood, 1987). Collagen can also be used as a clarification agent to remove the colloidal suspensions during production of alcoholic drinks and fruit juices (Courts, 1977). 13.6.2 Food applications of gelatin Water desserts Gelatin's largest single use is in water gel desserts. Gelatin desserts can be traced back to 1845 when a US patent was issued for `portable gelatin' in

296

Maximising the value of marine by-products

desserts. The current US market for gelatin desserts exceeds 100 million pounds (approximately 45 000 tons) annually. Gelatin desserts consist of mixtures of gelatin powder, sweeteners, acids and compounds to offer the desirable flavor and color. Some other biopolymers, such as agar and carrageenan, can also form thermally reversible gels with water. However, the main difference between gelatin and the polymers from red seaweed is the low melting point of gelatin gels, which is usually lower than the body temperature and makes gelatin gels melt-in-the-mouth. The former study from our laboratory has suggested that gelatin dessert made of gelatin from warm-water fish, which has a lower melting point than mammalian gelatin, showed a better release of aroma and gave a stronger flavor than one made of pork gelatin (Choi and Regenstein, 2000). Furthermore, gelatins of high viscosity give chewier jellies than gelatins of low viscosity, which are more brittle (Jones, 1977). Dairy products Gelatin is used in dairy products as a stabilizer and a texturing agent. It is widely used in yogurt, ice cream and other dairy products. Gelatin is added to yogurt to reduce syneresis and to increase firmness. Gelatin is an ingredient compatible with milk proteins, and gives a fat-like sensory perception because of its unique property of melt-in-the-mouth. It also masks the product flavor less than some other gums (Jones, 1977). Potentially, the use of different concentrations of gelatin would make it possible to obtain a wide range of textures, from the creamy, slightly gelled texture of yogurt to the firm, `moldable' gel of curd. In ice cream, gelatin is used to prevent the formation of coarse ice crystals, and to give body and a firm smooth texture (Jones, 1977). The gelatin concentration required for ice cream depends on its bloom strength and other factors such as melting point (Jones, 1977). Although gelatin has unique characteristics and is widely used in dairy products, there are continuing attempts to replace it with food polysaccharides. This is because the gelatin used in current dairy products is mainly from pig or non-religious slaughtered beef and is unacceptable to Jewish and Muslim consumers. Based on the gelatin status, the dairy products in the market can be divided into two main groups: those where gelatin is replaced with pectin, modified starch, carrageenen, carob bean gum, or locus bean gum; and the other where it does contain a gelatin that is not permitted by any of the major US certifying agencies. Gelatins from kosher and halal fish species, which include both warm-water fish and cold-water fish, may be a promising solution. Some dairy products using fish gelatins have appeared in the market place. Furthermore, fish gelatins can have a broad range of melting points, which may contribute more choices in designing the texture and melting properties of dairy products. Other food products Gelatin has been used in confectionery products such as gummy-type products and marshmallows. Gummy-type products contain gelatin as the main gelling

Collagen and gelatin from marine by-products 297 agent, because it offers the right texture and mouth feel. Marshmallow usually contains about 2±3% gelatin, in which it serves as a stabilizer and whipping agent (Jones, 1977). In recent years, marshmallows made from fish gelatin have become commercially available. Gelatin can also be applied in wine fining and juice clarification, in meat products to absorb meat juices, and to give form and structure to products that would otherwise fall apart (Gelatin Manufacturers Institute of America, 1993). Further information about food applications can be obtained from the reviews by Jones (1977), Johnston-Banks (1990), and Poppe (1997).

13.7

Non-food applications

The non-food uses of collagen and gelatin are in the pharmaceutical industry, photographic industry, and other technical fields such as paper manufacture and printing processes (Gelatin Manufacturers Institute of America, 1993). 13.7.1 Pharmaceutical applications Pharmaceutical gelatin accounts for a significant proportion of the total production (Wood, 1977), and it is used in the manufacture of capsules, tablets and pastilles. The use of gelatin to produce capsules accounts for the largest usage in pharmaceutical applications. Hard gelatin capsules are usually made from high bloom strength gelatin with a small quantity of edible dyes; while soft gelatin capsules are made from a medium grade gelatin with added plasticizers such as propylene glycol, sorbitol, glycerin and other approved mixtures (Wood, 1977; Gelatin Manufacturers Institute of America, 1993). Gelatin can act as a binding agent in tablets; and it is also used for tablet coating to reduce dusting, mask unpleasant tastes, and allow for printing and color coatings for product identification (Gelatin Manufacturers Institute of America, 1993). Glycerinated gelatin or gelatin/gum arabic is used as a base for pastilles, serving as a binder of the ingredients (Wood, 1977). In addition, gelatin is used to produce microencapsulated oils for various pharmaceutical applications; to form a sterile and water insoluble sponge to control bleeding during surgery; and to serve as an adhesive in protective dressings. 13.7.2 Photographic applications Gelatin has been used for photographic emulsions for more than 100 years, and is still the principal constituent of the binder in most commercial photographic films and papers (Kragh, 1977). During the preparation of photographic products that are based on silver halide technology, gelatin prevents flocculation of silver halide crystals in solutions of high ionic strength, facilitates washing of the emulsion, controls the digestion process so that high photographic speeds can be obtained with low fog, and serves many other functions (Kragh, 1977).

298

13.8

Maximising the value of marine by-products

Improving the quality of collagen and gelatin

13.8.1 Extraction process optimization As mentioned earlier in the manufacture section, the quality of collagen and gelatin is influenced not only by the species or tissue from which it is extracted, but also by the extraction process. For each specific source, an optimization of the extraction procedure would be necessary to improve the quality of extracts. One important step for process optimization is to determine the critical control variables. In an earlier study on cod skin gelatin extraction, Gudmundsson and Hafsteinsson (1997) suggested that the alkaline and acid concentrations during pretreatments would affect gelatin quality. In a subsequent study on megrim, GoÂmez-GuilleÂn and Montero (2001) also suggested that the type of acids used in the extraction might affect the gel properties of gelatin from megrim skin. With a fractional factorial design, Zhou and Regenstein (2004) further confirmed the importance of the pretreatment alkaline and acid concentrations on the quality of gelatin extracts. To obtain a high quality gelatin extract, the alkaline concentration should be high enough to remove the noncollagenous proteins and exclude the effect of proteases, and the acid concentration should be in a proper range to offer an optimal weak acid extraction medium (Zhou and Regenstein, 2004, 2005). Too strong an alkaline or acid extraction medium can cause significant degradation of the peptide chains, and result in an extract with poor quality. To guarantee a reasonable yield, the pretreatment temperature and the extraction temperature should also be controlled within a proper range. 13.8.2 Enzymatic modification Collagen and gelatin from fish, especially the cold-water fish, have low gelling temperatures and melting points. However, many applications require gelatin to gel and keep its gel form at room temperature. A possible way of improving the characteristics of a given fish gelatin is to use transglutaminase (TGase). The enzyme TGase can cross-link gelatin chains by catalyzing the reaction between a lysine residue and a glutamine residue, and make covalent chemical crosslinkages within the gelatin network (Babin and Dickinson, 2001). Several studies have been done on the effects of TGase on fish gelatins from megrim, cod or hake (GoÂmez-GuilleÂn et al., 2001; FernaÂndez-DõÂaz et al., 2001; Kolodziejska et al., 2004). The significance of TGase influence varies between different gelatins, and also depends on the enzyme quantity, gelatin concentration, and whether cross-linking occurs predominantly before or after the gelatin gel develops. After covalent cross-linking by TGase, the gelatin gel's characteristic thermo-reversible character is partly or completely lost. To obtain the enhanced gel strength for the final product, TGase should not be added over an appropriate enzyme concentration, and to get the desired cross-linking generally requires that the reaction occurs during or after gel development. Adding a high concentration of TGase or cross-linking before gelation would be detrimental to gel strength (Babin and Dickinson, 2001). Kolodziejska and

Collagen and gelatin from marine by-products 299 coworkers (2004) also suggested that gelatin solutions with a high protein concentration could form better gels using TGase modification than those with a low concentration. Thus, modification with TGase under certain conditions, could improve fish gelatin's properties and produce a gelatin gel at room temperature, even for the cold-water fish gelatin. But for each gelatin, a thorough study on the enzyme activity and optimizing the reaction is necessary. 13.8.3 Removing impurities The extracted marine collagen and gelatin may contain some impurities, such as insoluble particles, inorganic salts, pigments, and compounds responsible for the unpleasant fish flavors. The insoluble particles can be removed by centrifugation and/or filtration; inorganic salts are traditionally removed by ion exchange technology (Gelatin Manufacturers Institute of America, 1993), but they can also be removed by ultrafiltration, which concentrates the extract at the same time (Chakravorty and Singh, 1990; Simon et al., 2002); and pigments and some of the compounds causing the unpleasant fish flavors might be removed by activated charcoal.

13.9

Sources of further information and advice

This chapter only covers a limited introduction to collagen and gelatin from marine by-products. For a more thorough understanding of collagen and gelatin, additional references should be consulted. The oldest review on gelatin was a book entitled Glue and Gelatin by Alexander (1923). Half a century later, Ward and Courts (1977) edited an excellent book of contributed chapters, The Science and Technology of Gelatin, which covers almost every area related to gelatin and is still a very useful source of information for current collagen and gelatin producers and users, and for researchers. The book edited by Pearson, Dutson and Bailey (1987), on the other hand, gives a solid introduction to collagen. The book by Veis (1964) mostly focuses on the chemical properties of collagen and gelatin. For those who are interested in the rheological properties of gelatin, the review article by te Nijenhuis (1997) would be very helpful. There are also several brief reviews on gelatin, including those by Johnston-Banks (1990) and Poppe (1997). In addition, the article prepared by the Gelatin Manufacturers Institute of America (1993) would give a good introduction to gelatin from the point of view of the gelatin manufacturers.

13.10

References

(1923), Glue and Gelatin, New York, NY The Chemical Catalog Company. and GILDBERG A (2002), `Preparation and characterization of gelatine from the skin of harp seal (Phoca groendlandica)', Biores Technol, 82, 191±194.

ALEXANDER J ARNESEN J A

300

Maximising the value of marine by-products

and DICKINSON E (2001), `Influence of transglutaminase treatment on the thermoreversible gelation of gelatin', Food Hydrocolloids, 15, 271±276. BALIAN G and BOWES J H (1977), `The structure and properties of collagen', in Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 1±30. CHAKRAVORTY B and SINGH D P (1990), `Concentration and purification of gelatin liquor by ultrafiltration', Desalination, 78, 279±286. CHOI S S and REGENSTEIN J M (2000), `Physicochemical and sensory characteristics of fish gelatin', J Food Sci, 65, 194±199. COURTS A (1977), `Uses of collagen in edible products', in Ward A G and Courts A, The Science and Technology of Gelatin, New York, NY Academic Press, 395±412. DEVICTOR P, ALLARD R, PERRIER E and HUC A (1995), `Unpigmented fish skin, particularly from flat fish, as a novel industrial source of collagen, extraction method, collagen and biomaterial thereby', US Patent, 5,420,248. EASTOE J E and LEACH A A (1977), `Chemical constitution of gelatin', in Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 73±107. FAO (2002), World Fishery Production, Rome, FAO Fisheries Department. FENNEMA O R (1996), Food Chemistry, New York, Marcel Dekker. Â NDEZ-DIÂAZ M D, MONTERO P and GO Â MEZ-GUILLEÂN M C (2001), `Gel properties of FERNA collagens from skins of cod (Gadus morhus) and hake (Merluccius merluccius) and their modification by the coenhancers magnesium sulphate, glycerol and transglutaminase', Food Chem, 74, 161±167. GELATIN MANUFACTURERS INSTITUTE OF AMERICA (GMIA) (1993), Gelatin, New York, GMIA. GELATINE MANUFACTURERS OF EUROPE (GME) (2004), Market data, http:// www.gelatine.org, date of visiting: 12 Dec. 2004. GILBERT L, RICHARD B, GEORGES T and JACKY D (2002), `Process for the preparation of fish gelatin', US Patent, 6,368,656. Â MEZ-GUILLEÂN M C and MONTERO P (2001), `Extraction of gelatin from megrim GO (Lepidorhombus boscii) skins with several organic acids', J Food Sci, 66, 213±216. Â MEZ-GUILLEÂN M C, SARABIA A I, SOLAS M T and MONTERO P (2001), `Effect of microbial GO transglutaminase on the functional properties of megrim (Lepidorhombus boscii) skin gelatin', J Sci Food Agric, 81, 665±673. Â MEZ-GUILLEÂN M C, TURNAY J, FERNA Â NDEZ-DIÂAZ M D, ULMO N, LIZARBE M A and MONTERO P GO (2002), `Structural and physical properties of gelatin extracted from different marine species: a comparative study', Food Hydrocolloids, 16, 25±34. GROSSMAN S and BERGMAN M (1992), `Process for the production of gelatin from fish skins', U.S. Patent, 5,093,474. GUDMUNDSSON M and HAFSTEINSSON H (1997), `Gelatin from cod skins as affected by chemical treatment', J Food Sci, 62, 37±47. HINTERWALDNER R (1977), `Raw material', in Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 295±314. HOOD L L (1987), `Collagen in sausage casings', in: Pearson A M, Dutson T R and Bailey A J, Advances in Meat Research, Collagen as a Food, New York, Van Nostrand Reinhold, 109±129. JOHNS R and COURTS A (1977), `Relationship between collagen and gelatin', in Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 137±177. BABIN H

Collagen and gelatin from marine by-products 301 (1990), `Gelatine', in: Harris P, Food Gels, New York, Elsevier Applied Science, 233±289. JONES N R (1977), `Uses of gelatin in edible products', in: Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 365±394. KIMURA S (1983), `Vertebrate skin type I collagen: comparison of bony fishes with lamprey and calf', Comp Biochem Physiol, 74B, 525±528. KIMURA S and MATSUI R (1990), `Characterization of two genetically distinct type I-like collagens from hagfish (Eptatretus burgeri)', Comp Biochem Physiol, 95B, 137±143. KIMURA S and OHNO Y (1987), `Fish type I collagen: tissue-specific existence of two molecular forms, (1)22 and 123, in Alaska pollock', Comp Biochem Physiol, 88B, 409±513. KIMURA S, MIYAUCHI Y and UCHIDA N (1991), `Scale and bone type I collagens of carp (Cyprinus carpio)', Comp Biochem Physiol, 99B, 473±476. KIMURA S, OMURA Y, ISHIDA M and SHIRAI H (1993), `Molecular characterization of fibrillar collagen from the body wall of starfish Asterias amurensis', Comp Biochem Physiol, 104B, 663±668. KOLODZIEJSKA I, KACZOROWSKI K, PIOTROWSKA B and SADOWSKA M (2004), `Modification of the properties of gelatin from skins of Baltic cod (Gadus morhua) with transglutaminase', Food Chem, 86, 203±209. KRAGH A M (1977), `Swelling, adsorption and the photographic uses of gelatin', in Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 439±474. MATSUI R, ISHIDA M and KIMURA S (1991), `Characterization of an 3 chain from the skin type I collagen of chum salmon (Oncorhynchus keta)', Comp Biochem Physiol, 99B, 171±174. MUYONGA J H, COLE C G B and DUODU K G (2004a), `Characterisation of acid soluble collagen from skins of young and adult Nile perch (Lates niloticus)', Food Chem, 85, 81±89. MUYONGA J H, COLE C G B and DUODU K G (2004b), `Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin', Food Hydrocolloids, 18, 581±592. NAGAI T and SUZUKI N (2000), `Isolation of collagen from fish waste material ± skin, bone and fins', Food Chem, 68, 277±281. NAGAI T and SUZUKI N (2002), `Preparation and partial characterization of collagen from paper nautilus (Argonauta argo, Linnaeus) outer skin', Food Chem, 76, 149±153. NAGAI T, OGAWA T, NAKAMURA T, ITO T, NAKAGAWA H, FUJIKI K, NAKAO M and YANO T (1999), `Collagen of edible jellyfish exumbrella', J Sci Food Agric, 79, 855±858. NAGAI T, YAMASHITA E, TANIGUCHI K, KANAMORI N and SUZUKI N (2001), `Isolation and characterization of collagen from the outer skin waste material of cuttlefish (Sepia lycidas)', Food Chem, 72, 425±429. NAGAI T, ARAKI Y and SUZUKI N (2002), `Collagen of the skin of ocellate puffer fish (Takifugu rubripes)', Food Chem, 78, 173±177. TE NIJENHUIS K (1997), `Thermoreversible networks: viscoelastic properties and structure of gels', Advances in Polymer Science, 130, 1±267. NOMURA Y, SAKAI H, ISHII Y and SHIRAI K (1996), `Preparation and some properties of type I collagen from fish scales', Biosci Biotech and Biochem, 60, 2092±2094. NORLAND R E (1990), `Fish gelatin', in: Voigt M N and Botta J R, Advances in Fisheries Technology and Biotechnology for Increase Profitability, Lancaster, PA, Technomic Publishing, 325±333. JOHNSTON-BANKS

302

Maximising the value of marine by-products

and KIMURA S (1996), `Occurrence of fibrillar collagen with structure of (1)22 in the test of sea urchin Asthenosoma ijimai', Comp Biochem Physiol, 115B, 63±68. OSBORNE R, VOIGT M N and HALL D E (1990), `Utilization of lumpfish (Cyclopterus lumpus) carcasses for the production of gelatin', In: Voight M N and Botta J K, Advances in Fisheries Technology and Biotechnology for Increased Profitability, Lancaster, PA, Technomic Publishing, 143±153. PEARSON A M, DUTSON T R and BAILEY A J (1987), Advances in Meat Research, Collagens as a Food, New York, Van Nostrand Reinhold. PIEZ K A, EIGNER E A and LEWIS M S (1963), `The chromatographic separation and amino acid composition of the subunits of several collagens', Biochem, 2, 58±66. POPPE J (1997), `Gelatin', in: Imeson A, Thickening and Gelling Agents for Food, New York, Blackie Academic & Professional, 144±168. REGENSTEIN J M (2004), `Total utilization of fish', Food Technol, 58, 28±30. REGENSTEIN J M, LU X W, HERZ J and HOLTZER D (1996), `Kosher/halal fish gelatin', Activities Report of the R & D Associates, 48, 277±278. REGENSTEIN J M, CHAUDRY M M and REGENSTEIN C E (2003), `The kosher and halal food laws', Comprehensive Rev Food Sci Food Safety, 2, 111±127. RIGBY B J (1968), `Amino-acid composition and thermal stability of the skin collagen of the Antarctic ice-fish', Nature, 219, 166±167. SADOWSKA M, KOLODZIEJSKA I and NIECIKOWSKA C (2003), `Isolation of collagen from the skins of Baltic cod (Gadus morhua)', Food Chem, 81, 257±262. SAITO M, KUNISAKI N, URANO N and KIMURA S (2002), `Collagen as the major edible component of sea cucumber (Stichopus japonicus)', J Food Sci, 1319±1322. Â MEZ-GUILLEÂN M C and MONTERO P (2000), `The effect of added salts on the SARABIA AI, GO viscoelastic properties of fish skin gelatin', Food Chem, 70, 71±76. SHAHIDI F (1995), `Seafood processing by-product', in: Shahidi F and Botta J R, Seafood: Chemistry, Processing Technology and Quality, New York, Kluwer Academic Publishers, 320±334. SIMON A, VANDANJON L, LEVESQUE G and BOURSEAU P (2002), `Concentration and desalination of fish gelatin by ultrafiltration and continuous diafiltration processes', Desalination, 144, 313±318. SIVAKUMAR P and CHANDRAKASAN G (1998), `Occurrence of a novel collagen with three distinct chains in the cranial certilage of the squid Sepia officinalis: comparison with shark cartilage collagen', Biochim Biophys Acta, 1381, 161±169. SIVAKUMAR P, SUGUNA L and CHANDRAKASAN G (2003), `Similarity between the major collagens of cuttlefish cranial cartilage and cornea', Comp Biochem Physiol, 134B, 171±180. VEIS A (1964), The Macromolecular Chemistry of Gelatin, New York, Academic Press. WAINEWRIGHT F W (1977), `Physical tests for gelatin and gelatin products', in: Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 507±534. WARD A G and COURTS A (1977), The Science and Technology of Gelatin, New York, Academic Press. WOOD P D (1977), `Technical and pharmaceutical uses of gelatine', in: Ward A G and Courts A, The Science and Technology of Gelatin, New York, Academic Press, 413±437. YOSHIMURA K, TERASHIMA M, HOZAN D and SHIRAI K (2000), `Preparation and dynamic viscoelasticity characterization of alkali-solubilized collagen from shark skin', J OMURA Y. URANO N

Collagen and gelatin from marine by-products 303 Agric Food Chem, 48, 685±690. and LORIMER J W (1960), `The acid-soluble collagen of cod skin', Arch Biochem Biophys, 88, 373±381. YOUNG G E and LORIMER J W (1961), `A comparison of the acid-soluble collagens from the skin and swim bladder of the cod', Arch Biochem Biophys, 88, 183±190. ZHOU P and REGENSTEIN J M (2004), `Optimization of extraction conditions for pollock skin gelatin', J Food Sci, 69, 393±398. ZHOU P and REGENSTEIN J M (2005), `Effects of alkaline and acid pretreatments on Alaska pollock skin gelatin extraction', J Food Sci, 70, C392±396. YOUNG G E

14 Seafood flavor from processing by-products C. M. Lee, University of Rhode Island, USA

14.1

Introduction

In recent years, there has been a significant decrease in fishery resources worldwide due mainly to overfishing coupled with poor management. In response, serious efforts have been made to utilize underexploited resources and processing by-products. In the last few years, several conferences (Keller, 1990; Bechtel, 2002) have been held in the United States to review the progress and developments in the seafood processing by-product utilization. The following is a product list of commercial importance or in development. Finfish: · Fish meal, oil and bone meal · Fish hydrolysate (produced by acid or enzyme) ± Aquaculture feeds ± Silage for animal feeds ± Flavour extracts · Pet foods · Hydrolyzed fish protein (HFP) ± Functional food ingredients ± Aqua-feed for immatured digestion system of juvenile. Shellfish: · Shell meal (e.g., squid, shrimp) for aqua-feeds · Chitin and chitosan from shell · Enzymatic hydrolysate for flavor extracts · Mechanical recovery of shell meat.

Seafood flavor from processing by-products 305 The utilization strategy should take several important factors into consideration. They include resource availability at a low cost, market demand for end products, affordable production cost, and technology availability. In general, the processing by-products make up from a half to two-thirds of the incoming raw material of finfish to shellfish. One of the viable approaches to convert these by-products into commercially profitable products is `seafood flavor manufacturing' since flavor is considered a high value product and good quality seafood flavors are in high demand. The production of natural seafood flavor extracts from the process by-products has been an industrial practice in France and Japan. As a common industrial practice, the natural seafood flavors are reformulated by adding other ingredients and artificial flavors for flavor characteristics desired. Seafood flavors are being used in seafood sauces, chowders, soups, bisques, instant noodles, snacks and surimi seafoods. In seafood entreÂe, the use of good flavored sauce is critical to the acceptability of the meal. An analogy is one may not eat salad without dressing of one's choice. Generally, there are three basic process routes for making seafood flavors from finfish and shellfish processing by-products, namely, aqueous extraction, fermentation, and enzymatic hydrolysis.

14.2

Aqueous extraction

This process has been widely employed since it is simple, generates a good quality flavor, and is often done as part of the existing process. Raw material ! (homogenize) ! cooking ! (press) ! raw extract ! concentration

The steps in parenthesis can be omitted depending upon the process employed. The cooking can be done under either atmospheric or elevated pressure. The good examples of aqueous extraction process are extracts which are generated during cooking of clam, crab or lobster. The drawbacks of this process are a low yield, its dependency on cooking juice, and its high salt level upon concentration. However, it does provide the highest level of flavor and aroma retention of the original material. Ochi (1980) stressed that the freshness of the raw material is the most important requirement in ensuring consistent and high quality finished products. Preparation and product characterization of water-extracted flavors in the form of cooking juice or processing wash water have been reported for clam (Joh and Hood, 1979; Burnette et al., 1983; Reddy et al., 1989).

14.3

Fermentation

Fermentation has been a traditional practice of producing various fish sauces in Asia (Lee et al., 1993). The process involves enzymatic hydrolysis by endogenous enzymes with some level of flavor-producing microbial activity. This makes the flavors derived from fermentation different in flavor characteristics from

306

Maximising the value of marine by-products

other flavor production processes. It requires a very high level of salt; as high as 30% to control the growth of any pathogenic microorganisms (Gildberg, 1993). salt/pH adjustment/starter culture # Raw material ! autolysis (proteolytic digestion) ! (press) ! raw extract ! filtration/clarification

A special interest has been taken to shorten the maturation period from 6±12 months to 1±2 months by lowering pH and salt concentration which hasten proteolysis as well as microbial fermentation. To vary the flavor characteristics, starter cultures of interest can be added at the beginning of fermentation. The resulting extract from the low salt fermentation can be stabilized by concentration or dehydration and used as an umami-giving seafood flavor, not as fish sauce for soup and sauce applications.

14.4

Enzymatic hydrolysis

Enzymatic hydrolysis is a process which is being employed for the production of most commercial natural seafood flavor extracts. enzyme (protease) # Raw stock ! homogenize (pasteurize) ! hydrolysis ! enzyme inactivation by heating ! filtration for raw extract ! concentration/dehydration

As more high-performance commercial enzymes become available, highly acceptable seafood flavors can be mass-produced through hydrolysis of various seafood processing by-products. Most successful products in the market are clam, crab, shrimp, and lobster extracts. Also available are flavors from various fish species such as tuna, bonito, salmon and mackerel. The type of enzyme and hydrolysis conditions used for the fish protein hydrolysate and seafood flavor production may differ from each other. Endoprotease, such as Alcalase (Novozymes North America, Franklinton, NC) prepared from Bacillus spp, is widely accepted for the production of fish protein hydrolysate which prefers a high degree of hydrolysis (DH) for high solubility and digestibility. The technical information on the use of enzyme for the crustacean and fish flavor production is limited. According to the seafood flavor production at Isnard Lyraz (Queven, France) (In, 1990a,b), the process consists of liquefaction, separation, and concentration. Liquefaction is essentially an enzymatic hydrolysis which releases naturally occurring flavoring substances from the raw material and facilitates a separation process by solubilizing insoluble proteins, the main constituent of the seafood process by-products. Separation is to remove shells and bones and is done by filtration, but sometimes accompanied by chromatography which is employed to remove undesirable components such as off-flavors and pigments (In, 1990b). Both endoprotease

Seafood flavor from processing by-products 307 and exopeptidase have to be considered in flavor-producing enzymatic hydrolysis. Enzymatic hydrolysis changes the original amino acid profile and concentration. It also generates peptides of different molecular weights. Amino acids and peptides can further react with reducing sugars or other aldehydes and carbonyl groups in the solution and contribute many novel volatile compounds to the hydrolysate. Thus, enzymatic hydrolysis has a significant effect on both taste and aroma. Enzyme selection and hydrolysis optimization based on the degree of hydrolysis (DH) were studied in the crayfish (Baek and Cadwallader, 1995) and crab processing by-product (Baek and Cadwallader, 1999). Production of protein hydrolysate for flavor from lobster body was studied by Vieira et al. (1995a,b). The tyrosine yield by enzymatic hydrolysis was used as a response in evaluating the effect of different hydrolysis conditions, which included reaction time, enzyme type and enzyme concentration. They concluded that the use of enzymatically-produced hydrolysates as flavorants was potentially promising. Overall, the available technical information on crustacean flavor production from the processing by-products is limited despite ongoing industrial production. The advantages of enzymatic hydrolysis over other methods are high yield, yet good quality with less off-flavor generated, and control of flavor characteristics through variation of enzyme reactions. The key consideration is the selection of the right type of enzyme for a given raw material. In the past, the main problem with enzymatic production of seafood flavor was the formation of the bitter flavor. Now, with new generation enzyme system that comes with both endoprotease and exopeptidase, a flavor can be enzymatically produced without bitterness. Several seafood flavor manufacturers have been successfully producing seafood flavors through enzymatic hydrolysis. Among them are Isnard Lyraz (France), Hasegawa (Japan-USA), Takasago (Japan-USA), and Givoudan Roure (USA). 14.4.1 Source of enzymes for hydrolysate production After evaluation of several commercial proteases for their performance in terms of the organoleptic quality of produced hydrolysates and the rate of hydrolysis, Protamex and Flavourzyme (Novozyme North America, Franklinton, NC) were chosen for further study (Yang and Lee, 2000). Others tested were Alcalase, Neutrase (Novozyme North America, Franklinton, NC), Optimase, and HTProteolytic 200 (Solvay Enzymes, Elkhart, IN). Protamex is a Bacillus protease complex developed for hydrolysis of food proteins. In contrast to most other endoproteases, Protamex is claimed to produce non-bitter protein hydrolysates. It had declared activity of 1.5 Anson units per gram (AU/g) and optimal reaction conditions at pH 5.5±7.5 and 40±60ëC. Protamex can also be used together with Flavourzyme for extensive protein hydrolysis. For this purpose, the dosage of Protamex suggested by the enzyme supplier is 6±15 kg per ton of protein (0.6± 1.5%) at 55ëC for 15±30 min before Flavourzyme is added. Flavourzyme is produced by a strain of Aspergillus oryzae and consists of several enzymes, both

308

Maximising the value of marine by-products

endoproteases and exopeptidases, each with different activities and pH optima. The exopeptidase activities remove terminal amino acids that may cause bitterness. Flavourzyme is best used under neutral or slightly acidic conditions, and its optimal pH and temperature are in the range of 5.0±7.0 and 50±55ëC, respectively. It has a declared activity of 1,000 leucine aminopepeptidase units per gram (LAPU/g), and its suggested usage level is 5±10 LAPU/g protein (0.5± 1.0%) for flavor generation. For extensive protein hydrolysis, a dosage of 10±50 LAPU/g (1±5%) is recommended. 14.4.2 Bitterness generation from enzymatic hydrolysis Flavor production by enzymatic hydrolysis comes with a problem associated with generation of bitterness which is known to be caused by the exposure of the hydrophobic peptides and amino acids. Some type of exopeptidase such as Flavourzyme can break up bitter-causing peptides. To be effective in hydrolyzing and debittering, the enzyme should contain both endoprotease for hydroysis of protein and exopeptidase for breaking down bitter peptides (In, 1990b). 14.4.3 Source of raw material The sources of raw material for enzymatic production of seafood flavor are: Finfish whole or process by-products · white lean fish: fish frames or underutilized species · dark flesh fish: tuna, bonito, mackerel, anchovy, and others Shellfish process by-products · crab, shrimp, clam and lobster The raw material should be of food grade and its quality has to be carefully monitored not to produce inferior finished products. Freshness should be maintained from the point of collection through processing with low microbial counts and histamine content by handling the raw material in the same manner as fresh fish is handled. For this very reason, the term `by-product' rather than `waste' is more appropriate.

14.5 Enzyme-assisted seafood flavors from processing by-products At the University of Rhode Island, a process development was initiated for seafood flavor production from processing by-products including lobster body, red hake frame mince and clam belly, which are available in the New England region. Other species under consideration are crab and shrimp processing byproducts and other underutilized species such as low fat herring and mackerel.

Seafood flavor from processing by-products 309 The process development work focused on seafood flavor production from lobster bodies, red hake frames, and clam processing by-products along with process schemes and variables used for process optimization. The general process consisted of removal of organs and bones, homogenization, enzymatic hydrolysis, filtration to remove unhydrolyzed fractions, and concentration or dehydration. 14.5.1 Lobster flavor extracts from lobster bodies More than 160 million pounds (approximately 73 000 tons) of American lobsters (Homarus americanus) are being landed each year in the United States and Canada combined, of which about 40 million pounds (approximately 18 000 tons) are used to produce canned lobster products (Richardson, 1993; Holliday and O'Bannon, 1997). After removal of the tail and claw meat (56.2%) for canning, the rest (lobster body) constituting 43.8% is usually discarded as process waste. An alternative way to utilize the valuable components remaining in the lobster body is to convert the body meat to hydrolysate as a flavor extract employing enzymatic hydrolysis. As for lobster bodies, 100 kg lobsters yield 44 kg bodies after removal of claws and tails or 21 kg cleaned bodies after removal of carapaces, gills and internal organs. The process of flavor extraction from cleaned bodies consists of grinding to homogenate, proteolytic hydrolysis to liberate flavor-giving free amino acids, enzyme inactivation, filtering, and concentration or dehydration (Fig. 14.1). Variables used for process optimization were selection of a suitable enzyme system, the reaction condition, the degree of hydrolysis, and yield. The flavor quality of the hydrolysate was assessed using free amino acid profile, the ratio of hydrophilic (Gly + Arg + Ala + Pro) to hydrophobic amino acids (Val + Met + Leu + Ile), umami, sweetness, and bitterness. The ratio of hydrophilic to hydrophobic free amino acids was determined as a characteristic free amino acid profile related to the flavor property. Those hydrophilic free amino acids are associated with the characteristic lobster flavor (Yang and Lee, 2000). When applied singly, Flavourzyme (Novozyme North America) generated more free amino acids, higher hydrophilic to hydrophobic amino acid ratio, and less bitter short-chain peptides with a higher overall acceptability than other enzyme systems evaluated. A desirable lobster flavor was produced by pre-extracting the cooking juice and then combining it with the hydrolysate of the juice-removed body homogenate prepared with 0.5% Flavourzyme (on a homogenate weight basis) at 55ëC for 3±5 h. The enzyme used had a declared activity of 1,000 LAPU/g with a suggested usage level of 5±10 LAPU/g protein in substrate (equiv. to 0.5±1.0%). The predominant free amino acids in the hydrolysate of juice-removed body homogenate were Gly, Ala, Arg, and Leu, while those in unhydrolyzed, juiceretaining body homogenate were Arg, Gly, Ala and Pro (>100 mg/100 ml) (Table 14.1). The GMP (guanosine 50 -monophosphate) content was about 0.9 mg/100 g, while small amount of AMP (adenosine 50 -monophosphate) and

310

Maximising the value of marine by-products

Fig. 14.1 A general scheme of enzymatic production of lobster flavor extract and powder. The optimum hydrolysis condititon: 0.5% Flavourzyme (homogenate weight basis) at 55ëC for 5 h.

IMP (inosine 50 -monophosphate) were detected in the concentrated product (0.2 and 0.1 mg/100 g, respectively; ~30% solids). No significant differences were found in the desirability of the hydrolysates prepared by 0.15, 0.5 and 0.83% Flavourzyme, except that the saltiness increased with an increase in the enzyme concentration due to the presence of NaCl in the enzyme preparation. While the hydrolysate prepared with 0.15% Flavourzyme had a significantly chalky mouthfeel, probably due to insufficient hydrolysis at a low enzyme concentration, the one prepared with either 0.5 or 0.83% Flavourzyme did not. Since there was no clear difference in flavor quality between hydrolysates prepared with 0.5 and 0.83%, the 0.5% level of Flavourzyme was chosen for economic reasons. On the other hand, the levels between 0.15 and 0.5% were not examined. It is conceivable that some levels between 0.15 and 0.5% may be as good as 0.5%. For this reason, a further screening could be necessary. Hydrolysis time beyond 5 h decreased the flavor quality. The 9 h hydrolysate at any enzyme concentration had lower acceptability with detectable off-flavor, while 5 h hydrolysate did not have any detectable off-flavor. From the results, it was concluded that the 5 h hydrolysis at 0.5% Flavourzyme and 55ëC gave the highest acceptability. The yield of hydrolysate from body homogenate was 37.8% on a dry weight basis or 84.8% in the protein recovery. The low yield was due to the presence of remaining shell and other insoluble matters.

Seafood flavor from processing by-products 311 Table 14.1 Free amino acid concentration (mg/100 g) of lobster body homogenate cooking juice and the 5 h hydrolysate of juice-removed lobster body homogenate Amino acids Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Ile Leu Phe Lys

Cooking juice

Hydrolysate of juice-removed meat

6.9 33.8 15.1 234.5 15.5 431.4 15.6 147.8 119.7 18.2 22.3 21.1 12.6 31.3 11.3 19.5

24.22 78.43 50.93 145.53 38.01 112.81 55.43 114.56 45.94 52.36 58.83 31.26 50.98 106.64 66.66 93.27

The cooking juice was prepared from lobster body homogenate without enzyme treatment. The hydrolysate was prepared with 0.5% Flavourzyme at 55 ëC for 5 h (n ˆ 2).

Enzymatic hydrolysis released flavor-imparting free amino acids and shortchain peptides. The extent of release and the type of products are determined by the type of enzyme and hydrolysis conditions used. Even though aqueous extraction provides the highest flavor and aroma retention of the original material, its protein recovery is very low without enzymatic hydrolysis, meaning a low product yield. Among factors studied, type and concentration of enzyme had the most significant effects on the free amino acid profile and the TCA (trichloroacetic acid) soluble peptide concentration. The hydrolysate prepared by Flavourzyme generated more free amino acids with a higher hydrophilic to hydrophobic free amino acid ratio and a better acceptability than other commercial enzymes evaluated. The lobster flavor with good quality and high yield was produced by incorporation of the lobster body cooking juice into the hydrolysate from the juice-removed body homogenate (Table 14.2). It was thought that the enzymatic hydrolysis done on the whole homogenate (body meat plus juice) may lead to an adverse change in the flavor profile of the juice during the prolonged hydrolysis process at an elevated temperature, resulting in a flavor quality inferior to that prepared by combining the hydrolysate of juiceremoved body meat with juice. In an effort to bring the free amino acid profile close to that of cooking juice, the appropriate amounts of hydrophilic free amino acids (Gly, Arg, Ala and Pro) were supplemented to the hydrolysate. Such a compensation significantly increased the sweetness and the overall desirability. Sensory scores of the free amino acid-compensated hydrolysate and the non-compensated one are

312

Maximising the value of marine by-products

Table 14.2 Comparison of sensory properties of cooking juice, juice-retaining and removed hydrolysate, and juice added hydrolysate of lobster body homogenate Sweetness Bitterness Cooking juice Juice-retaining hydrolysate Juice-removed hydrolysate Juice-removed hydrolysate + cooking juice (1:1)

Umami

Lobster taste

Off-flavor Desirability

5.9a

1.8a

4.3a

6.9a

1.0a

7.2a

4.7b

2.3b

4.3a

4.8b

1.9b

5.6b

2.6c

1.2a

2.6b

4.8b

1.2a

4.7c

6.5a

1.0a

6.4c

7.4a

1.0a

7.6a

All samples were freeze-dried and prepared with water at 5% concentration before serving. The hydrolysate was prepared with 0.5% Flavourzyme. The intensity or hedonic score was 1 to 9 in which 1 was very weak or very poor and 9 was very strong or excellent. The number of panelists was 8. Different superscripts in the same column indicate significant differences (p < 0:05).

compared in Table 14.3. The same data is presented in a schematic flavor profile (Fig. 14.2). The same processing technique can be adopted for other crustaceans such as crab and shrimp. As for flavor extract from the crab processing by-product, the same processing steps can be employed using Flavourzyme under the similar conditions. 14.5.2 Fish flavor from white fish frames The production of seafood flavor from underutilized fish species through protein hydrolysis is somewhat challenging due to the difficulty of ensuring high organoleptic quality. The hydrolysis of protein often accompanies flavor defects such as bitterness and off-flavor along with the formation of desirable flavor Table 14.3 Comparison of sensory properties of lobster body hydrolysate, hydrolysate compensated with Gly, Arg, Ala and Pro, and cooking juice

Hydrolysate Compensated hydrolysate Cooking juice

Sweetness

Bitterness

Umami

Lobster taste

Off-flavor

Desirability

4.4a

2.3

4.3

4.6a

1.9a

5.6a

5.7b 5.9b

1.8 1.8

4.3 4.3

5.0a 6.9b

1.5a 1.0b

6.6b 7.2b

The hydrolysate was prepared from the cooked lobster body homogenate (juice retaining). All samples were freeze-dried and prepared with water at 5% concentration before serving. The hydrolysate was prepared with 0.15% Flavourzyme. The intensity or hedonic score was 1 to 9 in which 1 was very weak or very poor and 9 was very strong or excellent. The number of panelists was 16. Different superscripts in the same column indicate significant differences (p < 0:05).

Seafood flavor from processing by-products 313

Fig. 14.2 Schematic flavor profile of lobster body hydrolysates with and without free amino acid compensation. Compensation was done to match the free amino acid profile of hydrolysate with that of cook juice. Scored on 1±9 point scale (1 being very weak or very poor and 9 very strong or excellent) (same data given in Table 14.3).

(Kilara, 1985). Among lean fish species, red hake (Urophycis chuss) is found to be a kind of species that produces a uniquely mild pleasant flavor with low fat (0.8%, on a wet weight basis) (Imm and Lee, 1999). Red hake is one of the underutilized fish species found in Northern and Mid-Atlantic coasts of the United States and it is usually marketed only in the fresh form (Gendron, 1980) because of the development of extensive texture hardening during frozen storage. The use of filleting by-products (frame mince) would potentiate a commercial prospect of flavor manufacturing from red hake. Flavor quality of hydrolysates depends on several parameters. The use of fresh raw material is crucial in ensuring good quality flavor, as stressed previously. Fatty fish species are not desirable because of their high susceptibility to lipid oxidation and high cost of removing excess fat (Ritchie and Mackie, 1982). However, the lipid content in fatty fish species varies greatly with season. The fish with a low lipid content (< 5%) prior to spawning can be used. The extent of hydrolysis also determines sensory quality and is dependent upon the specificity of protease, level of enzyme, water-to-substrate ratio, pH and temperature. These parameters affect not only the quality of flavor, but also the yield of hydrolysate. Currently, several commercial proteases are available for the production of protein hydrolysates, and their optimum processing conditions are generally suggested by the manufacturers. However, the selection of suitable proteolytic enzymes and the extent of hydrolysis need to be refined according to the nature of application.

314

Maximising the value of marine by-products

Protein hydrolysate as a flavor extract was prepared from red hake frame mince as well as from the head-and-gutted (H&G) mince using Flavourzyme. A general process flow is given in Fig. 14.3. A water to fish ratio of 1:2 can be used for hydrolysate production at a natural pH of fish without significant loss of yield. The addition of 1.5% NaCl and 0.4% sodium tripolyphosphate (STPP) after 30 min enzymatic hydrolysis improved flavor quality of the hydrolysate by masking bitterness and off-flavor. Hydrolysate produced from the mince on a pilot plant production scale by 3 h hydrolysis at 50ëC with 2% (w/w on a sample protein weight basis) or 0.3% (mince weight basis) Flavourzyme had a highly acceptable quality, suggesting that a good quality fish flavor can be produced from unutilized frame mince (Imm and Lee, 1999). The predominant amino acids were Leu and Arg. The spray-dried or freeze-dried fish mince hydrolysate can be used as flavor supplement for various seafood products such as seafood sauce and chowder. The extensive hydrolysis (degree of hydrolysis, DH > 40%) resulted in increased bitterness intensity, thus necessitating the control of DH below 40% (< 6 h hydrolysis at 50ëC) to obtain an acceptable hydrolysate flavor extract. The comparisons for the sensory quality between frame and H&G mince, salted and unsalted, and hydrolyzed and unhydrolyzed frame mince are given in Table 14.4. In salted hydrolysate preparation from H&G and frame mince, control (cooking juice) showed a slightly higher overall liking score with no significance differences between control and mince hydrolysate, and between

Fig. 14.3 A general process scheme of enzymatic flavor production from red hake mince. The optimum hydrolysis condititon: 2% Flavourzyme (protein weight basis or 0.3% on a mince weight basis) at 50ëC for 3 h and 1:2 water to mince with addition of 1.5% salt and 0.4% STPP.

Seafood flavor from processing by-products 315 Table 14.4 Comparison of sensory properties of hydrolysates prepared from H&G and frame mince of red hake with and without salt added H&G mince Unsalted

Frame mince

Salt mixture1

Salt mixture

Attribute

Con2

Fla3

Con

Fla

Con

Fla

Unhyd4

Fish chowder flavor5 Off-flavor Bitterness Umami Overall liking

7.35ab 5.00a 2.40a 5.30a 4.75a

9.30b 2.43a 2.39a 6.46b 6.38b

10.36a 2.36a 1.34a 7.45 7.00a

11.01a 1.65a 1.44a 7.34a 6.75a

10.29a 2.10a 1.15a 5.83a 6.75a

9.24a 2.40a 2.13a 6.96a 6.38a

6.78b 3.03b 2.80a 3.73b 4.25b

Values are means of scores provided by 8 panelists. 1 A mixture of salt (1.5%) and STPP (0.4%) was added after 30 min hydrolysis. 2 Freeze-dried cooking juice. 3 Freeze-dried hydrolysate prepared with Flavourzyme for 6 h hydrolysis. 4 Unhydrolyzed frame mince was prepared following the same procedure except enzyme addition. 5 Liking as fish chowder flavor. a,b Means with different letters in a row within the same group are significantly different (p < 0:05).

H&G and frame mince. The salt-added mince hydrolysate showed a significantly higher overall acceptability than the unsalted mince hydrolysate. The hydrolysate prepared from frame mince has a flavor quality comparable to that of H&G mince hydrolysate, suggesting that a good quality fish flavor can be produced from unutilized frame mince. The hydrolysate solution has a slightly chalky mouthfeel which affects the overall acceptability. However, this will not be a problem in commercial products because the chalky mouthfeel can be easily masked by other added ingredients. Table 14.4 also shows that the hydrolysate received significantly higher scores in overall liking than the unhydrolyzed frame mince. This is probably due to the difference in the umami score which reflects the presence of umami-giving free amino acids generated by hydrolysis. There was a slight increase in bitterness after hydrolysis without any adverse impact on the overall liking. Barzana and Garcia-Garibay (1994) suggested that the intensity of bitterness depends on the DH and protease specificity because hydrophobic amino acids responsible for bitterness can be liberated by endopeptidase. In order to reduce undesirable sensory attributes such as offflavor and bitterness, a salt mixture was added during hydrolysis. The addition of salt mixture improved sensory quality of hydrolysates by effectively reducing off-flavor and bitterness scores and thus increasing the overall acceptability. This result was consistent with the finding of Gillete (1985) that addition of sodium chloride enhanced fullness and balance of perception while decreased bitterness and off-flavor note. The addition of 0.4% STPP reduced off-flavor and oxidation during storage (Matlock et al., 1984).

316

Maximising the value of marine by-products

14.5.3 Clam flavor production from sea clam processing by-product (clam belly) Sea clam (Spisula solidissima) processing by-product (CPB) is produced as shown in Fig. 14.4. It consists of residual meat (body), belly and viscera remaining after collection of clam juice and meat. The CPB contains 17.13% solid of which 63.7% (or 10.9% on a total CPB weight) is protein. The cooking juice prepared from the CPB still has a mild and sweet clam flavor. There are no critical differences in off-flavor and bitterness of the cooking juice between CPB and clam meat. However, the CPB juice has a slight astringency and less clam flavor and umami taste than the clam meat juice. The hydrolysate strictly from CPB lacks a good clam taste and has a green color and a chalky mouthfeel with a slight bitterness. It is not considered acceptable as a clam flavor. However, a mixture of three parts clam juice and two parts hydrolysate was found to be a right combination in terms of flavor quality and overall liking when it was presented in the chowder form for evaluation. The optimum hydrolysis was achieved with 2% Flavourzyme-2% Nutrase at 55ëC for 4 h which gave complete liquefaction (>15% DH) with 69% yield and the highest umami score without bitterness (Imm and Lee, 2000). As shown in the flow chart (Fig. 14.4), CPB was chopped to facilitate enzymatic hydrolysis and then subjected to heat treatment in a steam cooker. The internal temperature of the sample reached 85ëC in 35 min and final temperature was about 86.5ëC after cooking for 45 min. This precooking is a step to reduce some off-flavor and inactivate undesirable microorganisms. After precooking, samples were cooled down to 55ëC and water (1/5 part of CPB) was added to avoid high viscosity development. The pH of the sample before

Fig. 14.4 Preparation of clam flavor from clam processing by-product. The optimum hydrolysis condititon: 2% Flavourzyme-2% Nutrase at 55ëC for 4 h. The most desirable clam chowder flavor was obtained by blending 3 clam juice and 2 clam belly hydrolysate. Clams shell on (100): soft flesh (37)-shell (63); soft flesh (22 meat: 14 belly).

Seafood flavor from processing by-products 317 hydrolysis was 6.43 and it was close to the optimum pH range of enzymes under evaluation (Flavourzyme: 5.0±7.0, Neutrase: 5.5±7.5 and Alcalase: 6.5±8.5). In order to find the optimum enzyme level and combinations, the DH was determined at different enzyme combinations. For complete sample liquefaction within 4 h hydrolysis, at least 2% (based on sample protein weight) of Flavourzyme was required and the estimated point for complete liquefaction was around 15% DH. The mouthfeel of filtered hydrolysate varied with the particle size of hydrolysate. The hydrolysate was filtered and separated according to the particle size using different sizes of screen sieves. The noticeable chalkiness was effectively controlled after filtering through 250 m sieve. In the preparation of enzyme combinations, 2% Flavourzyme (FZ) (on a sample protein weight basis) was used as the principal enzyme source. Other commercial enzymes, Alcalase (AC) and Nutrase (NT) (Novozyme North America), were evaluated for their performance in assisting hydrolysis reaction in order to increase the yield and reduce the processing time. Based on the yield results, three hydrolysis conditions (FZ (4%), FZ (2%) + NT (2%), and FZ (2%) + AC (1%)) were chosen and the sensory qualities of the resulting hydrolysates were examined. A preliminary sensory evaluation indicated that hydrolysate prepared using 2% FZ +2% NT had a better sensory quality than the other two combinations. The 2% FZ+ 1% AC hydrolysate showed a slightly higher bitterness score while the hydrolysate prepared with 4% FZ alone had a lower umami score. As a major application of CPB hydrolysate, an actual clam chowder was prepared using hydrolysate (2% FZ+ 2% NT) to evaluate its potential to replace clam juice in clam chowder preparation (Table 14.5). When clam chowder was prepared with hydrolysate, a significant difference was found in acceptability in terms of clam chowder flavor and overall liking. The main reason for such a difference was probably due to off-flavor remaining in the hydrolysate. The offflavor of hydrolysate added `fishy taste' to chowder and reduced acceptability. Table 14.5 Comparison of sensory properties of hydrolysate, cooking juice and hydrolysate-cooking juice blend prepared from clam

Hydrolysate2 Clam juice3 Hydro-juice4

Chowder flavor1

Umami

Saltiness

Offflavor

Overall liking

4.2b 6.1a 6.8a

5.8 5.7 5.6

4.4 4.6 4.4

3.6a 1.5b 2.4b

4.3b 6.6a 6.5a

Values are the means of the sensory scores provided by 10 panelists. 1 Acceptability as clam chowder flavor. 2 Hydrolysate prepared from clam belly by Flavourzyme (2%) and Nutrase (2%) for 4 h. 3 Commercial clam juice from Blount Seafood, Warren, RI. 4 A mixture of clam juice and clam-belly hydrolysate at a ratio of 3:2 (v/v). a,b Means with different letter under each attribute are significantly different (p < 0:05).

318

Maximising the value of marine by-products

This off-flavor might have originated from the organ because CPB mainly consists of belly and viscera. The chowder prepared from the hydrolysate showed a significantly higher off-flavor score than others. The chowder prepared with clam juice and hydrolysate blend (3:2 v/v) also had a slightly higher off-flavor than the chowder prepared with clam juice. However, the hydrolysate provided the same level of umami taste and saltiness. When the clam chowder was prepared with a hydrolysate-juice blend, all evaluated sensory characteristics were not different from those of clam juice. The blend even showed a slightly higher score in acceptability as clam chowder flavor. This result suggests that the hydrolysate might have enough background clam flavor and blend well with clam juice without the loss of desirable characteristics. The proximate composition of CPB hydrolysate showed a markedly higher lipid (9.0%) content than clam meat (3.9%) and slightly higher protein (78.5% over 68.5%) on a solid weight basis. Predominant taste active free amino acids (TAFAA) found in hydrolysates were Arg, Ala, Gly, Glu and Met. TAFAA reflects the overall liking of hydrolysates when tested in the form of clam chowder. Identified taste active free amino acids can be appropriately adjusted to provide the most desirable flavor profile. When the cooking juice of clam meat and CPB were prepared by heating at 85ëC for 30 min and free amino acids were determined by the method of Sekiwa et al. (1997), clam meat cooking juice contained more free amino acids than CPB cooking juice. Collection of cooking juice prior to the removal of CPB might be the reason for smaller amounts of free amino acids in CPB. The predominant free amino acids in clam meat cooking juice were Arg, Ala, Gly, Glu and Met. The CPB cooking juice lacked Met in the profile of free amino acids but contained a significant amount of Thr. The overall proportion of free amino acids in clam meat cooking juice, CPB cooking juice and the concentrated commercial clam juice were similar to each other. Based on the above results, Arg, Ala, Gly, Glu and Met are characteristic free amino acids imparting distinct clam flavor and these free amino acids are considered as clam taste active free amino acids. The flavor profile can be characterized by the proportion of taste active free amino acids to the total free amino acids or by the distribution of free amino acids representing sweet, bitter and umami taste. The proportion of calm taste active components linearly increased as the concentration of Flavourzyme increased. When Flavourzyme was used as the only enzyme source, there was no noticeable change in the proportion of sweet or bitter taste free amino acids. The umami taste gradually increased with increasing Flavourzyme concentration. In the production of taste of soup stocks, the umami substance of glutamic acid and 50 -nucleotide in clams play a significant role through the synergistic effect between the two substances. Among the 50 -nucleotide, IMP, GMP and AMP have this synergistic function with glutamic acids (Fuke and Ueda, 1996). When the 50 -nucleotide compounds were determined by the method of McKeag and Brown (1978), four nucleotides including UMP (uridine 50 -monophosphate), AMP, CMP (cytidine 50 -monophosphate) and GMP were found in clam meat whereas only UMP and CDP (cytidine 50 -diphosphate) were detected in CPB.

Seafood flavor from processing by-products 319 CPB contained UMP less than one-third of that in clam meat but contained more CDP than clam meat. The same kinds of nucleotide compounds were found in the corresponding cooking juices. The optimum hydrolysis was achieved with 2% Flavourzyme-2% Nutrase (on a substrate protein weight basis) at 55ëC for 4 h, where Nutrase was required for a higher yield (> 65%) without reducing the desirable flavor profile.

14.6

Flavor-imparting compounds and chemistry

14.6.1 Lobster In both lobster body and meat homogenates, Gly, Arg, Ala and Pro were found to be predominant amino acids, and constituted 86 and 82% of the total detected free amino acids, respectively. Hydrophobic amino acids, such as Met, Val, Leu and Ile, were found to be in low concentrations, and constituted only 5% and 7% of the total detected free amino acids in lobster body and meat, respectively. Glutamic acid (including glutamine) concentration was lower than the predominant amino acids but higher than other amino acid concentrations in the lobster body. When nucleotides were measured in the spray-dried sample (95% solids), the concentrations of AMP, GMP and IMP were found to be 0.73, 2.80 and 0.39 mg/ 100 g, respectively. The 1:7 ratio of IMP to GMP was in agreement with our earlier work on a UF/RO concentrated lobster extract (Jayarajah and Lee, 1999). ATP and ADP, on the other hand, were not detected. Overall, the nucleotide concentrations were very low or undetectable in the product, indicating that nucleotides might not play a significant role in the lobster flavor quality. The importance of free amino acids and short-chain peptides in lobster flavor quality should be noted. In the muscle extract of the Japanese spiny and shovelnosed lobster, 25 different free amino acids were identified (Shirai et al., 1996). Among them, Tau, Gly, Arg, Ala and Pro were predominant, while the hydrophobic amino acids, such as Met, Val, Leu and Ile, were very low. An omission test showed that Gly, Arg, Ala, and Pro were essential for the taste of both species. While the hydrophobic amino acids (Met, Val, Leu and Ile) at low concentrations were essential for the taste of shovel-nosed lobster. These four hydrophobic amino acids contributed bitterness and thick mouthfeel to the shovel-nosed lobster. The concentrations of Gly and Arg in lobster and prawn vary with the season. Hujita et al. (1972a,b) recognized that the decrease in the acceptability of the prawn (Penaeus japonicus) in fall was accompanied by the decrease in glycine. They also noticed that the increase in the acceptability of prawn in winter paralleled the increase in the glycine content. From these results, they assumed that glycine was one of the most important contributors to the acceptability of prawns and lobsters. Shimizu and Hujita (1954) and Hujita et al. (1972a,b) found that lobster containing more free glycine was more palatable. Moreover, they suggested that the other three sweet amino acids (Ala, Pro and Ser) might contribute to the acceptability of the species to some extent,

320

Maximising the value of marine by-products

because the sum of these four amino acids and the palatability were highly correlated. The American lobster has high concentrations of Gly, Arg, Ala and Pro, and a low concentration of Ser. When the juice-removed lobster body homogenate was subjected to enzymatic hydrolysis, the concentration of hydrophobic amino acids increased quickly from their initial low concentrations. Thus the ratio of hydrophilic amino acids to hydrophobic amino acid concentrations decreased, and correspondingly, the acceptability of the hydrolysate decreased. By increasing the hydrophilic to hydrophobic amino acid ratio, the acceptability of the lobster body hydrolysate flavor significantly increased. It indicates that the hydrophilic to hydrophobic amino acid ratio played an important role in the flavor quality of the lobster body hydrolysate. However, it is difficult to preserve the original lobster flavor in the hydrolysate because other factors, such as the short-chain peptides, play an important role in taste quality. The formation of volatile compounds during enzyme hydrolysis also plays an important role in the acceptability of the hydrolysate. The dipeptides or tripeptides, which are soluble in the 7.5% TCA solution are known to be responsible for the bitterness of protein hydrolysate. Matoba and Hata (1972) showed that hydrophobic amino acids give the strongest bitterness when positioned in the interior of the peptide. It gave a slightly weaker bitterness when in the terminal position, and the lowest bitterness intensity as free amino acid. Enzymatic hydrolysis also had a significant impact on the volatile compound profile. Pyrazine concentration increased significantly after enzymatic hydrolysis of crayfish process by-products. The concentrations of dimethyl disulfide, dimethyl trisulfide, and benzaldehyde also increased after enzymatic hydrolysis (Baek and Cadwallader, 1996). Free amino acids and peptides act as precursors of the volatile compounds which are formed through the Maillard reaction. Each amino acid and peptide has its specific contribution to the volatile compound profile (Hayashi et al., 1990; Izzo and Ho, 1992). 14.6.2 Red hake flavor There is no critical difference in the compositions of free amino acids between mince and frame mince. Leu and Arg are dominant free amino acids in mince and frame mince control. Mackie (1982) reported that the amino acids composition is barely changed by digestion except some loss of the sulfur-containing amino acids such as Cys and Met depending on the hydrolysis conditions. However, the composition of free amino acids might be changed by enzyme hydrolysis because enzymes can cleave peptide bonds specifically and liberate amino acids. These free amino acids might be more meaningful than overall amino acids composition in terms of sensory quality because free amino acids contribute to the flavor with their own flavor characteristics. The proportion of, Arg, His and Leu in the hydrolysates decreased while that of Ile, Glu, Lys, Met, Pro, Ser, Thr, and Val increased approximately more than twofold by enzymatic hydrolysis.

Seafood flavor from processing by-products 321 14.6.3 Cooking juice vs. hydrolysate in lobster and crab flavor production Ochi (1980) stated that aqueous extraction is the best way to obtain the original taste and aroma. The purpose of using enzymes was to maximally release the original lobster flavor and increase the yield. In achieving this, one strategy is to extract the cooking juice first and hydrolyze the juice-removed remaining portion, mostly the water insoluble components. The cooking juice and the hydrolysate from the juice-removed body homogenate are combined and subjected to dehydration or concentration. In this way, the `freshness' of cooking juice can be preserved by not subjecting it to a prolonged enzymatic hydrolysis at an elevated temperature. The hydrolysate should not introduce offflavor and other negative properties, such as bitterness and chalkiness. Although water insoluble components in the hydrolysate are not important for flavor generation, they may play an important role in the mouthfeel of the hydrolysate. 14.6.4 Hydrolysis conditions Many studies have been conducted on the optimization of hydrolysis conditions for fish protein hydrolysate, such as the optimum temperature, pH, enzyme concentration, hydrolysis time, and substrate concentration. The purpose of these studies was to find the optimum conditions at which a high DH could be achieved (Baek and Cadwallader, 1995; Hoyle and Merritt, 1994; Martin and Porter, 1995; Shahidi et al., 1995; Vieira et al., 1995a,b). Generally, the higher the enzyme concentration and the longer the hydrolysis time, the higher will be the DH. A typical hydrolysis curve of changes in DH with hydrolysis time is shown in Fig. 14.5. The optimum temperature and pH could usually be obtained from the enzyme manufacturers. Based on the results of the factorial screening (Yang and Lee, 2000), among enzymes tested, Flavozyme was preferred because the hydrolysate had a better flavor quality, and was thicker but not chalky. The Flavourzyme hydrolysate also had a lower TCA soluble peptide concentration, and higher free amino acid concentration and hydrophilic to hydrophobic amino acid ratio. The juice-removed lobster body homogenate had a neutral pH which was within the optimum pH range for Flavourzyme. The optimum temperature was suggested at 50±55ëC by the enzyme manufacturer (Novozyme North America). The protein concentration in the juice-removed lobster body homogenate was 6.3%, in which the concentration of the total TCA soluble peptides was very low. This means that the major substrate for Flavourzyme is TCA insoluble meat proteins. Since it is below 10%, the usual protein concentration, further dilution is not necessary. Thus, the two remaining factors to be considered are enzyme concentration and hydrolysis time. The enzyme usage level is usually suggested by the enzyme supplier. However, due to the difference of the substrate and the purpose of hydrolysis, the optimum enzyme concentration has to be determined. Enzyme concentration and hydrolysis time rendered significant effects on free amino acid concentration, hydrophilic-hydrophobic amino acid ratio and TCA soluble peptide concentration in the lobster body hydrolysate (Fig. 14.6).

322

Maximising the value of marine by-products

Fig. 14.5 Changes in the DH of clam belly meat treated with Flavourzyme at different concentrations. The chopped belly meat was mixed with water (1/5 of meat weight) and hydrolyzed with Flavourzyme at 55ëC for varying time periods.

14.6.5 Concentration and drying methods on flavor quality When hydrolysis reached the optimum time, the enzyme was inactivated by heating to 85ëC and holding for 5 min. The hydrolysate was filtered through a 150 m screen to separate the shell and other residues. Filtrate and cooking juice were combined and dehydrated by freeze-drying, spray-drying, or concentrated by rotary evaporation. Spray-drying was done using an Anhydro spray-dryer (AVP Crepaco, Inc., Tonawanda, NY) at inlet air temperature of 220±230ëC, outlet air temperature of 80±90ëC, feed temperature of 40±50ëC, and a flow rate of 50±60 ml/min. Freeze-drying was carried out using a Virtis freeze-dryer (GPC-2, Virtis Co., Gardiner, NY) at a plate temperature of 30ëC. Rotary evaporation was conducted on a Labconco rotary evaporator (Labconco, Inc., Kansas City, MI) at 45ëC in a water bath and under vacuum which allowed gentle bubbling. Concentration was continued until the mixture of cooking juice and hydrolysate became thick (around 30% solid). Results of the sensory evaluation showed that the freeze-dried and rotary evaporated products had a quality similiar to that of the freeze-dried cooking juice in terms of sweetness, bitterness, umami, and lobster taste (Table 14.6). The overall desirability of the freeze-dried hydrolysate-juice was slightly better than that of the freeze-dried cooking juice and rotary evaporated hydrolysate-

Seafood flavor from processing by-products 323

Fig. 14.6 Changes in the total hydrophilic and hydrophobic free amino acid concentrations with hydrolysis time in the juice-removed lobster body homogenate at different Flavourzyme concentrations at 55ëC. Hydrophilic group (Gly, Arg, Ala and Pro): (ú) 0.15% Flavourzyme (on a homogenate weight basis); (4) 0.5%; () 0.83%; hydrophobic group (Val, Met, Leu, Ile): (n) 0.15% Flavourzyme; (s) 0.5%; (l) 0.83%.

juice. The spray-dried hydrolysate-juice had some burned off-flavor. This is typical of spray-dried product due to the high inlet air temperature and the occurrence of a Maillard reaction, both responsible for being less sweet and more bitter. The products prepared by freeze-drying and rotary evaporation received significantly higher sweetness, umami, lobster taste and overall desirability scores with less bitterness and off-flavor scores than the product Table 14.6 Comparison of sensory properties of the freeze and spray dried, and rotorevaporator concentrated lobster body hydrolysates and freeze-dried lobster body cooking juice Sweetness Bitterness Freeze-dried Spray-dried Rotary-evaporated Freeze-dried cooking juice

Umami

Lobster taste

Off-flavor Desirability

7.2 5.2 6.7

1.4 3.0 1.4

6.9 5.1 6.3

7.5 5.1 6.7

1.0 2.8 1.2

8.3 4.8 7.4

7.8

1.2

6.9

7.0

1.0

7.9

The hydrolysate was prepared from juice-extracted lobster body homogenate with 0.5% Flavourzyme at 55ëC for 5 h and combined with cooking juice before drying or concentrated. All samples were prepared with water at 5% concentration before serving. The intensity or hedonic scale was 1 to 9 in which 1 was very weak or very poor and 9 was very strong or excellent. The number of panelists was 8.

324

Maximising the value of marine by-products

prepared by spray drying. Should the hydrolysate be spray-dried, the mix may require thermoprotective and anti-Maillard reaction agents. Both freeze-dried and spray-dried powders were hygroscopic and thus required stabilizing agents which retard the development of hygrocopicity and allow free flowing during and after an extended storage period. 14.6.6 Species-specific taste active compounds Each species offers its own characteristic flavor which is based on speciesspecific taste active compounds. Those compounds are identified from extractive components which are generally water soluble and low-molecular-weight in nature. They can be divided into nitrogenous and non-nitrogenous compounds. The former includes amino acids, nucleotides, and organic bases, while the latter includes sugars and organic acids (Fuke, 1994). According to Hujita et al. (1972b), glycine and glutamic acid are the most important taste active components which determine the acceptability of lobster and prawn. Alanine, proline and serine are also found be taste active. Konosu (1979) and Hayashi et al. (1981) found that Glu, Gly, Arg, AMP and GMP are the primary taste active components in snow crab with a taste synergism between Gly and 50 -ribonucleotides. The taste active components in clam were Glu, Gly, Arg, Tau, AMP and succinic acid (Fuke and Konosu, 1989). 14.6.7 Formulation of seafood flavor extract Unlike synthetic flavors, so called `natural seafood extracts' of a hydrolysate origin give rather dull and round flavor notes. The intensity of taste-active compounds is somewhat toned-down by the presence of other non-taste active components such as taste neutral amino acids and flavor-binding proteins. To make it distinctive and intense, the flavor needs to be heightened by addition of flavor enhancer, primarily sodium chloride and umami-giving compounds such as MSG (monosodium glutamate), ribonucleotides, or their combination. Nowadays, because of consumer preference for natural ingredients, the natural sources of umami substances rather than MSG are highly preferred. They are so called `natural savory flavors' such as yeast or soy protein hydrolysate. In addition, amino acid and nucleotide profiles as well as other flavor-imparting compounds can be matched with those found in the cooking juice by appropriate supplementation.

14.7

Future trends

The finished fraction of the current enzymatic hydrolysis contains non-flavor imparting components, primarily high molecular weight (MW) proteins, in addition to low MW flavor-giving components, mostly free amino acids, fatty acids and nucleotides. The intensity and purity of flavor can be enhanced by

Seafood flavor from processing by-products 325 removal of non-flavor imparting fractions, especially high MW proteins that tend to bind flavors and subsequently reduce the overall intensity. An additional refining step to remove non-flavor imparting fractions needs to be developed in an effort to produce a flavor that has a greater intensity and a better sensory quality. The non-flavor imparting fractions can also be examined for quantity and any potential commercial use. This refined flavor can be made available to those who need high quality and intense flavor in addition to unrefined hydrolysate flavor which will have its own market. This effort will require a new set of process design using processing units of a larger production capacity and subsequent modifications in processing conditions for process optimization, especially hydrolysis and refining steps.

14.8

References

and CADWALLADER K R (1995), `Enzymatic hydrolysis of crayfish processing by-products' J. Food Sci. 60, 929±935. BAEK H H and CADWALLADER K R (1996), `Volatile compounds in flavor concentrates produced from crayfish-processing byproducts with and without protease treatment' J. Agric. Food Chem. 44, 3262±3267. BAEK H H and CADWALLADER K R (1999), `Optimization of the enzymatic hydroysis of crab processing by-products using Flavourzyme', Food Sci. Biotechnol. 8, 43±46. BARZANA E and GARCIA-GARIBAY M (1994), `Production of fish protein concentrates' in Martin A M, Fisheries Processing, Biotechnological applications, London, Chapman & Hall, 207±222. BECHTEL P J (2002), Advances in Seafood Byproducts: 2002 Conference Proceedings, Fairbanks, Alaska Sea Grant College Program, BURNETTE J A, FLICK G J, MILES J R, ORY R L, ST. ANGELO A J and DUPUY H P (1983), `Characterization and utilization of ocean quahog (Arctica islandica) clam juice as a liquid and dehydrated flavoring agent', J. Food Sci. 48, 353±359. FUKE S (1994) `Taste-active components of seafoods with special reference to umami substances' in Shahidi F and Botta J R, Seafoods: Chemistry, Processing Technology and Quality, London, Blackie Academic & Professional, 115±139. FUKE S and KONOSU S (1989), `Taste-active components of a few species of bivalves' in Kawamura Y, Society for Research on Umami Taste '89 Forum, Tokyo, Society for Umami Research on Umami Taste, 85±91. FUKE S and UDEA Y (1996), `Interactions between umami and other flavor characteristics', Trends Food Sci. Technol. 7, 407±411. GENDRON I S (1980), `Markets for hake' Mar. Fish. Rev. 42, 50±54. GILDBERG A (1993), `Enzymatic processing of marine raw materials', Process Biochem. 28, 1±15. GILLETTE M (1985), `Flavor effects of sodium chloride' Food Technol. 39, 47±52, 56. HAYASHI T, YAMGUCHI K and KONOSU S (1981), `Sensory analysis of taste-active components in the extract of snowcrab meat' J. Food Sci. 46, 479±483, 493. HAYASHI T, ISHII H and SHINOHARA A (1990), `Novel model experiment for cooking flavor research on crab leg meat', Food Rev. Intern. 6, 521±536. HOLLIDAY M C and O'BANNON B K (1997), Fisheries of the United States, p.119, Silver BAEK H H

326

Maximising the value of marine by-products

Spring, MD. and MERRITT J H (1994), `Quality of fish protein hydrolysate from herring (Clupea harengus)', J. Food Sci. 59, 76±79, 129. HUJITA M, ENDO K and SHIMIZU W (1972a), `Studies on muscle of aquatic animalsXXXXVI. Free amino acids, trimethylamine oxide, and betaine in shrimp muscle' Memoirs Faculty of Agriculture, Kinki University 5, 60±67. HUJITA M, ENDO K and SHIMIZU W (1972b), `Studies on muscle of aquatic animalsXXXXVII. Seasonal variation of nitrogenous extractives in shrimp muscle' Memoirs Faculty of Agriculture, Kinki University 5, 70±73. IMM J Y and LEE C M (1999), `Production of seafood flavor from red hake (Urophycis chuss) by enzymatic hydrolysis' J. Agric. Food Chem. 47, 2360±2366. IMM J Y and LEE C M (2000), `Enzyme-assisted production and composition characteristics of clam flavor from clam processing by-product' Annual Meeting of Institute of Food Technologists, Dallas, TX, June 10±14. IN T (1990a), `Seafood flavorants produced by enzymatic hydrolysis' in Voigt M N and Botta J R, Advances in Fisheries Technology and Biotechnology for Increased Profitability, Lancaster, PA, Technomic Publishing, 425±436. IN T (1990b), `Seafood flavorants produced by enzymatic hydrolysis' in Keller S, Making Profits Out of Seafood Wastes. Proceedings of the International Conference on Fish-Products, Fairbanks, AK, Alaska Sea Grant College Program, 197±201. IZZO H V and HO C T (1992), `Peptide-specific Maillard reaction products: a new pathway for flavor chemistry' Trends Food Sci. Technol. 3, 253±257. JAYARAJAH C N and LEE C M (1999), `Ultrafiltration/reverse osmosis concentration of lobster extract' J. Food Sci. 64, 93±98. JOH Y and HOOD L F (1979), `Preparation and properties of dehydrated clam flavor from clam processing wash water', J. Food Sci. 44, 1612±1614. KELLER S (1990), Making Profits out of Seafood Wastes: Proc. International Conference on Fish By-Products, Fairbanks, Alaska Sea Grant College Program, April 25±27. KILARA A (1985), `Enzyme-modified protein food ingredients' Process Biochem. 20, 149± 158. KONOSU S (1979), `The taste of fish and shellfish', in Boudreau, Food Taste Chemistry, ACS Symposium Series, No 115, American Chemical Society, Washington DC, 185±203. LEE C-H, STEINKRAUS K H and ALAN REILLY P J (1993), Fish Fermentation Technology, Tokyo, United Natons University Press. MACKIE I M (1982), `General review of fish protein hydrolysates' Animal Feed Sci. Tech, 7, 113±124. MARTIN A M and PORTER D (1995), `Studies on the hydrolysis of fish protein by enzymatic treatment' in Ansterdam B V and Charalambous G, Food Flavors: Generation, Analysis and Process, Amsterdam, Elsevier Science, 1395±1404. MATLOCK R G, TERRELL R N, SAVELL J W, RHEE K S and DUTSON T R (1984), Factors affecting properties of precooked-frozen pork sausage patties made with various NaCl/ phosphate combinations' J. Food Sci. 49, 1372±1375. MATOBA T and HATA H (1972), `Relationship between bitterness of peptides and their chemical structres' Agric. Biolog. Chem. 36, 1423±1431. MCKEAG M and BROWN P R (1978), `Modification of high-pressure liquid chromatographic nucleotide analysis' J. Chromatography, 152, 253±254. OCHI H (1980), `Production and applications of natural seafood extracts' Food Technol, 34(11), 51±53, 68. HOYLE N

Seafood flavor from processing by-products 327 and BOARDMAN G D (1989) `Characterization and utilization of dehydrated wash water from clam processing plants as flavoring agents', J. Food Sci. 54, 55±59. RICHARDSON E J (1993), `American lobster (Homarus americanus) market study with analysis for a management' PhD Thesis. University of Rhode Island, Kingston, RI. RITCHIE A H and MACKIE I M (1982), `Preparation of fish protein hydrolysates' Animal Feed Sci. Technol. 7, 125±133. SEKIWA Y, KUBOTA K and KOBAYASHI A (1997), `Influence of free sugars by glycolysis on the formation of the characteristic flavor in the brew of cooked clam' J. Agric. Food Chem. 45, 2195±2198. SHAHIDI F, HAN X-Q and SYNOWIECKI J (1995), `Production and characteristics of protein hydrolysates from capelin (Mallotus villosus)' Food Chem. 53, 285±293. SHIMIZU W and HUJITA M (1954), `Studies on muscle of aquatic animals-XXI. On Glycine content in the extractive of shrimp, with special reference to their taste' Nippon Suisan Gakkaishi, 20, 720±725. SHIRAI T, HIRAKAWA Y, KOSHIKAWA Y, TOROSHI H, TERAYAMA M, SUZUKI T and HIRANO T (1996), `Taste components of Japanese spiny and shovel-nosed lobsters' Fish. Sci. 62, 283±287. VIEIRA G H F, MARTIN A M, SAKER-SAMPAIAO S, SOBREIRA-ROCHA C A and GONCALVES R C F (1995a) `Production of protein hydrolysate from lobster (Panulirus spp.)' in Charalambous G, Food Flavors: Generation, Analysis and Process Influence, Amsterdam, Elsevier Science, 1405±1415. VIEIRA G H F, MARTIN A M, SAKER-SAMPAIAO S, SOBREIRA-ROCHA C A, OMAR S and GONCALVES R C F (1995b) `Studies on the enzymatic hydrolysis of Brazilian lobster (Panulirus spp.) processing wastes' J. Sci. Food Agric. 69, 61±65. YANG Y and LEE C M (2000), `Enzyme-assisted bioproduction of lobster flavor from the process by-product and its chemical and sensory properties', in Shahidi F, Seafood in Health and Nutrition ± Transformation in Fisheries and Aquaculture: Global Perspectives, St. John's, Science Tech, 169±193. REDDY N R, FLICK G J, DUPUY H P

15 Fish and bone as a calcium source S.-K. Kim and W.-K. Jung, Pukyong National University, Republic of Korea

15.1

Introduction

Calcium is known to be an essential element required for numerous functions in our bodies including the strengthening of teeth and bones, nerve function and many enzymatic reactions that require calcium as a cofactor. It is also necessary for muscle contraction and regulation of the permeability of sodium ion across cell membranes including those of nerve cells. The concentration of calcium in the blood plasma remains almost constant and varies only slightly over time for a given individual (Allen, 1982; Anderson and Garner, 1996). In various industries such as food, electronics, leather, and others, calcium originates from dolomite, bone meal, and oyster shell and is utilized as an important ingredient. For example, calcium is used to produce acryl resin, make emulsion coagulant in rubber, produce additives in paper making, and make early strengthening agent (concrete strengthening agent and coating material coagulating agent, and others) in the construction industry. Especially in food and agricultural industries, calcium is utilized as a food stuff antiseptic to prevent putrefaction of fruits and vegetables and help the process of cheese making. Although most people are aware of calcium as an important element in their bodies, it is still severely deficient in most diets. Calcium deficiency in the United States has been considered a major cause of osteoporosis, affecting approximately 26 million people annually (Melton, 1995). In 1994, the National Institute of Health (NIH) Consensus Panel revised the recommendations for calcium intake (NIH Consensus Development Conference, 1994). As shown in Table 15.1, the optimal calcium intake has been recommended to be 800 mg/day during childhood below five years of age, 800±1200 mg/day for children from

Fish and bone as a calcium source 329 Table 15.1

Recommended calcium intake for various population groups

Age (years)

Calcium needs

Children Adolescents Adults Elderly <65 on hormonal replacement therapy

800±1200 mg/d 1200±1500 mg/d 1000 mg/d 1500 mg/d 1000 mg/d

Source: NIH Consensus Development.

age six to ten, 1200±1500 mg/day for adolescents or young adults from age 12± 24 and pregnant or lactating women, 1000 mg/day from age 25 to the time of estrogen deprivation or age 65, and 1500 mg/day for elderly people. Generally, the most common and trusted source of calcium (Table 15.2) is milk or other dairy products (Anderson and Garner, 1996). However, some people, especially Asian people, prefer not to consume milk due to lactose indigestion and intolerance which makes them allergic to milk. As an alternative, these people prefer to take calcium-fortified fruit-juice, calcium-rich foods and calcium salt supplements, such as calcium fumarate, citrate, lactate, carbonate, di- and tri-basic phosphate, and gluconate. These salts are available in ingredient forms, each with its own calcium content, solubility, taste and cost issues. The solubility and bioavailability of calcium-containing ingredients are especially important. Although the low pH condition in stomach renders all calcium into its ionic form, precipitation as insoluble calcium phosphate, depending on the amount of phosphate present, can occur in the intestine, where the pH range is 6 to 7. The human body cannot absorb the calcium in precipitated calcium phosphate. In order to improve solubility and bioavailability of calcium, various proprietary blends of calcium salts have been developed with milk protein, food acids and sugar, polysaccharides and calcium/amino acid chelate complexes like Ca-casein phosphopeptides (CPPs), as specific end-use products, depending on their final pH (Allen, 1982). Table 15.2

High-bioavailable calcium sources in foods

Food source

Serving size

Calcium (mg)

Milk and yogurt Cheese Bones in canned sardines and salmon Calcium fortified foods (i.e. orange juice, soy milk, tofu) Dark green, leafy vegetables Nuts and seeds

8 oz or 1 cup 3 ounces 3 ounces 8 ounces

300±450 300±450 181±325 200±300

1/2 cup cooked, 1 cup raw 1 ounce

50±100 25±75

Source: http//:ag.arizona.edu/pubs/ health/az1296.pdf

330

Maximising the value of marine by-products

Annually, more than 50% of the total fishery products (over 120 million tons per year) are discarded as inedible by-products such as bone, skin, fins, internal organs and head. Thus many studies have been performed to utilize a large amount of protein, oil, mineral, carbohydrate and nucleic acid originated from fishery by-products, and improve their functional properties (Nair and Gopakumar, 1982; Rodriguez-Estrada et al., 1994; Nagai and Suzuki, 2000; Kim et al., 2001; Shahidi and Kamil, 2001; Kim et al., 2003). However, the studies on the utilization of organic components or minerals in the fish bone are scarce (Larsen, 2000, 2003; Kim et al., 2003). Recently, as a part of a research to utilize large amounts of fishery byproducts from fish processing, many studies have been carried out to improve functional properties of fishery by-products such as oyster shell, hoki frame, and crab shell by enzymatic modification (Kim et al., 1997a,b,c, 1998a,b, 2001, 2003, 2005; Jeon et al., 2000, 2001a,b, 2002; Jung et al., 2002, 2003, 2005a,b,c, 2006a,b). This chapter focuses on biochemical property of fish bone and utilization of fish bone as a calcium supplement or fortifier.

15.2

Biochemical properties of fish bone

Fish bone consists of both organic and inorganic (mineral) parts. According to the report of Jung et al. (2005a), the organic portion (30.54% on dry basis) of hoki (Johnius belengerii) bone was composed of 28.04% protein, 1.94% lipid and 0.56% carbohydrate. Collagen represented at 86.21% of total protein, and noncollageneous protein content was 13.79%. As reported by Garner et al. (1996), 90% of organic components in the bone matrix are composed of type I collagen, and the remaining 10% consisted of non-collageneous proteins such as osteocalcin, osteopontin, osteonectin, fibronectin, thrombospondin, proteoglycan I/II and growth factors (IGF-1, PDGF, TGF- , etc.). These molecules are produced by osteoblast-like cells and their functions are related to bone formation and cell attachment. Carp (Cyprinus carpio) osteocalcin, known as bone formation factor, was detected and characterized by Nishimoto et al. (2003). The inorganic mineral portion (69.46% on dry basis) was mainly composed of 59.69% of calcium (Ca) and 35.81% of phosphorus (P) with the mole ratio of Ca/P of 1.67. The inorganic portion of vertebrate bone is primarily composed of hydroxyapatite (HA) crystals deposited within an organic matrix of cross-linked collagen fibrils (Anderson and Garner, 1996). The HA crystals make up approximately 60±65% of bone and the HA has an extremely complicated crystaline structure [(Ca2+)10-x(H3O+)2x(PO43-)6(OHÿ)2]. In vertebrate animals, the crystals are usually organized with x value range from 0±2 and 1.67 mole ratio of Ca/P. As reported by Hamada et al. (1995), the skeletal Ca/P mole ratio in 15 species of commercially processed marine teleosts, in Asia, varied within the range of 1.63±1.20 (Table 15.3), and these bones are organized as a combinational structure with hydroxyapatite and beta type Ca3(PO4)2.

Fish and bone as a calcium source 331 Table 15.3

Ca and P composition in various fish bones

Sample species Hokia Sea breamb Norse mackerelb Carpb Sharkb Sardineb Mackerelb Tilefishb Croakerb Trigger fishb Lizard fishb Spanish mackerelb Flying fishb Conger eelb Flat fishb Anchovyb Fowlb Cattleb Swineb Human a b

Ca (% w/w)a

P (% w/w)

Ca/P (molar ratio)

36.2 35.4 35.6 34.9 34.9 35.8 33.9 35.1 35.1 34.4 35.7 34.0 34.4 35.7 35.9 24.4 36.1 36.5 36.9 37.8

16.8 17.5 17.3 16.9 16.9 17.3 17.0 18.4 17.8 17.8 17.8 18.6 17.8 17.7 18.1 16.3 17.4 17.2 17.0 17.1

1.67 1.54 1.60 1.63 1.60 1.63 1.59 1.52 1.56 1.54 1.60 1.47 1.55 1.60 1.57 1.20 1.60 1.64 1.68 1.69

Minerals were calculated according to the ratio of a mineral/total ash. The data were cited in Hamada et al. (1995).

15.3

Utilization of fish bone calcium and organic compound

15.3.1 Degradation of fish bone Skeletal frames discarded from industrial processing of J. belengerii were digested by a heterogeneous enzyme extracted from the intestine of a carnivorous fish (also discarded from industrial processing), bluefin tuna (Thunnus thynnus), in order to utilize the bone to produce nutraceuticals with a high Ca bioavailability (Jung et al., 2005a). Fish bone with well-organized tissue structures containing hard minerals and organic fibers should be degraded. In a previous study (Jung et al., 2005a), fish skeletal frames discarded from industrial processing had been digested by a heterogeneous enzyme extracted from the intestine of a carnivorous fish (also discarded from industrial processing). Further, T. thynnus intestinal enzymes (TICE) could effectively biodegrade the hoki bone matrices composed of collagen, non-collageneous proteins, carbohydrates and minerals using enzymatic degradation system (Fig. 15.1). In addition, specific proteolytic activities of TICE were examined with various substrates; type I collagen as a natural substrate, N-benzoyl-L-tyrosine ethyl ester (BTEE) and N-acetyl-L-tyrosine ethyl ester (ATEE) as a synthetic ester type substrate of -chymotrypsin, N-benzoyl-L-arginine ethyl ester (BAEE) as a synthetic ester type substrate of trypsin and N-benzoyl-DL-argininepnitroanilide (BAPNA) as a synthetic nitroanilide type substrate of trypsin.

332

Maximising the value of marine by-products

Fig. 15.1 Enzymatic degradation system for preparation of soluble calcium from fish skeletal frames.

The TICE could hydrolyze four kinds of synthetic substrates. The activity of TICE was the highest for BAEE, approximately 2.5 U/mg; TICE exhibited potent activity (1.6 U/mg) against BTEE. Collagenolytic activity of TICE was determined as 16.5 U/mg type I collagen. The results indicated that TICE contained have considered tryptic and collangenic enzymes. In a previous study (Kim et al., 1997c), the specific activities of tuna pyloric caeca crude proteinase (TPCCP) were determined using casein as a natural substrate and the same kinds of synthetic substrates. TPCCP showed a specific activity of 0.54 U/mg casein, and its activity was also the highest for BAEE (2.8 U/mg). Some other proteolytic activities have also been elucidated from internal organs of other carnivorous fish species (mackerel, carp, cod, salmon and trout) as heterogeneous or homogeneous proteinases (Gudmundsson and Hafsteinsson, 1997; Shahidi and Kamil, 2001). These can be classified into two large families, serine and aspartic proteinases. Among well known proteinases, pepsin and chymosin are aspartic proteinases, and trypsin, chymotrypsin, collagenase and elastase are serine proteinases. Ramakrishna et al. (1987) reported that generally dogfish enzyme could hydrolyze collagen molecules more efficiently than bovine enzyme regardless of the solubility of collagen. Bezerra et al. (2000) reported that the highest proteolytic activity was found in the stomach, and alkaline activity was greatest in the pyloric caeca of tambaqui (Clolssoma macropomum). In the other methods (Young and Lorimer, 1960; Sato et al., 1989; Miura and Nakano, 1998), acetic acid and citric acid were often treated to recover Ca and protein-like gelatin and collagen from fish bone or bovine bone. Citric acid is widely used for food-grade gelatin from fish because it does not impart objectionable color or odor to the gelatin (Gudmundsson and Hafsteinsson, 1997).

Fish and bone as a calcium source 333 Because of an acid lability of cross-linking in the collagen matrix (Montero et al., 1995), a treatment with acidic solution should be enough to affect solubilization. In detail, acidic treatment can break non-covalent bonds to disorganize the protein structure, thus producing adequate swelling and cleavage of intra- and inter molecular bonds, leading to subsequent collagen solubilization. In particular, acidic treatment at high temperature can easily convert insoluble collagen to gelatin (Stainsby, 1987), as a result Ca can be extracted from its organic matrix. 15.3.2 Calcium solubilization using fish bone peptide Casein phosphopeptides (CPP) derived from the intestinal digestion of casein have been shown to enhance bone calcification in rats (Lee et al., 1980; Tsuchita et al., 1993). Calcium fortifiers like CPP, egg yolk phosphopeptide (phosvitin), and some organic ingredients (citrate, malate, acetate, etc.) have the capacity to chelate Ca ion and to prevent precipitation of Ca-phosphate salts at neutral intestinal pH (Berrocal et al., 1989), thereby increasing the amount of soluble Ca available for absorption across the mucosa (Yuan and Kitts, 1991, 1994). In the previous study (Jung et al., 2005a), fish bone hydrolysate liberated by TICE consisted of 13.36% collagen and 10.25% non-collageneous protein. Phosphoprotein was determined to be 16.65% of non-collageneous protein, and the content of soluble calcium liberated by the TICE 6.55%. Proteins in the hydrolysates were mainly composed of Gly, Thr, Glx, Ala, Asx, Ser, Hyp, and Arg. As reported by Jiang and Mine (2000), Ca-binding phosphoproteins such as osteocalcin, phosvitin and casein phosphoprotein mainly consist of Ser, Thr, Ala and Tyr residues phosphorylated or bound to Ca (Houben et al., 1999). Pro, Gly, Pro and Hyp residues are known as typical amino acids of collagen (Edwards and O'Brien, 1980). Calcium-binding phosphoproteins derived from noncollageneous materials in the bone have a high affinity to Ca2+ on the surface of hydroxyapatite (Hoang et al., 2003). As shown in Fig. 15.2, the fish bone phosphopeptides (FBP I < 1 kDa of MW; 1 < FBP II < 5 kDa; FBP III > 5 kDa) could inhibit the formation of insoluble calcium phosphate, as measured by the calcium contents of the supernatant after the formation of Ca-FBP complex. Calcium binding activity of the FBP II was similar to that of casein oligophosphopeptide (CPP). The solubility of Ca was dependent on the concentration of FBP, and 41.06 mg/l of Ca was obtained at a concentration of 250 mg/l. The pH of the reaction system was maintained at 7.8, because low pH could increase the solubility of the insoluble calcium salt. Various concentrations up to 500 mg/l were mixed with 5mM CaCl2 and 20 mM sodium phosphate buffer (pH 7.8). The mixture was stirred at 22ëC for 30 min, and the pH was maintained at 7.8 in the buffer system. When the pH changed, it was adjusted with 6 M HCl or NaOH and monitored by a pH meter. After removal of insoluble calcium phosphate salts by filtration with a 0.45 mm membrane, Ca contents of the supernatant fraction were determined by flame atomic absorption spectrometry. The experiments were performed in triplicate.

334

Maximising the value of marine by-products

Fig. 15.2 In vitro assay for calcium solubility.

Values are means, with standard deviations represented by vertical bars. (l), Control; ( ), FBP I; (t), FBP II; (5), FBP III; (n), casein phosphopeptide. As reported by Jiang and Mine (2000), the solubility of 36.3 mg/l of Ca was obtained at 200 mg/l of the oligophosphopeptide from egg yolk phosvitin with 35% phosphate retention, and the solubility was higher than that of commercial CPP II (Meiji Seika Co., Ltd., Tokyo, Japan). According to the data of amino acid composition of the FBP, the relative contents of Gly, Pro, Hyp, and known typical collageneous amino acids, were significantly lower than those of hoki bone hydrolysates. However, the contents of Thr, Ser, Glx, and Ala, which are phosphorylated or Ca2+-binding, showed remarkable increments as compared to the bone hydrolysates. Nishimoto et al. (2003) isolated and characterized an osteocalcin from carp Cyprinus carpio, and carp osteocalcin consisted of a high proportion of Ala, Tyr, Thr, Gln, and Asp. The calcium binding oligophosphopeptide prepared from hen egg yolk phosvitin by Jiang and Mine (2000) mainly consisted of Ser, Asx, Glx and Arg. They reported that phosphoseryl groups in the oligophosphopeptide played an essential role in Ca2+-phosphopeptide interaction. As reported by Hoang et al. (2003), specific residues of osteocalcin implicated in HA binding are located on the same surface of helix chain, coordinate five Ca2+ in an elaborate network of ionic bonds. These five Ca2+ are sandwiched between two crystallographically related osteocalcin molecules from bone tissue and show both monodentate and malonate modes of chelation with extensive bridging (Fig. 15.3).



Fish and bone as a calcium source 335

Fig. 15.3 Hypothetical pattern of calcium-peptide binding after enzymatic digestion. Crystallographic dimer interface. Light and dark distinguish the two molecules. Spheres and the broken lines represent Ca2+ ions and ionic bonds, respectively.

15.4 In vivo availability of soluble calcium complex from fish bone In vivo effects of FBP II on Ca bioavailability were further studied in the ovariectomized rats (Jung et al., 2006b). Menopause is a time when oestrogen deficiency leads to accelerated bone resorption and negative bone balance. The present study was undertaken to evaluate the beneficial effects of FBP as a Ca fortifier in osteoporosis induced by ovariectomy and a concurrent low-Ca diet. During the experimental period corresponding to the menopause with osteoporosis disease, the loss of bone mineral (Ca) was decreased by FBP II supplementation in the ovariectomized rats. After the low-Ca diet, the FBP II Table 15.4

Effects of calcium fortifier intakes in the ovariectomized rats

Experimental groups (n=8 per group)

Control Mean

Body weight gain (g/d) 14.4 Food intake (g/d) 12.9 Ca intake (mg/d) 54.8 Fecal Ca (mg/d) 53.3a Urinary Ca (mg/d) 0.9 Ca retention* (mg/d) 0.2a Femoral total Ca (mg) 143a Femur length (mm) 33.9 Femur wet weight (g) 1.12a Bone mineral density (BMD) 0.161a 2 of the distal femur (g/cm ) Breaking force (kg) 3.96a

SD

CPP Mean

1.5 14.2 0.9 13.0 5.9 54.2 3.4 44.8b 0.4 1.4 0.5 8.0b 7 153b 0.5 34.8 0.06 1.32b 0.018 0.229b 0.57

8.97b

FBP II SD

Mean

SD

2.5 13.9 1.9 1.3 12.5 1.5 4.8 54.4 5.5 4.2 46.5ab 3.6 0.5 1.5 0.5 1.2 6.4b 1.7 9 155b 10 0.4 34.3 0.6 0.05 1.22ab 0.06 0.029 0.213b 0.025 1.03

8.48b

0.97

a,b Mean values within a row with unlike superscript letters were significantly different (P < 0.05). * Calcium retention (balance) was calculated as: Ca intake ± fecal Ca ± urinary Ca.

336

Maximising the value of marine by-products

diet, including both normal levels of Ca and vitamin D, significantly decreased Ca loss in faeces and increased Ca retention as compared with the control (Table 15.4). The levels of femoral total Ca, bone mineral density, and breaking strength were also significantly increased by FBP II diet to a level similar to those of the CPP diet group (no difference; P < 0.05). It illustrates that increased Ca retention by FBP II intake led to the prevention of mineral loss in the osteoporosis-modelling rats. As reported by Larsen et al. (2000, 2003), the intake of small fish with bones can increase Ca bioavailability, and the small fish may be an important source of Ca, especially in population groups with low intakes of milk and dairy products. In the present study, the results proved the beneficial effects of fish-meal in preventing Ca deficiency due to increased Ca bioavailability by FBP intake. Furthermore, it is possible to provide a novel nutraceutical with a high bioavailability for Ca to oriental people with lactose indigestion and intolerance and Ca-fortified supplements, such as fruit juice or Ca-rich foods, as alternatives to dairy products.

15.5

Acknowledgements

This research was supported by a grant (p-2004-01) from Marine Bioprocess Research Center of the Marine Bio 21 Center funded by the Ministry of Maritime Affairs and Fisheries, Republic of Korea.

15.6

References

(1982). Calcium bioavailability and absorption: A review. Am. J. Clin. Nutr. 35, 738±808. ANDERSON, J. J. B. and GARNER, S. C. (1996). Calcium and phosphorous nutrition in health and disease. In Anderson, J. J. B. and Garner, S. C. (eds) Calcium and Phosphorous in Health and Disease, pp. 1±5. New York: CRC Press. BERROCAL, R., CHANTON, S., JUILLERAT, M. A., PAVILLARD, B., SCHERZ, J. C. and JOST, R. (1989). Tryptic phosphopeptides from whole casein. II. Physiochemical properties related to the solubilization of calcium. J. Dairy Res. 56, 335±341. BEZERRA, R. D. S., SANTOS, J. F. D., LINO, M. A. D. S., VIEIRA, V. L. A. and CARVALHO JR. L. B. (2000). Characterization of stomach and pyloric caeca proteinases of tambaqui (Colossoma macropomum). J. Food Biochem. 24, 189±199. EDWARDS, C. A. and O'BRIEN, JR. W. D. (1980). Modified assay for determination of hydroxyproline in a tissue hydrolysate. Clin. Chim. Acta 104 (2), 161±167. GARNER, S. C., ANDERSON, J. J. B. and AMBROSE, W. W. (1996). Skeletal tissues and mineralization. In Anderson, J. J. B. and Garner, S. C., Calcium and Phosphorous in Health and Disease, pp. 97±117. New York: CRC Press. GUDMUNDSSON, M. and HAFSTEINSSON, H. (1997). Gelatin from cod skins as affected by chemical treatments. J. Food Sci. 62 (1), 37±39. ALLEN, L. H.

HAMADA, M., NAGAI, T., KAI, N., TANOUE Y., MAE, H., HASHIMOTO, M., MIYOSHI, K., KUMAGAI, H.

and

SAEKI, K.

(1995). Inorganic constituents of bone of fish. Fisheries Science

Fish and bone as a calcium source 337 63(3), 517±520.

and YANG, D. S. C. (2003). Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425 (30), 977±980. HOUBEN, R., JIN, D., STAFFORD, D., PROOST, P., EBBERINK, R. and VERMEER, C. (1999). Osteocalcin binds tightly to the -glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins. Biochem. J. 344, 265±269. JEON, Y. J., BYUN, H. G. and KIM, S. K. (2000). Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes. Proc. Biochem. 35, 471±478. JEON, Y. J., PARK, P. J. and KIM, S. K. (2001a). Antimicrobial effect of chitoligosaccharides produced by bioreactor. Carbohydrate Polymers 44, 71±76. JEON, Y. J. and KIM, S. K. (2001b). Potential immuno-stimulating effect of antitumoral fraction of chitosan oligosaccharides. J. Chitin Chitosan 6, 163±167. JEON, Y. J. and KIM, S. K. (2002). Antitumor activity of chitosan oligosaccharides produced in ultrafiltration membrane reactor system. J. Microbiol. Biotechnol. 12, 503±507. JIANG, B. and MINE, Y. (2000). Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin. J. Agric. Food Chem. 48, 990±994. JUNG, W. K., JE, J. Y., KIM, H. J. and KIM, S. K. (2002). A novel anticoagulant protein from Scapharca broughtonii. J. Biochem. Mol. Biol. 35, 199±205. JUNG, W. K., PARK, P. J. and KIM, S. K. (2003). Purification and characterization of a lectin from the hard roe of skipjack tuna. Inter. J. Biochem. Cell Biol. 34, 255±265. JUNG, W. K., PARK, P. J., BYUN, H. G., MOON, S. H. and KIM, S. K. (2005a). Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase. Food Chem. 91, 333±340. JUNG, W. K., PARK, P. J., BYUN, H. G., MOON, S. H. and KIM, S. K. (2005b). Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase. Food Chem. 91, 333±340. JUNG, W. K., RAJAPAKSE, N. and KIM, S. K. (2005c). Antioxidative activity of low molecular peptide derived from the sauce of fermented blue mussel, Mytilus edulis. Eur. Food Res. Technol. 220, 535±539. JUNG, W. K., MENDIS, E., JE, J. Y., PARK. P. J., SON, B. W., KIM, H. C., CHOI, Y. K. and KIM, S. K. (2006a). Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chem. (doi:10.1016/j.foodchem.2004.09.048). JUNG, W. K., LEE, B. J. and KIM, S. K. (2006b). Fish bone peptide increases Ca solubility and bioavailability in ovariectomized rats. British Journal of Nutrition (doi: 10.1079/ BJN20051615). KIM S. K., LEE, C. K., BYUN, H. G., JEON, Y. J., LEE, E. H. and CHOI, J. S. (1997a). Synthesis and biocompatibility of the hydroxyapatite ceramic composites from tuna bone(I) ± The sintering properties of hydroxyapatite and hydroxyapatite-containing wollastonite crushed with dry milling process. J. Korean Ind. and Eng. Chem. 8(6), 994±999. KIM S. K., CHOI, J. S., LEE, C. K., BYUN, H. G., JEON, Y. J. and LEE, E. H. (1997b). Synthesis and biocompatibility of the hydroxyapatite ceramic composites from tuna bone(II) ± The sintering properties of hydroxyapatite treated with wet milling process. J. Korean Ind. and Eng. Chem. 8(6), 1000±1005. KIM, S. K., JEON, Y. J., BYUN, H. G., KIM, Y. T. and LEE, C. K. (1997c). Enzymatic recovery of cod frame proteins with crude proteinase from tuna pyloric caeca. Fisher. Sci. 63, 421±427. HOANG, Q. Q., SICHERI, F., HOWARD, A. J.

338

Maximising the value of marine by-products

and KIM, S. K. (1998a). Effect of calcium compounds from oyster shell bound fish skin gelatin peptide in calcium deficient rats. J. Korean Fish. Soc. 31(2), 149±159. KIM, S. K., CHOI, J. S., LEE, C. K., BYUN, H. G., JEON, Y. J., LEE, E. H. and PARK, I. Y. (1998b). Synthesis and biocompatibility of the hydroxyapatite ceramic composites from tuna bone(III) ± SEM photographs of bonding properties between hydroxyapatite ceramics composites in the simulated body fluid. J. Korean Ind. and Eng. Chem. 9(3), 322±329. KIM, S. K., KIM, Y. T., BYUN, H. G.., NAM, K. S., JOO, D. S. and SHAHIDI, F. (2001). Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin. J. Agri. Food Chem. 49 (4), 1984±1989. KIM, S. K., PARK, P. J., BYUN, H. G., JE, J. Y., MOON, S. H. and KIM, S-.H. (2003). Recovery of fish bone from hoki (Johnius belengerii) frame using a proteolytic enzyme isolated from mackerel intestine. J. Food Biochem. 27 (3), 255±266. KIM, S. K., PARK, P. J., YANG, H. P. and HAN, S. S. (2005). Subacute toxicity of chitosan oligosaccharide in Sprague-Dawley rats. Arzneim.-Forsch./Drug Res. 51, 769±774. LARSEN, T., THILSTED, S. H., KONGSBAK, K. and HANSEN, M. (2000). Whole small fish as a rich calcium source. Br. J. Nutr. 83, 191±196. LARSEN, T., THILSTED, S. H., BISWAS, S. K. and TETENS, I. (2003). The leafy vegetable amaranth (Amaranthus gangeticus) is a potent inhibitor of calcium bioavailability and retention in rice-based diets. Br. J. Nutr. 90, 521±527. LEE, Y. S., NOGUCHI, T. and NAITO, H. (1980). Phosphopeptides and soluble calcium in the small intestine of rat given a casein diet. Br. J. Nutr. 43, 457±467. MELTON, L. J. (1995). How many women have osteoporosis now? J. Bone Mineral Res. 10, 175±177. MIURA, T. and NAKANO, M. (1998). Calcium bioavailability of a total bone extract (TBE) and its effects on bone metabolism in rats. Biosci. Biotechnol. Biochem. 62, 1307± 1312. MONTERO, P., ALVAREZ, C., MARTI, M. A. and BORDERIAS, J. J. (1995). Plaice skin collagen extration and functional properties. J. Food Sci. 60(1), 1±3. NAGAI, T. and SUZUKI, N. (2000). Isolation of collagen from fish waste material-skin, bone and fins. Food Chem. 68, 277±281. NAIR, A. L. and GOPAKUMAR, K. (1982). Soluble protein isolate from low cost fish and fish wastes. Fishery Technol. 19, 101±103. NIH CONSENSUS DEVELOPMENT PANEL ON OPTIMAL CALCIUM INTAKE (1994). JAMA 272 (24), 1942±1948. NISHIMOTO, S. K., WAITE, J. H., NISHIMOTO, M. and KRIWACKI, R. W. (2003). Structure, activity, and distribution of fish osteocalcin. J. Biol. Chem. 278 (14), 11843±11848. RAMAKRISHNA, M., HULTIN, H. O. and ATALLAH, M. T. (1987). A comparison of dogfish and bovine chymotrypsin in relation to protein hydrolysis. J. Food Sci. 52, 1198±1202. RODRIGUEZ-ESTRADA, M. T., CHUNG, S. and CHINACHOTI, P. (1994). Solids extraction of cod frame and effects on ultrafiltration of the aqueous extract. J. Food Sci. 59, 799± 803. SATO, K., YOSHINAKA, R. and SATO, M. (1989). Hydroxyproline content in the acid-soluble collagen collagen from muscle of several fishes. Bulletin of the Japanese Society of Scientific Fisheries, 55, 1467. SHAHIDI, F. and JANAK KAMIL, Y. V. A. (2001). Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci. Technol. 12, 435±464. STAINSBY, G. (1987). GELATIN GELS. IN PEARSON, A. M., DUTSON, T. R. and BAILEY, A. J. (eds), KIM, G. H., JEON, Y. J., BYUN, H. G., LEE, C. K., LEE, E. H.

Fish and bone as a calcium source 339 Advances in Meat Research. Vol. 4. Collagen as a Food, pp. 209±222. New York: Van Nostrand Reinhold Co. Inc. TSUCHITA, H., SEKIGUCHI, I., KUWATA, T., IGARASHI, T. and EZAWA, I. (1993). The effect of casein phosphopeptides on calcium utilization in young ovariectomized rats. Z. ErnaÈhrungswiss 32, 121±130. YOUNG, E. G. and LORIMER, J. W. (1960). The acid-soluble collagen of cod skin. Arch. of Biochem. Biophys. 88, 373±381. YUAN, Y. V. and KITTS, D. D. (1991). Conformation of calcium absorption and femoral utilization in spontaneously hypertensive rats fed casein phosphopeptide supplemented diets. Nutr. Res. 11, 1257±1272. YUAN, Y. V. and KITTS, D. D. (1994). Calcium absorption and bone utilization in spontaneously hypertensive rats fed on native and heat-damaged casein and soyabean protein. Br. J. Nutr. 71, 583±603.

16 Chitin and chitosan from marine by-products F. Shahidi, Memorial University of Newfoundland, Canada

16.1

Introduction

Chitin is a major component of the exoskeleton of invertebrates, crustaceans, insects and the cell wall of fungi and yeast and acts as a supportive and protective component (Knorr, 1984; Lower, 1984; Tan et al., 1996). Chitin is the second most abundant natural polymer on Earth after cellulose (Brzeski, 1987; Ornum, 1992). At least 10 gigatonnes (1  1013 kg) of chitin are produced and hydrolyzed each year in the biosphere (Muzzarelli, 1999). Chitin, poly-(164)-Nacetyl-D-glucosamine, is a cellulose-like biopolymer found in a wide range of products in nature (Shahidi et al., 1999). Chitin is biosynthesized from polymerization of uridine diphosphate-N-acetyl-D-glucosamine by chitin synthase (EC 2.4.1.16) (Hirano, 1996). Chitosan, a copolymer of mainly D-glucosamine and a small proportion of Nacetyl-D-glucosamine with -(164) linkage, is obtained by alkaline or enzymatic deacetylation of chitin and is also an abundant polymeric product in nature. Chitosan was first discovered in the nineteenth century when chitin was heated to boiling in a concentrated KOH solution (Dunn et al., 1997). Chitosan is found in different morphological forms such as a primary, unorganized structure, crystalline and semicrystalline forms. For different reasons, especially problems of environmental toxicity, these two biopolymers are considered to be interesting substances (Sorlier et al., 2001). Owing to their unique structures, they possess high biological and mechanical properties as they are biorenewable, biodegradable, and biofunctional (Hirano et al., 2000). Both chemical and enzymatic methods are known for preparation of chitin, chitosan and their oligomers, with different degrees of deacetylation, polymerization and molecular weight. Chitin, chitosan

Chitin and chitosan from marine by-products 341 and their oligomers can be produced chemically using concentrated HCl followed by column chromatographic fractionation (Jeon et al., 2000). Three methods are known for modification of the process of isolation of chitin and chitosan oligomers (Jeon et al., 2000). These are acetolysis, fluorohydrolysis and sonolysis. Meanwhile, chitin and chitosan oligomers can be prepared through microbiological and fungal treatments (enzymatic preparation). Chitin and chitosan may be degraded by certain enzymes such as chitinases and chitosanases, respectively, and the process is environmentally friendly. Chitin, chitosan and their oligomers may be employed in medical uses as wound-healing agents, as dietary and hypocholesterolemic agents, anti-tumor and anti-ulcer compounds and as coating of artificial parts of the body such as leg, tooth and arm, among others. They may also be used in food preservation such as for seafoods (Shahidi et al., 1999) and fruits (El Ghaouth et al., 1992a,b) as well as for acidity adjustment (Scheruhn et al., 1999) and as antibacterial and antifungal agents (Shahidi et al., 1999). This chapter provides a cursory account of the chemistry and uses of chitin, chitosan and their oligomers.

16.2

Chemical characteristics

16.2.1 Structure and properties of chitin and chitosan Chitin is a high-molecular-weight polymer of 1000±3000 units of N-acetyl-Dglucosamine (NAG) linked together by -D (164) bonds (Lower, 1984). The chemical structure of chitin is the same as that of cellulose with the hydroxyl group at position C-2 replaced by an acetamido group (Fig. 16.1). Chitin can be deacetylated to produce chitosan, which is soluble in dilute acidic solutions and is highly viscous when dissolved; this makes it distinctly different from chitin (Jeon et al., 2000; Shahidi et al., 1999). Chitin and chitosan are different in their solubility characteristics. There are few solvents for chitin, whereas almost all aqueous acids dissolve chitosan. Most solvents, namely dimethylformamide, lithium chloride, hexafluoroacetone and hexafluoroisopropanol, among others used for dissolution of chitin are toxic and hence cannot be used in food processing applications. Nonetheless, when chitin is ground to a fine mesh, it could be used to increase viscosity of liquids. Solvents for chitosan, such as acetic acid, glutamic acid and glutamine acids, are generally safe to consume, allowing the formation of solutions that are appropriate for gel production. Thus, chitosan is better matched to the viscosity of foods (Winterowd and Sandford, 1995). The solubility characteristics of chitosan are generally dictated by the extent of N-acetylation, the distribution of acetyl groups, the pH and the ionic strength (Anthonsen et al., 1993). The amino group in chitosan has a pKa value of 6.2±7.0, which makes chitosan a polyelectrolyte at low pH values (Claesson and Ninham, 1992). Recently, it was reported that a highly deacetylated chitin with a number of acetylated chitin joining each other in a block and then a number of units with free amino groups

342

Maximising the value of marine by-products

Fig. 16.1

The chemical structures of chitin, chitosan and cellulose.

in a block could produce products which are soluble in water (Kristbergsson et al., 2003). However, no details are yet available in the non-proprietary literature in this regard. Solubility issues associated with chitin and chitosan may limit their use in physiological and functional foods. The intestines of most animals lack the ability to produce chitinase and chitosanase that are able to hydrolyze chitin and chitosan, respectively. Therefore, they will be excreted unchanged in the feces. On the other hand, chitin and chitosan oligomers are considered to have more physiological functions because they are water-soluble and their solutions are less viscous, so they are readily absorbed in the human intestine (Jeon et al., 2000).

Chitin and chitosan from marine by-products 343 Chitosan has many useful applications in different fields as summarized in Table 16.1. These are mainly due to the presence of amino groups at the C-2 positions, and also because of the primary and secondary hydroxyl groups at the C-3, and C-6 positions, respectively (Kurita, 1986). Chitosan is the simplest and the least expensive derivative of chitin (Ornum, 1992). Presence of positively charged amino groups repeatedly placed along the chitosan polymer chain allows the molecule to bind to negatively charged surfaces via ionic or hydrogen bonding (Muzzarelli, 1973; Rha, 1984; Shahidi, 1995). The term chitosan is favored when the nitrogen content of the molecule is higher than 7% by weight (Muzzarelli, 1985) and the degree of deacetylation is more than 70% (Li et al., 1992). Chitin and chitosan are weak bases and hence go through the usual neutralization reactions of basic compounds. The non-bonding pair of electrons on the primary amino group of the glucosamine unit accepts a proton, and thus becomes positively charged (Winterowd and Sandford, 1995). In addition, chitosan serves as a strong nucleophile because of the presence of non-bonding pair of electrons on its primary amino groups. Chitosan reacts readily with most aldehydes to produce imines (Kurita et al., 1988). It also reacts with acyl chlorides to form the corresponding acylated derivatives (Hirano et al., 1976). Chitin and chitosan are capable of forming complexes with many of the transition metals (Muzzarelli, 1973). The heavy metal complexes are supposed to form as a result of donation of non-bonding pair of electrons on the nitrogen and/or on the oxygen of the hydroxyl groups to a heavy metal ion. Cupric ion appears to form one of the strongest metal complexes with chitosan in the solid state (Domard, 1987; Kentaro et al., 1986; McKay et al., 1986). Ferrous ion has the ability of binding to chitosan (Koshijima et al., 1973). Under experimental conditions (100 mg of powdered chitosan mixed with a solution of ferrous nitrate (25 mg) in 50 ml water, at 30ëC, and reaction time of 74 h), about 28% of the ferrous ions were complexed with chitosan. The rate of formation and stability of these complexes are generally affected by the presence of counterions, competing heavy metal ions, temperature, and pH of the solution, as well as particle size, crystallinity, and the degree of N-acetylation of chitin and chitosan (Winterowd and Sanford, 1995). Chitin and chitosan are labile to acid- or alkaline-assisted degradation. Under acidic or basic conditions, acetic acid can be freed as N-acetyl groups at the C-2 positions of N-acetyl glucosamine units are released, leaving behind primary amine groups (Muzzarelli, 1977). In addition, presence of the primary amino groups in chitosan presents further potential for modification of the molecule such as N-acylation and N-alkylation, among others. Acidic conditions also cause partial depolymerization and degradation of the -glycosidic bonds (Madhaven and Ramachandran, 1974). Depolymerization under basic conditions occurs, but to a lesser extent, and chitosan can be hydrolyzed using nitrous acid (Allan and Peyron, 1989).

344

Maximising the value of marine by-products

Table 16.1 Applications of chitin, chitosan and their derivatives Area of application

Examples

Antimicrobial agent

Bactericidal Fungicidal Measure of mold contamination in agricultural commodities

Edible film

Controlled moisture transfer between food and the surrounding environment Controlled release of antimicrobial substances Controlled release of antioxidants Controlled release of nutrients, flavors and drugs Reduction of oxygen partial pressure Controlled rate of respiration Temperature control Controlled enzymatic browning in fruits Reverse osmosis membranes

Food additive

Clarification and deacidification of fruit juices Natural flavor extender Texture adjusting agent Emulsifying agent Food mimetic Thickening and stabilizing agent Color stabilization

Nutrition

Dietary fiber Hypocholesterolemic agent Livestock and fish feed additive Reduction of lipid absorption Production of single cell protein Antigastritis agent Infant food ingredient

Water treatment

Recovery of metal ions, pesticides, phenols and PCBs Removal of dyes, radioisotopes

Agriculture

Seed and fruit-covering Fertilizer Fungicide

Cosmetics

Skin and hair products

Biomedical and pharmaceutical materials

Artificial skin Surgical structures Contact lens Treating major burns Blood dialysis membranes Artificial blood vesicles

Others

Enzyme immobilization Encapsulation of nutraceuticals Chromatography Analytical reagent Synthetic fiber Chitosan-coated paper Manufacturing material for fiber Film and sponges

Chitin and chitosan from marine by-products 345 Natural chitin has a molecular weight exceeding 1,000 kDa while commercially available chitosan has a MW of 100±1200 kDa (Lower, 1984; Li et al., 1992). Numerous forces during commercial production may influence the molecular weight of chitosan. Factors such as high temperature (above 280ëC thermal degradation of chitosan occurs and the polymer chains quickly break down), dissolved oxygen concentration and shear stress may cause these changes to occur (Muzzarelli, 1977; Li et al., 1992). 16.2.2 Production of chitin and chitosan Two hydrolytic methods are known for preparation of chitin and chitosan. These are acid hydrolysis (chemical treatment) and enzymatic hydrolysis. Shahidi and Synowieki (1991) and Shahidi et al. (1999) reported isolation and characterization of chitin from shrimp and crab processing by-products. Different parts of crab shells contained varying amounts of chitin with the legs having the maximum amount. On a dry weight basis, 17.0 to 32.2% chitin was present in shrimp and crab section by-products (Shahidi and Synowiecki, 1991). The normal procedure for preparation of chitin from crustacean shells includes the use of sodium or potassium hydroxide for deproteination, hydrochloric acid for demineralization and agents to remove the remaining proteins, calcium, and color, respectively (Fig. 16.2). The chitin that is produced can then be deacetylated with concentrated base (40±50%) at high temperatures (100±130EC) to produce chitosan (Jaworska and Kowieczna, 2001; Tsai et al., 2002). Deacetylation proceeds rapidly during the first hour of treatment with 50% NaOH at 100ëC leading to 68% deacetylation (Oh et al., 2001). This is followed by a slower step and by the end of 5 h, about 78% deacetylation is achieved. Increasing time does not lead to any further deacetylation of chitin, but it lowers the molecular weight of the product. Chitosans produced from both chemical and enzymatic methods are different with respect to their degree of deacetylation (DD), distribution of acetyl groups, chain length, and conformational structure of chitin and chitosan molecules. These factors, together, will affect the characteristics of the products. Optimum conditions for chitosan pre-treatment (deacetylation by 45% alkali solution for 1 h) were studied by investigating the coagulation efficiencies of chitosan prepared under different conditions (Huang et al., 2000). The procedure involved crushing of crab shells to a powder and isolation of chitin. Subsequently, chitin was deacetylated using NaOH at 100ëC (Kamil et al., 2002) followed by multiple rinsing of the product with deionized water to reach pH 7, and finally drying at 80ëC for 48 h. The resulting chitosan was dissolved in different concentrations of acetic or hydrochloric acid, stirring at room temperature, until it was completely dissolved. As the concentration of acid increased, the viscosity of dissolved chitosan coagulants decreased due to binding of positively charged chitosan to the negatively charged acid anion in the solution. The conformation of chitosan polymers changes and becomes more

346

Maximising the value of marine by-products

Fig. 16.2

Flowsheet for preparation of chitin, chitosan, their oligomers and monomers from shellfish processing by-products.

compact in the acidic solution and thus lowers the viscosity of the solution; the best solution was obtained at pH 2.0. 16.2.3 Deacetylation and MW of chitosan and its activity Chitin with varying degrees of acetylation (DA), ranging from fully acetylated to totally deacetylated, may be procured. The degree of acetylation affects the physical properties of chitin; thus increasing the degree of acetylation leading to a decrease in the degree of solubility in different solvents. Oh et al. (2001) reported that the DD of chitosan is affected by the concentration of alkali, temperature, reaction time, prior treatment of chitin, particle size, and chitin concentration. Heux et al. (2000) reported that after partial deacetylation (to less than 50%), the product of chitin becomes soluble in acidified water. Therefore, chitosan is characterized by its degree of acetylation (DA), which is the average mole fraction/percentage of N-acetyl-D-glucosamine units within the macromolecular chain (Desbrieres, 2002); other methods may also be employed for this purpose

Chitin and chitosan from marine by-products 347 (Huang et al., 2000). Many different techniques are used to evaluate the average degree of acetylation of chitosan, such as infrared, solid state NMR, ultraviolet spectrometry and potentiometric titration, 1H liquid-state NMR, and elemental analysis (Heux et al., 2000), as well as 13C solid-state NMR and elemental analysis; these techniques do not require solubilization of the polymer. Techniques used for evaluation of DA of chitin and chitosan over the whole range of DA include 13C and 15N cross-polarization/magic angle spin (CP-MAS) solid-state nuclear magnetic resonance (NMR) and 1H liquid-state NMR. These methods afford results in good agreement, but the limitation of solid-state NMR is that it requires a detection threshold not higher than 5%. Meanwhile the15N CP-MAS technique was found to be a powerful technique to assess the acetyl content in the case of complex association of chitin and other polysacchrides (Heux et al., 2000). The degree of acetylation and the molecular weight of chitin/chitosan may be determined using circular dichroism and viscometric methods, respectively (Zhang and Neau, 2001). The degree of deacetylation has no effect on the acidbinding properties of chitosan (Scheruhn et al., 1999). Chitosans which have a relatively high degree of deacetylation enhance fibroblast proliferation, but those with lower levels of deacetylation exhibit less activity. However, the molecular weight and polymer chain length were of little consequence (Howling et al., 2001). The molecular weight of chitosan has an important effect on its activity. Chitosan preparations with a molecular weight of 5±50 kDa reduced serum cholesterol levels in rats (Ikeda et al., 1993). Meanwhile, chitosans with a molecular weight of 8 kDa were more effective as hypocholesterolemic agents in rats than chitosans with a MW of 2 or 220 kDa (Enomoto et al., 1992). Chitosan with a MW of 12 kDa (DDA, 87%) was most effective against L. fructivorans, and chitosan with MW of 32.5 kDa (DDA, 80%) was most effective against L. plantarum (Oh et al., 2001). The molecular weight of chitosan had no effect on S. liquifaciens. Thus, a relationship between the type of microorganism and antimicrobial activity of different MW chitosans is evident. Chitosans with average molecular weights of over 10 kDa had a positive effect on enhancing fecal excretion of neutral steroids. In addition, as the viscosity or the degree of deacetylaion of chitosan preparation increased, the effects on the apparent fat digestibility were more clear (Ylitalo et al., 2002). Thus an increase in the DD, and hence the number of NH2 groups, resulted in stronger antimicrobial activity of chitosan (Tsai et al., 2002). This result agrees with the findings of Chang et al. (1989), Darmadji and Izumimoto (1994), Simpson et al. (1997), and Wang (1992). 16.2.4 Depolymerization and N-acetylation Thermal depolymerization of chitosan chloride in solid state showed increasing of intrinsic viscosity with increasing degree of acetylation, which is an important parameter for thermal degradation (Holme et al., 2001). The presence of oxygen

348

Maximising the value of marine by-products

had no effect on the rate of chitosan degradation, but pH did affect the degradation of chitosan. Furthermore, acid hydrolysis was the primary mechanism involved in thermal depolymerization of chitosan chloride in the solid state (Holme et al., 2001). Chitosan, similar to other polysaccharides, is influenced by several degradation mechanisms, including oxidative-reductive free radical depolymerization, and acid-, alkaline- and enzymatically-catalyzed hydrolysis (Holme et al., 2001). In these, cleavage of glycosidic bonds is involved, hence it is important to control the depolymerization process in order to maintain other properties such as viscosity, solubility, and biological activity. Decomposition (release of material) of chitosan begins at 200ëC, but Holme et al. (2001) reported that chitosan chlorides were thermally degraded at 60, 80, 105, and 120ëC, the degradation rate of chitosan increasing with increasing temperature and degree of acetylation during acid hydrolysis (Holme et al., 2001). N-acylation of chitosan fibers upon treatment with a series of carboxylic acid anhydrides in methanol at room temperature was examined (Hirano et al., 2000). The N-acylation had little effect on mechanical properties of the resultant filaments such as tenacity and elongation values. Treatment of chitin fiber and chitin-cellulose mixed fiber with 40% NaOH at 95±100ëC for 4 h in suspension, afforded a chitosan fiber and a novel cellulose-chitosan mixed fiber, respectively. Novel N-acylchitosan fibers produced were N-acetyl-, N-propionyl-, Nbutyryl-, N-hexanoyl-, and N-octanoyl chitosans. These fibers were insoluble in water, aqueous basic and acidic solutions. 16.2.5 Comb-shaped chitosan Chitosan has a considerable advantage over chitin for modification purposes because it possesses free amino groups. N-substituted chitosan derivatives may be obtained using reducing sugars, aldehydes or ketones via reductive alkylation, which is a typical example of reactions of chitosan. Reductive alkylation of chitosan leads to production of comb-shaped chitosan derivatives with monoaldehydes from tri- and tetra (ethylene glycol) monosubstituted derivatives. The introduction of such branches clearly increased the affinity of molecules for both water and organic solvents without loosening the attractive characteristics of chitosan, such as metal ion adsorption capacity (Kurita et al., 1999). In order to prepare comb-shaped chitosan derivatives, chitosan may be completely deacetylated via treatment with monoaldehyde derived from tri(ethylene glycol) under homogeneous conditions in an acetic acid-methanol solution. Sodium cyanoborohydride was added to the solutions which afforded a weak gel. The resultant mixtures were then dialyzed against deionized water to afford clear solutions which were concentrated and freeze-dried in order to afford slightly yellowish solids (Kurita et al., 1999). 16.2.6 N-alkylation of chitin for improved solubility Lack of solubility of chitin in usual solvents, except in fluorinated solvents, N,Ndimethylacetamide/LiCl, and methanol/CaCl2 leads to preservation of chitin in

Chitin and chitosan from marine by-products 349

Fig. 16.3 Schiff base formation with aldehydes, reduction, and N-acetylation of chitin. Symbols are: Ac, Acetyl; Ac2O, acetic anhydride.

nature. Deacetylated chitin (50% random), and chitin derivatives having tosyl, iodo, trimethylsilyl and glycosyl groups are soluble in water or organic solvents (Kurita et al., 2002). The N-alkylation of chitin improves solvent affinity and lowers crystallinity of chitins. Thus, many experiments have been conducted to synthesize polymers with N,N-dimethylacetamide moieties in their backbone by ring-opening polymerization of 2-oxazolines. Introduction of methyl, ethyl, and pentyl groups into chitin at the nitrogen of C-2 acetamido moiety via an adjusted 5-step modification process was reported by Kurita et al. (2002) (Fig. 16.3). Chitosan was completely deacetylated and treated with formaldehyde (methanal), acetaldehyde (ethanal) or valenaldehyde (pentanal). The Schiff bases of chitosan were then reduced to N-alkylated chitosan using sodium cyanoborohydride (NaCNBH3). The N-alkyl chitosans were subsequently changed into corresponding N-alkyl chitins via acetylation using acetic anhydride followed by transesterification to eliminate partially formed O-acetyl groups. This synthetic pathway is direct and effective to provide well-defined novel chitin derivatives. The resulting Nmethyl-, N-ethyl-, and N-pentylchitins were amorphous and displayed a high affinity for solvents (Kurita et al., 2002). 16.2.7 Trapping-retention of heavy metals Chitinous materials are known to interact with metal ions, including radioactive products. However, chitosan derivatives are better known for their trapping-retention of heavy metal. Cardenas et al. (2001) described a method for preparing chitosan mercaptan derivatives with mercaptoacetic acid and

350

Maximising the value of marine by-products

Fig. 16.4 Production of mercaptan derivatives of chitosan via conversion with 1-chloro2,3-epoxypropane.

1-chloro-2,3-epoxypropane and evaluated their retention capacities using different concentrations of copper and mercury. These derivatives are shown in Fig. 16.4 and include N-hydroxy-3-mercaptopropylchitosan (chitosan 1), and N-(2-hydroxy-3-methylaminopropylchitosan, (chitosan 2). All chitosan derivatives tested were thermally stable; N-hydroxy-3-mercaptopropylchitosan showed the highest thermal stability at 314ëC compared with chitosan at 290ëC. The copper ion adsorption was less than that of mercury ions at either pH2.5 or pH4.5, suggesting a lower selectivity for Cu (Cardenas et al., 2001). Hydrolysis of chitin and chitosan may occur upon the action of chitinases, chitosanases, lysozymes, and cellulases (Fig. 16.5) (Shahidi et al., 1999). Tsigos et al. (2000) reported the necessity for pre-treatment (alkali treatment) of crystalline chitin before adding the enzyme to increase the rate of deacetylation in order to produce new polymers with new physical and chemical characteristics. The compounds are easily soluble if produced with different distribution of N-deacetylated residues. A synthetic procedure for chitin with N-acetyl-D-glucosamine and chitosan derivatives with Dglucosamine branches has been reported (Kurita et al., 2000). These resulting non-natural branched chitin and chitosan have extra amino sugars in branches that render them much improved properties in comparison with linear ones, such as the affinity for solvents and hygroscopicity. These characteristics would be of great interest in different applications, such as moisturizers for cosmetics and antimicrobial substances for fiber and textile treatment (Kurita et al., 2000). 16.2.8 Enzymatic hydrolysis and preparation of chitin and chitosan oligomers Several reports on chemical hydrolysis (including acid hydrolysis) for preparation of chitin oligomers have appeared (Bosso et al., 1986; Defaye et al., 1989; Inaba et al., 1984; Hirano and Nagano, 1989; Kendra et al., 1989; Kurita et al., 1993; Rupley, 1964; Sakai et al., 1990; Takahashi, 1995). A series of commer-

Chitin and chitosan from marine by-products 351

Fig. 16.5

Preparation of products from chitin.

cially available chitin oligomers, up to hexamer, has been prepared by partial hydrolysis of chitin with concentrated HCl, followed by fractionation using column chromatography (Rupley, 1964). The procedures for isolation of chitins and oligomers include acid hydrolysis, neutralization, demineralization, fractionation by charcoal-celite column, fractionation by HPLC (high performance liquid chromotography) and lyophilization. These methods suffer from several disadvantages, such as being time consuming, laborious, and environmentally unfriendly (Tsigos et al., 2000). In addition, these methods may afford a low yield of oligomers with a high degree of polymerization (Takahashi et al., 1995). To overcome drawbacks associated with the conventional methods, procedures such as acetolysis (Bosso et al., 1986; Defaye et al., 1989; Inaba et al., 1984; Hirano and Nagano, 1989; Kendra et al., 1989; Kurita et al., 1993; Rupley, 1964; Sakai et al., 1990; Takahashi, 1995), fluorosis (Bosso et al., 1986), fluorohydrolysis (Defaye et al., 1989), and sonolysis (Takahashi et al., 1995) (Fig. 16.6) have been considered. Acetolysis is a procedure for preparation of oligomers from chitin using acetic anhydride and sulfuric acid (Fig. 16.6). Beta-chitin from squid was used as a starting material for simple acetolysis, leading to the formation of

352

Maximising the value of marine by-products

Fig. 16.6 Mechanism for acid hydrolysis of chitin.

N-acetylchitooligosaccharide peracetate in high yields with reasonable reproducibility (Kurita et al., 1993). Fluorohydrolysis is another method used for preparation of chitin oligomers in which anhydrous HF is employed (Fig. 16.6). Defaye et al. (1989) reported that fluorohydrolysis of chitin in anhydrous hydrogen fluoride (HF) yields chitin oligomers ranging from 2 to 9 residues and chitin oligomer isomer ( -(166)linked acetamino-2deoxy-D-glycosyloligosaccharides) nearly quantitatively. In addition, sonolysis may be used for preparation of oligomers from chitin using hydrochloric acid hydrolysis under ultrasound irradiation (Fig. 16.6) (Takahashi et al., 1995). The above combined method that includes a mild acid hydrolysis and sonolysis was able to hydrolyze polymers independent of temperature of the bulk solution and degradation of chitin by HCl under ultrasound irradiation (Takahashi et al., 1995). This method saves time and does not require more than 2 h. However, caution should be exercised to avoid deacetylation of the acetamido group. Chitosan oligomers were first prepared by Horowitz et al. (1957). Acid hydrolysis of chitosan with concentrated HCl led to the production of chitosan oligomers with a low degree of polymerization (DP), but in a quantitative manner. Several studies have described the production of chitosan oligomers with a DP of less than 6 residues (Sakai et al., 1990; Takahashi et al., 1995; Tsukada and Inoue, 1981). On the other hand, Domard and Cartier (1989)

Chitin and chitosan from marine by-products 353 reported that a wide distribution of glucosamine oligomers could be easily produced and separated up to DP of 15 in the pure form. Defaye et al. (1994) prepared chitosan oligomers by fluorolysis in anhydrous hydrogen fluoride. They obtained oligomers with DP of 2±11. The majority of acidic hydrolysis methods have reported production of chitosan oligomers with a low DP, mainly from monomer to tetramer in quantitative amounts. The yields of relatively higher DP (pentamer to heptamer) oligomers were low. However, physiological function is rendered best by high DP oligomers. Chitin and chitosan oligomers can also be prepared by enzymatic methods. Enzymatic methods offer many benefits over chemical hydrolysis as they produce desirable oligomers with a high DP under milder conditions (Jeon et al., 2000). Jaworska and Konieczna (2001) investigated the effect of supplemental components (Fe+2, Co+2, Mn+2, trypsin, and chitin) on the in vivo activity of two enzymes (chitin synthase and chitin deacetylase) to produce chitosan from fungus Absidia orchodis. Manganese ions (Mn+2) and ferrous ions (Fe+2) gave rise to the highest increase in the amount of biomass rather than chitosan content in cell walls of the fungus. The effects of trypsin and chitin on biomass and chitosan content in cell walls were not significant, while Co+2 totally inhibited the growth of fungi. Ferrous ions decreased the activity of chitin deacetylase. Chitosan from fungi cultivated with ferrous ions had a higher DD (26±30%) than chitosan from unsupplemented medium (15%). The same trend was observed for Mn+2. The amount of chitosan from fungi cultivated in the presence of Mn+2 was higher (about 30%) than that produced in an uncultivated medium (15%). The effects of degree of deaceylation (DD) and preparation procedures for chitosan were evaluated for their antimicrobial activity (Tsai et al., 2002). Chitin was chemically (CH-chitin) and microbiologically (MO-chitin) prepared from shrimp shells. The resulting chitins were subsequently deacetylated chemically to produce chitosan with DD ranging from low (47±53%) to medium (74±76%) to high (95±98%). The antimicrobial activities of both chemically and microbiologically prepared chitin/chitosan were the same, and in both cases the activity increased with increasing DD. Moreover, the size and conformational characteristics of chitin and chitosan were crucial for their antimicrobial function. In general, chitosan has a stronger effect against bacteria than fungi. Chitosan with a high DD (98%) efficiently inhibited various bacteria (Tsai et al., 2002). Therefore, chitosan displays potential for increasing the shelf life of refrigerated fish fillets (Shahidi et al., 1999). Furthermore, Uchida et al. (1989) showed that enzymatic hydrolysis produced a high amount of high DP oligomers from chitin and chitosan when compared to acid hydrolysis.

16.3

Applications of chitin, chitosan and their oligomers

Different applications of chitins, chitosan and their oligomers have been summarized in Table 16.1. As indicated, there is a myriad of areas in which such products could be used (see below).

354

Maximising the value of marine by-products

16.3.1 Medical applications Chitin and chitosan have both material and biological properties that might be beneficial to enhance wound-repair. As well, both of them have great influence on different stages of wound-healing in experimental animal models (Howling et al., 2001). Howling et al. (2001) found that chitosan polymers can interact with and modulate the migration behavior of neutrophils and macrophages modifying subsequent repair processes such as fibroplastia and reepithelialization. Chitin and chitosan have both stimulatory and inhibitory effects on proliferation of human dermal fibroblasts and keratinocytes (Howling et al., 2001). They also have enhancing effects on the survival function of osteoblasts and chondrocytes (Lahiji et al., 2000). The procedure for promoting wound-healing by chitosan was tested as follows: chitosan was coated onto plastic coverslips that had been filled into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips. The incubation temperature of the culture was 37ëC and under 5% CO2 for 7 days. By using a fluorescent molecular probe, cell viability was judged. Reverse transcriptase± polymerase chain reaction and immunocytochemistry were used for phenotyping expression of osteoblasts and chondrocytes. The results showed that the chondrocytes and osteoblasts appeared spherical and refractile of the chitosancoated coverslips, while 90% of the cells on the plastic coverslips were elongated and spindle shaped after this period of incubation (Lahiji et al., 2000). It was reported that the wound recovering material composed of polyelectrolytic complexes of chitosan and sulfonated chitosan that speeded up wound healing and afforded a good-looking skin surface (Lahiji et al., 2000). Chitosan has the ability to promote wound-healing; this is due to the tendency to form polyelectrolyte complexes with polyanion heparin, which possesses anticoagulant and angiogenic properties (Lahiji et al., 2000). By forming a complex with heparin and acting to lengthen the half-life of growth factors, chitosan supports tissue growth and helps wound-healing. Other studies have examined the effect of chitin and chitosan samples with different deacetylation levels and polymer chain length on the proliferation of human dermal fibroblasts in vitro (Howling et al., 2001). It was found that chitosans with a high degree of deacetylation strongly motivated fibroblast proliferation; meanwhile, samples with lower degrees of deacetylation showed less activity. Cho et al. (1999) used water-soluble chitin (WSC) prepared at room temperature through depolymerization by ultrasonication after alkaline treatment of chitin. The degree of deacetylation and molecular weight were controlled. Chitin with DD of 8.60%, chitosan with DD of 83.9% and WSC were embedded to the wounded backs of rats after full thickness skin cuts. It was noticed that the WSC had the highest efficiency in recovering strength of the wounded skin due to the hydrophilicity and high biodegradability of WSC that maximized its activity as a wound-healing accelerator. In addition, the arrangement of the collagen fibres in the wound was the same as that of the normal skin. Hirano and Zhang (2000) described the preparation of a novel blend fiber. This fiber is a mixture of

Chitin and chitosan from marine by-products 355 cellulose with each of hyaluronate (HA), heparin (Hep), chondroitin 4-sulfate, chondroitin 6-sulfate and a chitin-chondroitin 6-sulfate blend using an aqueous 10% sulfuric acid solution containing 40±43% ammonium sulfate as a coagulating solution. These blend fibers could be used as covering materials for the wound-healing tissues of animals and plants. Recently, bandages made of chitosan were investigated in the military field in the new war in Iraq (Brown, 2003). Z-Medica, a small company supplied these products to the US ground troops in Iraq and Afghanistan (Becker, 2003). These bandages were used immediately after injury to control bleeding at this critical time and were found to save numerous lives (Becker, 2003). Arterial bleeding was stopped in about a minute when these bandages were applied with pressure to a wound (Brown, 2003). The use of such bandages was approved by the Food and Drug Administration (FDA) in November 2002 (Mientka, 2003). They called it `shrimp' bandage that contains chitosan. This bandage can stop capillary bleeding and stanch severe arterial hemorrhaging (Mientka 2003). Mientka (2003) reported that chitosan bandages had the ability to stop bleeding at a rate of 600 mL per minute. Moreover, there was no sign of alergenicity for use of these bandages in soldiers who were allergic to shrimp (Mientka, 2003). Dietary applications Chitosan may be considered as a dietary supplement for reducing body weight in humans. Industrial production of chitosan tablets (Muzzarelli et al., 2000) and chitosan dietary fibers (Hughes, 2002) has occurred. In addition, Schiller et al. (2001) reported that a rapidly-soluble chitosan (LipoSan Ultra that has a higher density and solubility than chitosan itself) facilitated weight loss and reduced body fat. This effect was due to the fact that this chitosan was able to prevent dietary fat absorption in overweight and mildly obese individuals that consumed a high-fat diet. Chitosans have also been used to prevent body weight increase in animals (Hughes, 2002). Meanwhile, negative results were recorded regarding chitosan effectiveness in this field (Hughes, 2002). During a high-fat diet and chitosan supplementation, no increase in fecal fat content was noticed, meaning that chitosan had no effect on fat absorption (Hughes, 2002). Use of chitosan in the diet has been questioned by some researchers for individuals suffering from allergic reaction to crustaceans (Ylitalo et al., 2002). Although the reactivity of chitosan toward lipids is not clear, it is claimed that chitosan, due to its cationic nature, binds to appropriate bile and fatty acids and brings about their excretion (Muzzarelli et al., 2000; Ylitalo et al., 2002). This claimed efficacy of chitosan in reducing the body weight, hypercholesterolemia, and hypertension stimulated production of chitosan tablets. The capacity of chitin, chitosan, N-lauryl chitosan and N-dimethylaminopropyl chitosan on sequestering steroids was investigated (Muzerelli et al., 2000). They reported that chitin might be more effective in holding olive oil and enriching the retained oil fraction with steroids sequestering than chitosan. As well, chitin derivatives were able to distinguish between different lipids. These results put into question

356

Maximising the value of marine by-products

the need for high cationity for sequestering cholesterol. The use of chitosan monomer, glucosamine sulfate, for joint building is also commonplace. Antihypercholesterolemic agent Chitosan has been reported to render a significant hypocholesterolemic activity in different experimental animals (Hirano et al., 1990; Sugano et al., 1978, 1980; Ylitalo et al., 2002). Sugano et al. (1988) noted that chitosan oligomers did not exhibit this effect. The studies were carried out on rat groups fed on a diet rich in cholesterol to find the effect of chitosan hydrolyzates with different molecular weights and viscosity on the hypocholesterolemic activity. The lower the molecular weight of chitosan, the better was its cholesterol-lowering potential. The mechanism of antihypercholesterolemic activity of chitosan has been described by Ylitalo et al. (2002). In the stomach, the acidic condition protonates the amino groups. Fats, fatty acids (oleic, linoleic, palmatic, stearic and linolenic acids) and other lipids as well as bile acids, due to their negative charge (X-COOÿ), attach themselves strongly to the positively charged amino groups (ÿNH3+) of chitosan. This binding might inhibit their absorption and recycling from the intestine to the liver. However, this interruption of enterohepatic circulation of cholic acid and other bile acids can lead to an increase in the biosynthesis of cholic acid from cholesterol in the liver. The cholesterol content of liver cells is thus decreased and this may lead to subsequent activation of LDL-receptor expression, and could further increase LDL uptake via LDLreceptors in the liver (Ylitalo et al., 2002). We have previously, reported that production of dietary cookies, potato chips and noodles enriched with chitosan is commonplace in certain countries. The products enriched with chitosan are expected to render hypocholesterolemic effects. As well, vinegar products containing chitosan are produced and sold in Japan because of their cholesterollowering ability (Shahidi et al., 1999). Anti-tumor activity Suzuki (1996) reported that chitin and chitosan oligomers have the ability to act as inhibitors of growth tumor cells via their immuno-enhancing effects. Suzuki et al. (1985) found that chitin oligomers from (GlcNAc)4 to (GlcNAc)7 have strong attracting responses to peritoneal excudate cells in BALB/c mice. However, chitooligosaccharides from (GlcN)2 to (GlcN)6 did not exhibit such an effect. With regard to hexamers, both (GlcNAc)6 and (GlcN)6, were reported to process growth inhibitory effects against allogenic and syngeneic mouse systems (Suzuki et al., 1986a). These results indicate that the effect was not by direct cytocidal action on tumor cells, but was in fact host-mediated. Anti-ulcer agent Ito et al. (2000) reported that chitosans with different molecular weights had ulcer healing actions. The effects of low molecular weight (LMW) chitosan,

Chitin and chitosan from marine by-products 357 high molecular weight chitosan (HMW), and chitin on ethanol-induced gastric mucosal injury and on the healing of acetic acid-induced gastric ulcers in rats were compared. It was found that orally administrated LMW chitosan could prevent ethanol-induced gastric mucosal injury. Repeated oral administration of LMW chitosan, in a dose-dependent manner, accelerated the gastric ulcer healing. The effects of HMW chitosan and chitin on gastric ulcer healing were less than those of LMW chitosan. A coating agent for prosthetic articles (artificial parts of the body) Muzzarelli et al. (2000) described a method for coating prosthetic articles with chitosan-oxychitin. Plates of Ti (titanium) and its alloys were plasma-sprayed with hydroxyapatite and glass layers, and subsequently a chitosan coat was deposited on the plasma-sprayed layers using chitosan acetate. These layers were treated with 6-oxychitin to form a polyelectrolytic complex. This complex was optionally contacted with 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide at 4ëC for 2 h to form amide links between the two polysaccharides, or acetylation with acetic anhydride in methanol to obtain a chitin film. In all cases, the modified coats were insoluble, uniformly flat and smooth. Prosthetic materials coated with chitosan-oxychitin were capable of provoking colonization by cells, osteogenesis and osteointegration. There were two main reasons behind the selection of chitosan-oxychitin coated orthopedic plates. Firstly, chitosan enhances the integration of the implant and secondly, chitosan stimulates bone regeneration. 16.3.2 Food applications of chitin, chitosan and their oligomers New applications of chitin and its oligomers led to over 50 patents in the 1930s and the early 1940s. However, commercialization of these products was hindered by inadequate manufacturing services and competition from synthetic polymers (Averbach, 1981). However, after the 1970s industrial utilization of chitin and its oligomers has increased (Kaye, 1985). Furthermore, improvement in research and small-scale production of chitin and chitosan has extended the number and the varieties of potential applications of chitinous materials. In addition, environmental problems and cost of disposal of shellfish processing disards have increased the urgency for development of environmentally safe alternatives for numerous plastic or polymeric products (Ashford et al., 1976; Berkeley, 1979; Shahidi and Synowiecki, 1991). Some food applications of chitin, chitosan and their olgomers are summarized in Table 16.1 on page 344. Chitin, chitosan and their derivatives offer a wide range of applications (Table 16.1) including bioconversion for the production of value-added food products, preservation of foods from microbial spoilage, formation of biodegradable films, recovery of waste material from food processing discards, purification of water as well as clarification and deacidification of fruit juices (Shahidi et al., 1999).

358

Maximising the value of marine by-products

Antimicrobial activity Chitin, chitosan and their derivatives have antimicrobial activity against bacteria, yeast and fungi (Yalpani et al., 1992). The exact mechanism of antimicrobial action of chitin and chitosan and their derivatives remains illusive, but different mechanisms have been proposed (Shahidi et al., 1999). Chitosan has the ability to produce phytoalexins, cell wall phenols and callose (Tsai et al., 2002). Chitosan is considered to be a soluble chelating agent and activator due to the presence of a positive charge on the C-2 of its glucosamine monomer at pH values below 6. This characteristic gives it a higher antimicrobial activity than chitin (Chen et al., 1998). A leakage of proteineous and intercellular components occurs due to the interaction between the positively charged chitosan molecules and the negatively charged microbial cell membranes (Chen et al., 1998; Papineau et al., 1991; Sudharshan et al., 1992; Young et al., 1982). This is affected by the molecular weight of chitosan (Tsai et al., 2002). Being a chelating agent, chitosan has the ability to selectively bind trace metals, which prevents production of toxins and microbial growth (Cuero et al., 1991). Chitosan is also an activator for several defence processes in the host tissue (El Ghaouth et al., 1992c), having the ability to bind water and inhibit various enzymes (Young et al., 1982). Tsai et al. (2002) studied the effects of degree of deacetylation (DD) and preparation methods for chitin and chitosan on their antimicrobial activity. It was found that chemically-(CH-chitin) and microbiologically-prepared chitin (MO-chitin) could undergo further chemical deacetylation to produce chitosan with different DDs. However, MO-chitin that was deacetylated by various proteases had no antimicrobial activity (Tsai et al., 2002). However, for chitosan, as the DD increased, its antimicrobial effect on bacteria increased, even to a greater extent than that on fungi. Genetically, chitosan can enter the nuclei of a microorganism and bind with DNA. This binding inhibits the mRNA and protein synthesis (Hadwiger et al., 1985; Sudharashan et al., 1992). The effect of concentration of chitosan for complete inactivation of certain types of bacteria has been reported (Shahidi et al., 1999). Wang (1992) observed that a much higher concentration of chitosan (1±1.5%) was required for complete inactivation of Staphylococcus aureus after two days of incubation at pH 5.5 or 6.5 in the medium. Furthermore, Chang et al. (1989) found that chitosan concentrations of  0.005 were sufficient to elicit complete inactivation of S. aureus. This was in accordance with the findings of Darmadji and Izumimoto (1994) on the effect of chitosan in meat preservation. Different concentrations of chitosan and their effect on the growth of different cultures of bacteria on raw shrimp was examined (Simpson et al., 1997). Bacillus cereus required chitosan concentrations of 0.02% for anti-bacterial effect, while Escherichia coli and Proteus vugaris exhibited minimal growth at 0.005% and growth was inhibited at  0.0075%. The effect of different concentrations of chitosan on Escherichia coli growth has also been studied. Wang (1992)

Chitin and chitosan from marine by-products 359 reported complete inhibition after 2 days incubation with 0.5 or 1% chitosan at pH 5.5. It was also reported that if chitosan concentration increased by about 1% in the broth, it could afford complete inactivation. However, Darmadji and Izumimoto (1994) reported that growth inhibition of E. coli required a 0.1% chitosan concentration. Simpson et al. (1997) found that only 0.0075% chitosan was required to inhibit the growth of the same species. Existing differences in the degree of acetylation of chitosans employed might explain the observed variations (Shahidi et al., 1999). Iida et al. (1987) and Nishimura et al. (1984) have reported that if chitin is partially deacetylated, especially at 70%, it has the ability to stimulate nonspecific host resistance against E. coli and Sendai virus infection in mice. Meanwhile, chitin and chitosan have the ability to increase the number of mouse peritoneal exudate cells that generate reactive oxygen intermediates and then display candidacidal activities (Suzuki et al., 1984). Suzuki et al. (1986b) reported that chitin hexamer (GlcNAc)6 had a strong candidacidal activity. Chitosan was found to decrease the in vitro proliferation of many fungi with the exception of Zygomycetes (Allan and Hadwiger, 1979). Chitosan acts as an antifungal agent via the formation of gas permeable coats, interference with fungal growth, and stimulation of many defence processes, including accumulation of chitinases, production of proteinase inhibitors, and lignifications and stimulation of callous synthesis (Bai et al., 1988; El Ghaouth et al., 1992c). Antifungal effect of chitosan on in vitro growth of common post-harvest fungal pathogens in strawberry fruits was reported by El Ghaouth et al. (1992b). Chitosan with a 7.2% NH2 significantly decreased the eradial proliferation of Botrytis cinerea and Rhizopus stolonife, with a greater impact at higher concentrations. Fang et al. (1994) reported the preservative influence of chitosan on low-sugar candied Kumquat (fruit). Chitosan (at 0.1±5 mg/ml) inhibited the growth of Aspergillus niger, whereas chitosan at less than 2 mg/ml was ineffective in inhibiting mold proliferation and aflatoxin synthesis by Aspergillus parasiticus. Cuero et al. (1991) conducted a similar study and observed that N-carboxymethylchitosan decreased aflatoxin formation in A. flavus and A. Parasitius by more than 90% while fungal growth was decreased to less than half. Furthermore, Savage and Savage (1994) reported that apples coated with chitosan reduced the rate of molds occurring on them over a period of 12 weeks. Cheah and Page (1997) found that chitosan coating of carrot with a 2 or 4% chitosan solution considerably reduced their Sclerotinia rotting by 28 to 88%. El-Katatny et al. (2001) reported the characterization of a chitinase and endo -1,3-glucanase from Trichoderma harzianum strain Rifai T24. These two enzymes are the key enzymes in the lyses of cell walls during their mycoparasitic effect against plant diseases caused by fungi, including S. rolfsii. The chitinase from T. harzianum was purified in two steps using ammonium sulfate precipitation followed by hydrolytic interaction chromatography. SDS-PAGE showed that the enzyme exhibited a single band at 43 kDa. The -1,3-glucanase

360

Maximising the value of marine by-products

was purified and found to have a molecular mass of 74 kDa. The optimum pH of both enzymes was 4.5. The optimum temperature of the T24 chitinase was 40ëC, whereas the optimum temperature of -1, 3-glucanase was 50±60ëC. Both the T. harzianum T24 chitinase and -1,3-glucanase were strongly inhibited by Hg+2, suggesting that sulfhydryl groups are involved in the catalytic reaction (ElKatatny et al., 2001). A mixture of these enzymes, which showed thermostability and low effective dose (ED50) values against S. rolfsii, may be considered a potential tool for controlling of plants' pathogens. Chitin may be used to determine the total content of mycelium based on chitin (Bishop et al., 1982; Donald and Mirocha, 1977). Bishop et al. (1982) used chitin to evaluate the presence of mold in tomato products, ketchup, paste and puree. They noticed variations in chitin content among different fungal species depending upon the cultural age and growth conditions; values ranged from 5.7 to 43 g of glucosamine per mg dry weight. Preservation of foods Chitosan can be used for food preservation in order to inhibit the growth of spoilage microorganisms (Oh et al., 2001). By treating crude chitin with various NaOH concentrations (45, 50, 55, and 60% w/v), four kinds of chitosans were prepared (chitosan-45, -50, -55 and -60, respectively) (Ylitalo et al., 2002). Four species of food spoilage microorganisms were treated with chitosans in order to examine their effects on microbial activity (Oh et al., 2001). These were Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquifaciens and Zygosacchaomyces bailii. Chitosan had a biocidal effect; the number of cells grown was clearly reduced. It has been found that after an extended phase, some strains recovered and started to grow. As the concentration of chitosan increased, the activities of these strains increased. It was noticed that chitosan-50 was most effective against L. fructivorans; meanwhile, the inhibition of L. plantarum growth was mostly by chitosan-55 and no difference was found among the chitosans tested against S. liquefaciens and Z. bailii. Thus, for mayonnaise, during its storage at 25ëC, the addition of chitosan decreased the viable cell counts of L. fructivorans and Z. bailii. Seafood and meat preservations A 1% solution of chitosan with a high degree of deacetylation increased shelf life of fish from 5 to 9 days. Kamil et al. (2002) showed that chitosans prepared from snow crab shells had different viscosities, closely correlated to the time of deacetylation. Different viscosity chitosans (14, 57, 360 cP chitosans) were prepared and used to examine the impact of chitosan covering on fish quality during refrigerated storage. This study showed the potential of chitosan as a protective coating for herring and cod in decreasing or preventing moisture loss, lipid oxidation, and microbial growth. Cod samples coated with 57 and 360 cP chitosans demonstrated a considerably (p < 0:05) lower relative moisture loss in comparison with those of uncoated samples and those coated with 14 cP chitosan

Chitin and chitosan from marine by-products 361 throughout the storage period. Furthermore, crab chitosan showed a medium to high viscosity-dependent protective effect in both fish model systems. In general, 360 cP chitosan exhibited a better preservative effect in comparison with 57 and 14 cP chitosans in both systems at 4  1ëC. The chitosan showed antioxidant activity in cooked comminuted fish model system, as revealed in their peroxide value and content of 2-thiobarbituric acid-reactive substances (TBARS) which were reduced in a concentration-dependent manner. However, the antioxidant efficiency of relatively high viscosity chitosan in both model systems was lower than that of the low viscosity chitosan at the same concentration. The mechanism of action appears to be a result of chelation of metal ions found in fish muscle proteins, gas exchange adjustment (particularly oxygen) between fish meat and the surrounding environment, and bactericidal effect of chitosan itself. Thus, chitosan as an edible coating would enhance the quality of seafoods during storage (Jeon et al., 2002). Weist and Karel (1992) studied the effect of using chitosan powders in a fluorescence sensor for monitoring lipid oxidation in muscle foods. The efficiency of chitosan powders was explained to be due to the ability of the primary amino groups of chitosan to form a stable fluorosphere with volatile aldehydes such as malonaldehyde which is derived from the breakdown of fats (Weist and Karel, 1992). On the other hand, chitosan was used to improve the preservation of vacuum-packaged processed meats stored under refrigerated conditions (Quattar et al., 2000). These authors used chitosan matrix to produce antimicrobial films by adding acetic or propionic acid (with or without addition of lauric acid or cinnamaldehyde) to this matrix (Quattar et al., 2000). These films were applied on to bologna, regular cooked ham, or pastrami. The amounts of antimicrobial agents found in the chitosan matrix were measured several times during storage. It was found that within the first 48 hours of application, propionic acid was nearly completely released from the chitosan matrix, whereas 2±22% of acetic acid remained in chitosan even after 168 hours of storage. With regard to the presence of lauric acid, but not cinnamaldehyde, it was found that the release of acetic acid was reduced significantly and more limited on to bologna than on to ham or pastrami (Quattar et al., 2000). In another study, Li et al. (1996) found that addition of 3000 ppm of N-carboxymethylchitosan to cooked pork was sufficient to prevent the oxidative rancidity of the product. Fruit preservative Chitin, chitosan and their derivatives have been used as food wraps, due to their film-forming properties. The chitosan film controls moisture movement between food and the surrounding environment, thus decreasing the rate of metabolism, respiration, and rendering high impermeability to certain substances such as fats and oils, in addition to temperature. These would lead to a delay in ripening of fruits (Shahidi et al., 1999). The coating of fruits with chitosan delays the rate of ripening and the occurrence of decay in tomato (El-Ghaouth et al., 1992a), bell pepper and

362

Maximising the value of marine by-products

cucumber (El-Ghaouth et al., 1991a), and strawberries (El-Ghaouth et al., 1991b). The control of disease in fruits by chitosan could account for the antifungal activity of chitosan and its capacity to provoke defence enzymes and phytoalexins in the plant tissue or a combination of both (El Ghaouth et al., 1992c). Chitosan (7.2% NH2) inhibited the growth of post harvest pathogens, namely B. cinerea, A. alternata, C. gloesporioides, and R. stolonifer (El Ghaouth et al., 1992c). Among the fungi examined, R. stolonifer was least affected by chitosan (El Ghaouth et al., 1992c). While chitin did not influence the growth of any of the fungi tested, the growth delay of fungi provoked by chitosan increased with increasing of the degree of deacetylation (El Ghaouth et al., 1992c). The inhibitory effects of chitosan correlated with its cationic nature and the size of the polymers. Moreover, the importance of cationic groups and the length of the polymer chain was demonstrated by the low fungicidal activity displayed by N,O-carboxymethyl chitosan compared to that of chitosan, and by the improved activity of chitosan with increasing levels of deacetylation. The antifungal effects may, in part, account for the capacity of chitosan to enhance membrane permeability and result in cellular leakage. Three mechanisms may be involved in the action of chitosan as an antifungal agent in the preservation of post harvest crops. Firstly, the treatment of potato with chitosan, challenged with Erwinia carotovora (the soft rot pathogen of potato), showed a declined count of bacteria and tissue maceration, thus resulting in an increase in cell viability. Secondly, potatoes treated with chitosan showed an inhibition in bacterial reproduction as well as secretion of pectic enzymes (produced by pathogenic bacteria capable of attacking the plant tissue). The third mechanism of chitosan action is by controlling the pH. The pathogenic bacteria that cause decay of crops such as potatoes after harvesting secrete macerating enzymes (negatively charged proteins), leading to an outflow of protons and cations from the cell wall of the plant and hence a pH increase in the cell and cell wall. Acidity adjustment Chitosan could be used for deacidification of fruit juices because chitosan salts carry strong positive charge that could interact with proteins and hence act as dehazing agents in fruit juice (Shahidi et al., 1999). Scheruhn et al. (1999) reported that treating of coffee drinks with chitosan increased the pH and decreased the acid content of the coffee drinks due to the acid binding properties of chitosan in the coffee. This treatment depended on the concentration of chitosan and the acid content of the drinks; in addition to the raw material of chitosan and its processing (Scheruhn et al., 1999). Antioxidant activity Muscle food products containing a high content of unsaturated lipids are highly labile to off-flavor and rancidity development. Warmed-over flavor is developed in cooked poultry and uncured meat upon storage, resulting in the loss of

Chitin and chitosan from marine by-products 363 attractive meaty flavor (Shahidi et al., 1999). Darmadji and Izumimoto (1994) noticed that 1% chitosan added to meat resulted in a decline of 70% in the 2thiobarbituric acid (TBA) values after three days of storage at 4ëC. St. Angelo and Vercellotti (1989) reported that N-carboxmethylchitosan was effective in preventing the formation of warm-over flavor (WOF) over a broad range of temperature. Moreover, ground beef treated with 5000 ppm of N-carboxymethylchitosan exhibited 93 % inhibition of TBA values and 99% reduction in hexanal content. Furthermore, Shahidi et al. (1999) reported that N,O-carboxymethylchitosan (NOCC) and its lactate, acetate and pyrrolidine carboxylate salts were effective in controlling the oxidation and off-flavor development in cooked meat stored for nine days at refrigerated temperature. The mechanism by which this inhibition occurred was thought to be related to the chelation of free iron, which was released from hemoproteins during heat processing. These results were further confirmed by Li et al. (1996) who found that addition of 3000 ppm N-carboxmethylchitosan to cooked pork was sufficient for inhibiting the development of oxidative rancidity in the product. 16.3.3 Agricultural applications and retention of nutrients Smither-Copperl (2001) found that chitin exhibits several functions, including retention of nutrients, in the soil. Chitin contributes to the cycling of nutrients such as nitrogen. When chitin decomposes, it produces ammonia, which takes part in the nitrogen cycle. Furthermore, chitin is a main constituent in geochemical recycling of both carbon and nitrogen. Fungi, arthropods, and nematodes are the major contributor of chitin in the soil. Among these the fungi provide the largest amount of chitin in the soil (6±12% of the chitin biomass which is in the range 500±5000 kg/ha). In another study, Kokalis-Burelle (2001) reported that chitin contributes significantly to soil enrichment. It was found that chitin could control plant pathogens, pathogenic nematodes and provoke the development of host plant resistance against these pathogens. Chitin led to an increase in microorganism population; this sharp increase could shift and prompt their action as anti-plant pathogens in two ways. Firstly, the microorganism may act as parasite for plant pathogens. Secondly, they can kill or inhibit these pathogens through production of toxins or metabolites or enzymes. Furthermore, the increase in microorganism numbers increases the number of non-parasitic nematodes, which results in a decline in the number of pathogenic nematodes. 16.3.4 Industrial applications and water purification Treatment of industrial wastewater is necessary before their use or disposal because of the environmental and health difficulties associated with heavy metals and pesticides and their deposit through the food chain (Shahidi et al., 1999). Traditional methods for the elimination of heavy metals from industrial

364

Maximising the value of marine by-products

wastewater may be inefficient or costly, particularly when metals are present at low concentrations (Deans and Dixon, 1992; Volesky, 1987). Recovering of metal ions from discards can be achieved using a chelation ion exchange process. Biopolymers, such as chitin and chitosan, have the ability to lower the concentration of transition metal ions to parts per billion levels. These biopolymers should be ecologically safe, commercially available and bear a number of different functional groups, such as hydroxyl and amino groups in their backbones (Deans and Dixons, 1992). Chitosan can be used for treatment of wastewater because it has a good sorption capacity (Jeuniaux, 1986). In Japan, chitin and chitosan have been used for water purification due to their ability to complex metal ions via their amino groups (Simpson et al., 1994). Chitosan powder and dried films of it have free amino groups above the pKa of their NH2 groups. Therefore, chitosan powder and dried films have potential use in complexing metal ions (Tirmizi et al., 1996). The United States Environmental Protection Agency (USEPA) has approved the use of commercially available chitosan for wastewater treatment up to a maximum level of 10 mg/L (Knorr, 1984). Muzzarelli et al. (1989) have demonstrated the effectiveness of cross-linked N-carboxymethylchitosan in removing lead and cadmium from drinking water. Micera et al. (1986) have shown that chitosan has a high binding capacity for metals such as copper and vanadium. Deans and Dixons (1992) observed that unfunctionalized chitosan was efficient in eliminating Cu2+, but not Pb2+. Thome and Daele (1986) examined the ability of chitosan to remove polychlorinated biphenyls (PCB) from polluted stream water. The authors showed that chitosan was highly effective, compared to activated charcoal, for purification of (PCB) of polluted water. Use of chitosan for purification of potable water is also in practice.

16.4

Safety and regulatory status

Chitosan has many industrial, agricultural, food, pharmaceutical, and cosmetic applications. Consequently, safety and toxicological studies have been performed on chitosan in order to address issues related to its regulatory status. Rao and Sharma (1997) reported no toxicity for 2% chitosan solution in acetic acid, when applied on punctured bleeding capillaries in mice, rabbits and guinea pigs. These researchers further observed that eye irritation tests in rabbits and skin irritation tests in guinea pigs did not produce any toxic effect due to chitosans. Similar results were obtained by Mou et al. (2003) who reported no obvious toxic reaction using a mixture of polylactic acid and chitin as a basic scaffold material in tissue engineering. Chitosan received the `Generally Recognized As Safe' (GRAS) status by the Food and Drug Administration (FDA) in the United States in 1983 for use as animal feed component; its use in pet food was also reported by Shepherd et al. (1997). The use of chitosan for purification of potable water was approved by the US Environmental Protection Agency (EPA),

Chitin and chitosan from marine by-products 365 up to a maximum concentration of 10 mg/L (Knorr, 1986). In 1992, Japan's Health Department approved the use of chitin and its derivatives as functional food ingredients. Based on their definition of functional foods, chitin and chitosans possess most of the required attributes related to enhancement of immunity, prevention of illness, delaying of aging, recovery for illness and control of biorhythm (Subasinghe, 1999). Thus, the use of chitosan in foods such as potato chips has been in commercial practice for some time. Therefore, regulatory status of chitosan varies from country to country and its use in food requires further studies in order to address issues of concern (Lenz and Hamilton, 2004).

16.5

References and further reading

and L.A. HADWIGER, The Fungicidal Effect of Chitosan on Fungi of Varying Cell Wall Composition, Exp. Mycol. 3: 285±287 (1979). ALLAN, G.G. and M. PEYRON, The Kinetics of the Depolymerization of Chitosan by Nitrous Acid. In Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties, and Applications. Eds. G. Skjak-Braek, T. Anthonsen, and D. Sandford, Elsevier Applied Science, New York, 443±466 (1989). ANTHONSEN, M.W., K.M. VARUM and O. SMIDSROD, Solution Properties of Chitosans: Conformation and Chain Stiffness of Chitosans with Different Degree of Nacetylation. Carbohydr. Polym. 22: 193±201 (1993). ASHFORD, N.A., D.B. HATTIS, A.E. MURRAY and K. SEO, Industrial Applications of Chitin and Chitin Derivatives. Inter Ocean, 76: 1160±1170 (1976). AUSTIN, P.R., Solvents and Purification of Chitin, US Patent 3,892,731 (1975). AVERBACH, B.L., Chitin-Chitosan Production for Utilization Shellfish Wastes. In Seafood Waste Management in the 1980s: Conference Proceedings, September 23±25, Orlando, FL, Ed. W.S. Otwell, Gainesville, Florida, Marine Advisory Program, Florida Cooperative Extension Service, University of Florida, FL, pp. 285±300 (1981). BAGNARA-TARDIF, C., C. GAUDIN, A. BEHAICH, P. HOEST, T. CITARD and J.P. BELAICH, Sequence Analysis of a Gene Cluster Encoding Cellulases from Clostridium cellulolyticum. Gene 119: 17±28 (1992). BAI, R. K., M. Y. HUANG and Y. Y. JIANG, Selective Permeabilities of Chitosan-Acetic Acid Complex Membrane for Oxygen and Carbon Dioxide, Polymer Bull. 20, 83±88 (1988). BECKER C. July 22, 2003. Bloodless Coup-Revolutionary Bandage That Stanches Heavy Bleeding. Available at: http://www.noblood.com/forum/showthread.php?t=460. BERKELEY, R.C.W., Chitin, Chitosan and Their Degradative Enzymes. In Microbial Polysaccharides and Polysaccharases, Eds. R.C.W. Berkeley, G.W. Gooday and D.C. Ellwood, Academic Press, London, pp. 174±189 (1979). BISHOP, R.H., C.L. DUNCAN, G.M. EVANCHO, and H. YOUNG, Estimation of Fungal Contamination on Tomato Products by A Chemical Assay for Chitin. J. Food Sci. 47: 437±439 (1982). BOSSO, C., J. DEFAYE, A. DOMARD, A. GADELLE and C. PEDERSON, The Behaviour of Chitin ALLAN, C.R.

366

Maximising the value of marine by-products

Toward Anhydrous Hydrogen Fluoride Preparation of -1-4-linked 2 acetamido-2Deoxy-D-Glucopyranosyloligosaccharides, Carbohydr. Res. 156: 57 (1986). BROWN, D. March 24, 2003; Page A18, The War Against Battlefield Wounds. Available at: http://www.hemcon.com/WashPost.pdf. BRZESKI, M.M. Chitin and Chitosan ± Putting Waste to Good Use. Infofish Int. 5: 31±33 (1987). CAPOZZA, R.C., Enzymically Decomposable Biodegradable Pharmaceutical Carrier, Ger. Patent 2,505,305 (1975). CARDENAS, G., P. ORLANDO and T. EDELIO, Synthesis and Applications of Chitosan Mercaptanes as Heavy Metal Retention Agent. Inter. J. Biol. Macromol. 28: 167±174 (2001). CHANG, D.S., H.R. CHO, H.Y. GOO and W.K. CHOE, A Development of Food Preservation with the Waste of Crab Processing. Bull. Korean Fish Soc. 22: 70±78 (1989). CHEAH, L.H. and B.B.C. PAGE, Chitosan Coating for Inhibition of Sclerotinia Rot of Carrots, in New Zealand J. Crop Hort. Sci. 25: 89±92 (1997). CHEN, C., LIAU, W. and TSAI, G., Antibacterial effects of N-sulfonated and N-sulfobenzoyl Chitosan and Application to Oyster Preservation. J. Food Protect. 61: 1124±1128 (1998). CHO, Y.-W., Y.-N. CHO, S.-H. CHUNG, G. YOO and S.-W. KO, Water-soluble Chitin as a Wound Healing Accelerator. Biomaterials 20: 2139±2145 (1999). CLAESSON, P.M. and B.W. NINHAM, pH-Dependent Interaction Between Adsorbed Chitosan Layers. Langmuir 8: 1406±1412 (1992). CUERO, R.G., G. OSUJI and A. WASHINGTON, N-Carboxymethyl Chitosan Inhibition of Aflatoxin Production: Role of Zinc. Biotechnol. Lett. 13: 441±444 (1991). DARMADJI, P. and M. IZUMIMOTO, Effect of Chitosan in Meat Preservation. Meat Sci. 38: 243±254 (1994). DEANS, J. R. and B. G. DIXON, Bioabsorbents for waste-water Treatment. In Advances in Chitin and Chitosan, Eds. C.J. Brine, P.A. Sandford, J.P. Zikakis. Elsevier Applied Science, Oxford, pp. 648±656 (1992). DEFAYE, J., A. GADELLE and C. PEDERSON, Chitin and Chitosan. Eds. G. Skjak-Braek, T. Anthonsen, and P. Sandford, Elsevier, London, pp. 415±429 (1989). DEFAYE, J., A. GADELLE and C. PEDERSEN, Synthesis of Cyclohexakis- and Cycloheptakis(1!4)-(7-amino-6,7-dideoxy-alpha-D-glucoheptopyranosyl), homoanalogues of 6-amino-6-deoxy-cyclomaltooligosaccharides. Carbhydr. Res. 261±267 (1994). DESBRIERES, J., Viscosity of Semiflexible Chitosan Solutions: Influence of Concentration, Temperature, and Role of Intermolecular Interactions. Biomacromolecules 3: 342± 349 (2002). DOMARD, A., pH and CD Measurements on A fully Deacetylated Chitosan: Application to Copper (II) Polymer Interactions. Int. J. Boil. Macromol. 9: 98±104 (1987). DOMARD, A. and N. CARTIER, Glucosamine Oligomers: Preparation and Characterization. In Chitin and Chitosan. Eds. Skjak-Braek, G. T. Anthonsen, and P. Sandford, Elsevier, London, 287±383 (1989). DONALD, W.W. and C.J. MIROCHA, Chitin as a Measure of Fungal Growth in Stored Corn and Soybean Seed. Cereal Chem. 54: 466±474 (1977). DUNN, Q.L.E.T., E.W. GRANDMAISON and M. F GOOSON, Application and Properties of Chitosan. Ed. M.F.A. Goosen. Technomic Publishing Co., Lancaster, PA, (1997). EL-GHAOUTH, A., J. ARUL and R. PONNAMPALAM, Use of Chitosan Coating to Reduce Water Loss and Maintain Quality of Cucumber and Bell Pepper Fruits. J. Fruit Proc. Preserv. 15: 359±368 (1991a).

Chitin and chitosan from marine by-products 367 and M. BOULET, Chitosan Coating Effect on Storing and Quality of Fresh Strawberries, J. Food Sci. 56: 1618±1620 (1991b). EL-GHAOUTH, A., R. PONNAMPALAM, F. CASTAIGNE and J. ARUL, Chitosan Coating to Extend the Storage Life of Tomatoes, Hortscience 27: 1016±1018 (1992a). EL-GHAOUTH, A., J. ARUL, A. ASSELIN and N. BENHAMOU, Antifungal Activity of Chitosan on Two Post-Harvest Pathogens of Strawberry Fruits, Phytopathology 82: 398±402 (1992b). EL-GHAOUTH, A., J. ARUL, A. ASSELIN and N. BENHAMOU, Antifungal Activity of Chitosan on Post-harvest Pathogens: Induction of Morphological and Cytological Alterations an Rhizopus stolonifer. Mycol. Res. 96: 769±779 (1992c). EL-KATATNY, M.H., M. GUDELJ, K.-H. ROBRA, M.A. ELNAGHY and G.M. GUBITZ, Characterization of a chitinase and an Endo- -1,3-glucanase from Trichoderma harzianum Rifai T24 Involved in Control of the Phytopathogen Sclerotium rolfsii. Appl. Microbiol. Biotechnol. 56: 137±143 (2001). ENOMOTO, M., M. HASHIMOTO and T. KURAMAE, Low molecular weight chitosan as anticholesterolemic. Jpn. Kokai Tokkyo Koho, 117: 104±168 (1992). FANG, S.W., C.F. LI and D.Y.C. SHIHI, Antifungal Activity of Chitosan and its Preservative Effect on Low- sugar Candies Kumquat, J. Food Protect. 56: 136±140 (1994). HADWIGER, L.A., D.F. KENDRA, B.W. FRISTENSKY and W. WAGONER, Chitosan both Activates Genes in Plants and Inhibits RNA Synthesis in Fungi. In Chitin in Nature and Technology, Eds. R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday, Plenum Press, New York, 209±222 (1985). HEUX, L., J. BRUGNEROTTO, J. DESBRIERES, M.-F. VERSALI and M. RINAUDO, Solid state NMR for Determination of Degree of Acetylation of Chitin and Chitosan. Biomacromolecules 1: 746±751 (2000). HIRANO, S. Chitin biotechnological applications. Biotechnol. Ann. Rev. 2: 237-258 (1996). HIRANO, S. and N. NAGANO, Effects of Chitosan, Pectic Acid, Lysozyme and Chitinase on The Growth of Several Phytopathogens. Agric. Biol. Chem. 53: 3065±3066 (1989). HIRANO, S. and M. ZHANG, Cellulose-acidic Glycosaminoglycan Blend Fibers Releasing a Portion of the Glycosaminoglycans in Water. Carbohydrate Polymers 43: 281± 284 (2000). HIRANO S., Y. OHE and H. ONO, Selective N-acetylation of Chitosan, Carbohydr. Res. 47: 315 (1976). HIRANO, S., C. ITAKURA, H. SEINO, Y. AKIYAMA, I. NONAKA, N. KANBARA and T. KAWAKAMI, Chitosan as an Ingredient for Domestic Animal Feeds. J. Agric. Food Chem. 38: 1214±1217 (1990). HIRANO, S., M. ZHANG, B.G. CHUNG and S.K. KIM, The N-acylation of Chitosan Fibre and The N-deacetylation of Chitin Fibre and Chitin-Cellulose Blended Fibre at a Solid State. Carbohydrate Polymers 41: 175±179 (2000). HOLME, H.K., H. FOROS, H. PETTERSEN, M. DORNISH and O. SMIDSROD, Thermal Depolymerization of Chitosan Chloride. Carbohydrate Polymers 46: 287±294 (2001). HOROWITZ, S. T., S. ROSEMAN and H.J. BLUMENTHAL, The Preparation of Glucosamine Oligosaccharides Separation. J. Am. Chem. Soc. 79: 5046±5049 (1957). HOWLING, G.I., P.W. DETTMAR, P.A. GODDARD, F.C. HAMPSON, M. DORNISH and E.J. WOOD, The Effect of Chitin and Chitosan on the Proliferation of Human Skin Fibroplasts and Kratinocytes in vitro. Biomaterials 22: 2959±2966 (2001). HUANG, C., S. CHEN and J.R. PAN, Optimal Condition for Modification of Chitosan: a Biopolymer for Coagulation of Colloidal particles. Wat. Res. 34: 1057±1062 (2000). EL-GHAOUTH, A., J. ARUL, R. PONNAMPALAM

368

Maximising the value of marine by-products

Chitosan and Dietary Fibers. Prepared Foods: NS11±NS14 (2002). and I. AZUMA, Stimulation of Non-Specific Host Resistance Against Sendai Virus and Escherichia coli by Chitin Derivatives in Mice. Vaccine 5: 270±274 (1987). IKEDA, I., M. SUGANO and K. YOSHIDA, Effects of Chitosan Hydrolysates on Lipid Absorption and on Serum and Liver Lipid Concentration in rats. J. Agric. Food Chem. 41: 431±435 (1993). INABA, T., T. OHGUCHI, T. IGA and E. HASEGAWA, Synthesis of 4-Methylcoumarine-7-Yloxy Tetra-N-Acetyl- -Chitotetraoside, a Novel Synthetic Substrate for the Fluorometric Assay of Lysozyme. Chem. Pharm. Bull. 32: 1597±1603 (1984). ITO, M., A. BAN and M. ISHIHARA, Anti-ulcer Effects of Chitin and Chitosan, Healthy Foods, in Rats. Jpn. J. Pharmacol. 82: 218±225 (2000). JAWORSKA, M.M. and E. KONIECZNA, The Influence of Supplemental Components in Nutrient Medium on Chitosan Formation by the Fungus Absidia orchidis. Appl. Microbiol Biotechnol. 56: 220±224 (2001). JEON, Y.-J., F. SHAHIDI and S.-K. KIM, Preparation of Chitin and Chitosan Oligomers and their Applications in Physiological Functional Foods. Food Rev. Int. 16: 159±176 (2000). JEON, Y.-J., J.-Y. KAMIL and F. SHAHIDI, Chitosan as an Edible Invisible Film for Quality Preservation of Herring and Atlantic Cod. J. Agric. Food Chem. 50: 5167±5178 (2002). JEUNIAUX, C., Chitosan as a Tool for Purification of Waters. In Chitin in Nature and Technology, Eds. R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday, Plenum Press, New York, pp. 551±570 (1986). KAMIL J.Y.V.A., Y-J JEON and F. SHAHIDI, Antioxidative Activity of Chitosans of Different Viscosity in Cooked Comminuted Flesh of Herring (Clupea harengus), Food Chem. 79: 69±77 (2002). KAWACHI, I., T. FUJIEDA, M. UJITA, Y. ISHII, K. YAMAGISHI, H. SATO, T. FUNAGUMA and A. HARA, Purification and Properties of Extracellular Chitinases from The Parasitic Fungus Isaria Japonica. J. Biosci. Bioeng. 92: 544±549 (2001). KAYE, R., Chitosan Markets and Quality Go Hand-in-hand. In Biotechnology of Marine Polysaccharides, Proceedings of the Third Annual MIT Sea Grant College Program Lecture and Seminar, Eds. R.R. Colwell, E.R. Pariser and A.J. Sinskey, Hemisphere Publishing Corporation, New York, 333±342 (1985). KENDRA, D.F., D. CHRISTIAN and L.A. HADWIGER, Chitosan oligomers from Fusarium solani/ Pea Interactions, Chitinase/( -glucanase Digestion of Sporelings and from Fungal Wall Chitin Actively Inhibit Fungal Growth and Enhance Disease Resistance. Physiol. Mol. Plant Path. 35: 215±230 (1989). KENTARO, K., Y. TETSUTARO, I. RUMI and K. ICHIRO, Basic Study on Metal Ion Uptake onto Chitosan Using Ion-selective Electrodes. Kyushu Kogyo Daigaku Kenkyu Hokoku, Kogaku, 53: 81±85 (1986). KNORR, D., Use of Chitinous Polymers in Food ± A Challenge for Food Research and Development. Food Technol. 38: 85±97 (1984). KNORR, D., Nutritional Quality, Food Processing and Biotechnology Aspects of Chitin and Chitosan: A Review. Process Biochem. 6: 90±92 (1986). KOKALIS-BURELLE, N., Chitin Amendments for Suppression of Plant Nematodes and Fungal Pathogens. Phytopathology 91: 5168±5175 (2001). KOSHIJIMA T., R. TANAKA, E. MURAKI, A. YAMADA and F. YAKU, Chelating Polymers Derived HUGHES, K.

IIDA, J., T. UNE, K. C. ISHIHATA, NISHIMURA, S. TOKURA, N. MIZUKOSHI

Chitin and chitosan from marine by-products 369 from Cellulose and Chitin .I. Formation of Complexes from Metal Ions. Cell. Chem. Technol. 7: 197±205 (1973). KRISTBERGSSON, K., J.M. EINARSSON, S. HAUKSSON, M.G. PETER and J. GISLASON, Recent Developments in Deacetylation of Chitin, and Possible Applications in Food Formulations. Proceedings of the First Joint Trans Atlantic Fisheries Technology Conference, Reykjavik, Iceland, June 10±14, 2003, p. 350. KURAKAKE, M., S. YO-U, K. NAKAGAWA, M. SUGIHARA and T. KOMAKI, Properties of Chitosanase from Bacillus cereus S1. Current Microbiology 40: 6±9 (2000). KURITA, K., Chemical Modification of Chitin and Chitosan. In Chitin in Nature and Technology, Eds. R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday, Plenum Press, New York, pp. 287±293 (1986). KURITA, K., M. ISHIGURO and T. KITAJIMA, Studies on Chitin. 17. Introduction of Long Chain Alkylidene Groups and the Influence of Properties. Int. J. Biol. Macromol. 10: 124±129 (1988). KURITA, K., K. TOMITA, S. ISHII, S. NISHIMURA and K. SHIMODA, Squid Chitin A Potential Alternative Chitin Source: Deacetylation Behaviour and Characteristics Properties. J. Poly. Sci.: Part A: Poly. Chem., 31: 485±491 (1993). KURITA, K., J. AMEMIYA, T. MORI and Y. NISHIYAMA, Comb-shaped Chitosan Derivatives Having Oligo (ethylene glycol) Side Chains. Polymer Bull. 42: 387±393 (1999). KURITA, K., T. KOJIMA, Y. NISHIYAMA and M. SHIMOJOH, Synthesis and Some Properties of Nonnatural Amino Polysaccharides: Branched Chitin and Chitosan. Macromolecules 33: 4711±4716 (2000). KURITA, K., S. MORI, Y. NISHIYAMA and M. HARATA, N-Alkylation of Chitin and Some Characteristics of the Novel Derivatives. Polymer Bull. 48: 159±166 (2002). LAHIJI, A., A. SOHRABI, D.S. HUNGERFORD and C.G. FRONDOZA, Chitosan Supports the Expression of Extracellular Matrix Proteins in Human Osteoblasts and Chondrocytes. J. Biomed. Mater. Res. 51: 586±595 (2000). LEE, H.-S., D.-S. HAN, S.-J. CHOI, S.-W. CHOI, D.-S. KIM, D.-H. BAI and J.-H. YU, Purification, Characterization, and Primary Structure of a Chitinase from Pseudomonas sp. YHS-A2. Appl. Microbiol. Biotechnol. 54: 397±405 (2000). LEE, J.-S., D.-S. JOO, Y. S.-Y. CHO, J.-H. HA and E.-H. LEE, Purification and Characterization of Extracellular Chitinase Produced by Marine Bacterium, Bacillus sp. LJ-25. J. Microbiol. Biotechnol. 10: 307±311 (2000). LENZ, T.L. and W.R. HAMILTON, Supplemental Products Used for Weight Loss, J. Am. Pharm. Assoc. 44: 59±67 (2004). LI, Q., E.T. DUNN, E.W. GRANDMAISON and M.F.A. GOOSEN, Applications and Properties of Chitosan. J. Bioac. Compat. Polym. 7: 370±397 (1992). LI, K., Y. HWANG, T. TSAI and S. CHI, Chelation of Iron Ion and Antioxidative Effect on Cooked Salted Ground Pork by N-Carboxymethylchitosan (NCMC). Food Sci. Taiwan 23: 608±616 (1996). LOWER, S.E., Polymers from The Sea Chitin and Chitosan. Manufac. Chem. 55: 73±75 (1984). MADHAVEN, P. and N.K.G. RAMACHANDRAN, Utilization of Prawn Waste. Isolation of Chitin and its Conversation to Chitosan. Fish Technol. 11: 50±56 (1974). MCKAY, G., H. BLAIR and A. FINDON, Kinetics of Copper Uptake on Chitosan. In Chitin in Nature and Technology. (R. Muzzarelli, C. Jeuniaux and G. W. Gooday, Eds.) Plenum Press, New York, pp. 559±583 (1986). MEINS F., J.-M. NEUHAUS, C. SPERISEN and J. RYALS, The Primary Structure of Plant-

370

Maximising the value of marine by-products

pathogenesis-related Glucanohydrolases and their Genes. In T. Boller, F. Meins. Jr, eds, Genes Involved in Plant Defense. Springer, New York, Berlin, pp. 245±282 (1992). MICERA, G., S. DEIANA, A. DESSI, P. DECOCK, B. DUBOIS and H. KOZLOWSKI, Copper and Vanadium Complexes of Chitosan. In Chitin in Nature and Technology, Eds. R.A.A. Muzzarelli, C. Jueuniaux, and G. W. Gooday, Plenum Press, New York, pp. 565±567 (1986). MIENTKA, M., May 2003. New Shrimp Bandage Could Reduce Tourniquet Reliance. Available at http://www.usmedicine.com/article.cfm?articleID=642&issueID=50 MOU, S.S., A-D. MA, M. TU, L.H. LI and C.R. ZHOU, Preparation of Polylactic Acid/Chitin Composite Material and its Safety Evaluation by Animal Experiments, Di-yi-junyi-da-xue-xue-bao, 23: 245±247 (2003). MUZZARELLI, R.A.A., Natural Chelating Polymers: Alginic acid, Chitin and Chitosan, Pergamon Press, Oxford, UK (1973). MUZZARELLI, R.A.A., Chitin, Pergamon Press, Oxford, UK (1977). MUZZARELLI, R.A.A., Chitin. In The Polysaccharides Vol. 3, Ed. G.O. Aspinall, Academic Press Inc., New York, pp. 417±450 (1985). MUZZARELLI, R.A.A., Native, Industrial and Fossil Chitins. In Chitin and Chitinases. Eds. P. Jolles and R.A.A. Muzzarelli, Birhhauser Verlag, Basel, Switzerland, 1±6 (1999). MUZZARELLI, R.A.A., M. WECKX and O. FILLIPINI, Removal of Trace Metal Ions from Industrial Waters, Unclear Effluents and Drinking Water, with the Aid of Crosslinked N-Carboxymethyl Chitosan, Carbohydrate Polymers 11: 293±296 (1989). MUZZARELLI, R.A.A., N. FREGA, M. MILIANI, C. MUZZARELLI and M. CARTOLARI, Interactions of chitin, chitosan, N-lauryl chitosan and Dimethylaminopropyl Chitosan with Olive Oil. Carbohydrate Polymers 43: 263±268 (2000). NISHIMURA, K., S. NISHIMURA, N. N. NISHI, I. SAIKI, S. TOKURA and I. AZUMA, Immunological Activity of Chitin and its Derivatives. Vaccine 2: 93±99 (1984). OH, H., Y.J. KIM, E.J. CHANG and J.Y. KIM, Antimicrobial Characteristics of Chitosan Against Food Spoilage Microrganisms in Liquid Media and Mayonnaise. Biosci. Biotechnol. Biochem. 65: 2378±2383 (2001). ORNUM, J.V., Shrimp Waste ± Must it be Wasted? Infofish Int. 6: 48±52 (1992). PAPINEAU, A.M., D.G. HOOVER, D. KNORR and D.F. FARKAS, Antimicrobial Effect of Watersoluble Chitosan with High Hydrostatic Pressure. Food Biotechnol. 5: 45±57 (1991). QUATTAR, B., R.E. SIMARD, G. PIETT, A. BEGIN and R.A. HOLLEY, Inhibition of Surface Spoilage Bacteria in Processed Meats by Application of Antimicrobial Films Prepared with Chitosan. Int. J. Food Microbiol. 62: 139±148 (2000). RAO, S.B. and C.P. SHARMA, Use of Chitosan as a Biomaterial: Studies on its Safety and Hemostatic Potential, J. Biomed. Mater. Res., 34: 21±28 (1997). RHA, C., Chitosan as a Biomaterial. In Biotechnology in the Marine Sciences, Proceedings of the First Annual MIT Sea Grant Lecture and Seminar, Eds. R.A. Colwell, A.J. Sinskey and E.R. Pariser, John Wiley and Sons, New York, pp. 177±189 (1984). RUPLEY, J.A., The Hydrolysis of Chitin by Concentrated Hydrochloric Acid, and the Preparation of Low-Molecular Substrate for Lysozyme. Biochem. Biophys Acta. 83: 245±255 (1964). SAKAI, K., F. NANJO and T. USUI, Production and Utilization of Oligosaccharides from Chitin and Chitosan. Denpun Kagaku, 37: 79±86 (1990). SAVAGE, P.J. and G. P. SAVAGE, The Effect of Coating Apples on the Quality of Stored

Chitin and chitosan from marine by-products 371 Apples, Proceed. Nutr. Soc. New Zealand 19: 129±133 (1994). and D. KNORR, Studies of Acid Binding Properties of Chitosan in Coffee Beverages. Nahrung 43: 100±104 (1999). SCHILLER, R.N., E. BARRAGER, A.G. SCHAUSS and E.J. NICHOLS, A Randomized, Double-Blind, Placebo-Controlled Study Examining the Effects of a Rabidly Soluble Chitosan Dietary Supplement on Weight Loss and Body Composition in Overweight and Mildly Obese Individuals, Am. Nutr. Assoc. 4: 34±41 (2001). SHAHIDI F., Role of Chemistry and Biotechnology in Value-added Utilization of Shellfish Processing Discards, Can. Chem. News 47: 25±29 (1995). SHAHIDI F. and J. SYNOWIECKI, Isolation and Characterization of Nutrients and Valueadded Products from Snow Crab (Chinoecetes opilio) and Shrimp (Pandalus Borealis) process is discard, J. Agric. Food Chem. 39: 1527±1532 (1991). SHAHIDI, F., J.K.V. ARACHCHI and Y.-J. JEON, Food Application of Chitin and Chitosan. Trends Food Sci. Technol. 10: 37±51 (1999). SHEPHERD, R., S. READER and A. FALSHOW, Chitosan Functional Properties, Glycoconjugate J. 14: 535±542 (1997). SIMPSON, B.K., N. GAGNE and M.V. SIMPSON, Bioprocessing of Chitin and Chitosan. In Fisheries Processing: Biotechnological Applications. Ed. A.M. Martin, Chapman and Hall, London, pp. 155±173 (1994). SIMPSON, B.K., N. GAGNE, I.N.A. ASHIE and E. NOROOZI, Utilization of Chitosan for Preservation of Raw Shrimp (Pandalus boreaslis). Food Biotechnol. 11: 25±44 (1997). SMITHER-COPPERL, M.L., Chitin as Biomass, its Origin and Role in Nutrient Cycling. Phytopathology 91: S167±S168 (2001). SORLIER, P., A. DENUZIERE, C. VITON and A. DOMARD, Relation Between the Degree of Acetylation and the Electrostatic Properties of Chitin and Chitosan. Biomacromolecules 2: 765±772 (2001). ST. ANGELO, A.J. and J.R. VERCELLOTTI, Inhibition of Warmed-over Flavour and Preserving of Uncured Meat Containing Materials, US Patent. 4,871,556 (1989). SUBASINGHE, S., Chiton for Shellfish Waste ± Health Benefits Overshadowing Industrial Uses. Infofish Int. 3: 58±65 (1999). SUDHARASHAN, N.R., D.G. HOOVER and D. KNORR, Antibacterial Action of Chitosan, Food Biotechnol. 6: 257 (1992). SUGANO, M., T. FUJIKAWA, Y. HIRATSUJI and Y. HASEGAWA, Hypocholesterolemic Effects of Chitosan in Cholesterol-fed Rats. Nutr. Rep. Int. 18: 531 (1978). SUGANO, M., T. FUJIKAWA, Y. HIRATSUJI, K. NAKASHIMA, N. FUKUDA and Y. HASEGAWA, A novel Use of Chitosan as a Hypocholesterolemic Agent in Rats. Am. J. Clin. Nutr. 33: 787 (1980). SUGANO, M., S. WATANABE, A. KISHI, M. IZUME and A. OHTAKARA, Hypocholesterolemic Action of Chitosans with Different Viscosity in Rats. Lipids 23: 187 (1988). SUZUKI, S., Studies on Biological Effects of Water Soluble Lower Homologous Oligosaccharides of Chitin and Chitosan. Fragrance J. 15: 61±68 (1996). SUZUKI, K., Y. OKAWA, K. HASHIMOTO, S. SUZUKI and M. SUZUKI, Protecting Effect of Chitin and Chitosan on Experimental induced Murine Candidiasis. Microbiol. Immunol. 28: 903±912 (1984). SUZUKI, K., A. TOKORO, Y. OKAWA, S. SUZUKI and M. SUZUKI, Enhancing Effects of N-acetyl Chitoligosaccharides on the Active Oxygen-Generating and Microbicidal Activities of Peritoneal Exudates Cells in Mice. Chem. Pharm. Bull. 33: 886± 888 (1985). SCHERUHN, E., P. WILLE

372

Maximising the value of marine by-products

and M. SUZUKI, Antitumor Effect of Hexa-N-acetylchitohexaose and Chitohexaose. Carbohydr. Res. 151: 403±408 (1986a). SUZUKI, K., A. TOKORO, Y. OKAWA, S. SUZUKI and M. SUZUKI, Effect of N-Acetylchitooligosaccharides on Activation of Phagocytes. Microbiol. Immunol. 30: 777±787 (1986b). TAKAHASHI, Y., F. MIKI and K. NAGASE, Effect of Sonolysis on Acid Degradation of Chitin to Form Oligosaccharides. Bull. Chem. Soc. Jpn. 68: 1851±1858 (1995). TAN, S.C., T.K. TAN, S.M. WONG and E. KHOR, The Chitosan Yield of Zygomycetes at their Optimum Harvesting Time. Carbohydr. Polym. 30: 239±242 (1996). THOME, J.P. and Y.V. DAELE, Adsorption of Polychlorinated Biphenyls (PCB) on Chitosan and Application to Decontamination of Polluted Stream Water. In Chitin in Nature and Technology, Eds. R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday, Plenum Press, New York, pp. 551±554 (1986). TIRMIZI, S.A., J. IQBAL and M. ISA, Collection Metal Ions Present in Water Samples of Different Sites of Pakistan Using Biopolymers Chitosan, J. Chem. Soc. Pakistan 18: 312±315 (1996). TSAI, G.J., W.-H. SU, H.C. CHEN and C.-L. PAN, Antimicrobial Activity of Shrimp Chitin and Chitosan from Different Treatments and Applications of Fish Preservation. Fisheries Sciences 68: 170±177 (2002). TSIGOS, I., A. MARTINOU, D. KAFETZOPOULOS and V. BOURIOTIS, Chitin Deacetylases: New, Versatile Tools in Biotechnology. Tibtech. 18: 305±312 (2000). TSUKADA, S. and Y. INOUE, Conformational Properties of Chitooligosaccharides-Titration, Optical Rotation and C-13 NMR-Studies of Chito-oligosaccharides. Carbohydr. Res. 88: 19±38 (1981). UCHIDA, Y., M. IZUME and A. OHTAKARA, Preparation of Chitosan Oligomers with Purified Chitosanase and its Application. In Chitin and Chitosan. Eds. G.T. Skjak-braek, Anthonsen and P. Sandford. Elsevier, London, pp. 373±382 (1989). UENO, K., T. YAMAGUCHI, N. SAKAIRI, N. NISHI and S. TOKURA, Advances in Chitin Science. Eds. A. Domard, G.A.F. Roberts, and K.M. Varum, Jacques Andre Publisher, Paris, 156±161 (1997). VOLESKY, B., Biosorbents for Metal Recovery, Trends Biotechnol. 5: 96±99 (1987). WANG, G., Inhibition and Inactivation of Five Species of Foodborne Pathogens by Chitosan. J. Food Protec. 55: 916±919 (1992). WEIST, J.L. and M. KAREL, Development of a Fluorescence Sensor to Monitor Lipid Oxidation. 1. Fluorescence Spectra of Chitosan Powder and Polyamide Powder after Exposure to Volatile Lipid Oxidation Products. J. Agric. Food Chem. 40: 1158±1162 (1992). WIN, N.N. and W.F. STEVENS, Shrimp Chitin as Substrate for Fungal Chitin Deacetylase. Appl. Microbiol. Biotecnol. 57: 334±341 (2001). WINTEROWD, J.G. and P.A. SANDFORD, Chitin and Chitosan, In Food Polysacchrides and their Applications. Ed. M.S. Alistair. Marcel Dekker Inc., New York, pp. 441±462 (1995). WOLFORM, M.L. and T.M. SHEN-HAN, The Sulfonation of Chitosan. J. Am. Chem. Soc. 81: 1764 (1959). YALPANI, M., F. JOHNSON and L.E. ROBINSON, Antimicrobial Activity of Some Chitosan Derivatives. In Advances in Chitin and Chitosan, Eds. C.J. Brine, P.A. Sandford, and J.P. Zikakis. Elsevier Applied Science, London, pp. 543±555 (1992) SUZUKI, K., T. MIKAMI, Y. OKAWA, A. TOKORO, S. SUZUKI

Chitin and chitosan from marine by-products 373 and T. LEHTIMAKI, Cholesterol-lowering Properties and Safety of Chitosan. Drug Res. 1: 1±7 (2002). YOUNG, D.H., H. KOHLE and H. KAUSS, Effect of Chitosan on Membrane Permeability of Suspension Cultured Glycine Max and Phaseolus vulgaris cells. Plant Physiol. 70: 1449±1454 (1982). ZHANG, H. and S.H. NEAU, In vitro Degradation of Chitosan by a Commercial Enzyme Preparation: Effect of Molecular Weight and Degree of Deacetylation. Biomaterials 22: 1653±1658 (2001). YLITALO, R., S. LEHTINEN, E. WUOLIJOKI, P. YLITALO

17 Marine enzymes from seafood by-products M. T. Morrissey and T. Okada, Oregon State University Seafood Laboratory, USA

17.1

Introduction

World fish and shellfish production has increased from 40 million metric tonnes (MMT) in 1962 to 133 MMT in 2002, with a concomitant increase in processed fish and production of fish by-products (Vannuccini, 2004). Frozen and fresh fish were the main fishery products accounting for over 60% of total production followed by canning/curing (7% and 4%, respectively) and non-food uses (26%). While the wild-caught fisheries production appears to have leveled off by the mid 1990s, aquaculture has shown considerable growth and now represents about 30% of the total production (Johnson, 2004). Currently, only about 50 to 60% of total catch is used for direct human consumption, and the annual discards from the world fisheries have been estimated to be approximately 25± 30 MMT of fish and shellfish caught each year (Sovik and Rustad, 2005). While much of these discards can be used to address increasing demands for fish meal there is also potential for the use of certain by-products for the extraction of unique compounds such as marine enzymes. Enzyme technology has been used extensively in the food processing industry including fish processing. Enzymes are used as processing aids and can be produced from by-product material from both new and traditional fish processing operations (Vecchi and Coppes, 1996). There are several excellent reviews on the utilization of enzymes from aquatic organisms (Shahidi and Kamil, 2001; Gildberg et al., 2000; Haard, 1998). Marine enzymes have drawn the attention of numerous researchers as they can possess unique specificity and characteristics. Enzyme recovery from seafood by-products could also help the

Marine enzymes from seafood by-products 375 environmental and ethical concerns surrounding discards and improve the bottom line for seafood companies wishing to exploit new technologies and markets. In this chapter, the current literature on the extraction and purification of various marine enzymes is reviewed and their potential use and limitations in their utilization are discussed. Extractable enzymes from seafood and seafood by-products include digestive and cellular proteinases, extracellular gastric proteinases, chitinases, lipases, phospholipases, transglutaminases, polyphenoloxidase and others. An excellent reference source on this topic is the text Seafood Enzymes by Haard and Simpson (2000). A partial list of enzymes which have been purified from fish and processing by-products include lipase from tilapia stomach (Taniguchi et al., 2001), amino peptidase, carboxypeptidases from tilapia intestine (Taniguchi and Takano, 2002, 2001), glycogenolytic and alpha glucosidase from Pacific mackerel, Japanese eel, black sea bream and frog flounder (Nakagawa et al., 1996), carbohydrases from butterfish, silver drummer, and marble fish, (Skea et al. 2005), transglutaminase from walleye pollock liver (Kumazawa et al., 1996) and oyster gill (Kumazawa et al., 1997), trypsin-like enzyme from shrimp (Honjo et al., 1990), protease from arrowtooth flounder (Wilson and Choudhury, 2004), myosin heavy chain-degrading proteinase from squid muscle (Ehara et al., 1994), cathepsin S from carp hepatopancreas (Pangkey et al., 2000), glucosidase, -glucosidase and amylase from digestive cecum of scallop (Nakai et al., 2005), amylases from hard clam viscera (Tsao et al., 2004), acid and alkaline phosphatase from intestine, liver, kidney of mackerel, sea robin, tongue sole, and others (Kuda et al., 2004), and cellulase and xylanase from crayfish and marine prawns (Crawford et al., 2005). Marine-based enzymes often have unique physical, chemical and catalytic properties compared to corresponding enzymes from terrestrial animals and plant sources (Shahidi and Kamil, 2001). Fish muscle, for example, contains approximately ten times more catheptic enzyme activities than mammalian muscles (Haard et al., 1994), and marine enzymes are generally more susceptible to hydrostatic pressure inactivation than their mammalian counterparts (Ashie and Simpson, 1996). Since the habitat of many marine animals tends to be in cold temperatures, their enzymes often have cold-adapted properties. They are known to be more catalytically active at low temperatures and possess lower thermal stability making them suitable for enzymatic applications in food processing as they can often be inactivated at lower temperatures (Gerday et al., 2000). Several enzymes have been shown to be salt tolerant as well, which can be an advantage in certain food applications (Caviccholi et al., 2002; Haard, 1998). Compared to their mesophilic analogues, cold-adapted enzymes tend to have a lower number of hydrogen bonds, less densely packed structures, increased surface hydrophilicity, a higher number of methionine residues, a different fold of the autolysis loop as well as the carboxyterminal region (Gudmundsdottir and Palsdottir, 2005). Enzyme extraction and utilization from seafood by-products include enzymes from both solid and liquid waste streams that are often discarded or traditionally

376

Maximising the value of marine by-products

processed into fish feed or fertilizer. Seafood by-products include viscera, skins, bones, heads, and frames from fish as well as shells and exoskeletons from shellfish in both traditional and modern processing operations. Fish and shellfish processing discards can consist of two thirds of incoming raw materials (Lee, 2000) depending on the process and species. Whether it is economically feasible to commercially extract enzymes from these by-products depends on the volume and quality of raw material, extraction technology and potential markets. The following sections will describe several different categories of marine enzymes and some of their unique properties. Extraction and purification methods will then be discussed followed by some examples of commercial operations as well as a discussion of limitations in the use of by-products for marine enzymes.

17.2

Marine enzymes

17.2.1 Proteolytic enzymes Among the extractable enzymes from seafood and seafood by-products, digestive proteolytic enzymes have received a considerable amount of interest from researchers over the last two decades due to the availability of raw materials such as viscera and their high rate of enzymatic activity. Proteases hydrolyze the peptide bonds in proteins and polypeptides and are characterized as endo(proteinases) and exo- (peptidases) proteases (Shahidi and Kamil, 2001). The increased production of value-added products such as fresh and frozen fillets and others has created large volumes of discards such as viscera that could be used as a raw material source. Proteolytic enzymes can also be found in muscle cells, the extracellular matrix or other organs and the hepatopancreas in shellfish. They can be extracted from seafood digestive tract and frame muscle as well as waste water from fish, shellfish and surimi processing. Digestive proteolytic enzymes include pepsin, trypsin, chymotryosin, elastase, gastricin and others. Pepsin can be categorized as aspartic proteinase and formed by an autocatalytic reaction from pepsinogen, which is normally located within the fish stomach and has peak activity at acidic conditions (pH 2.0 for most of substrates). Trypsin can be categorized as serine proteinase and it has a dual role in that it cleaves ingested proteins and activates the precursor forms of several other digestive proteinases including chymotrypsin. Trypsin is normally located in pyloric cecum and shows its activity at neutral and alkaline conditions (Naz, 2002). Muscle proteinases include catepsin A, B, C, D, H, and L, calpains and muscle collagenases, and they are mainly located in lysosomes, the sacroplasm, and extracellular matrix of the connective tissue (An and Visessanguan, 2000). Proteolytic enzymes have been purified and characterized from many species and seafood by-products. These include Atlantic cod (Amiza and Owusu Apenten, 2002; Jonsdottir et al., 2004), skipjack tuna, yellowfin tuna, tongol tuna (Suppashith et al., 2004), anchovy (Heu et al., 1995), white croaker skeletal muscle (Makinodan et al., 1987), salmon (Yamashita and Konagaya, 1995), decapoda muscle (Ehara et al., 1992), Pacific whiting (An et al., 1994), orange

Marine enzymes from seafood by-products 377 roughy stomach (Xu et al., 1996), Atlantic menhaden muscle (Choi et al., 1999a,b), Asian bony tongue (Natalia et al., 2003), mackerel white muscle (Aoki et al., 1995), gastric fluid of crab (Saborowski et al., 2004), Antarctic krill (Yoshitomi, 2005; Sjodahl et al., 2002) jumbo squid (Cardenas-Lopez and Haard, 2005), and surimi waste water (Mireles-DeWitt and Morrissey, 2002a; Benjakul et al., 1996, 1997). Proteolytic activity of Atlantic cod by-products from different fishing locations was studied by Sovik and Rustad (2005). They found that the proteolytic activity in viscera and frame muscle was the highest at pH 3 and 35ëC, whereas the proteolytic enzyme from liver showed its maximum activity at pH 3 and 50ëC. The proteolytic activity in viscera was 20 times higher than liver and 250 times higher than that of frame muscle. A significant difference in enzymatic activity was also reported for cod viscera harvested from different locations. There were seasonality differences in cod from the Icelandic and Barents Sea in April±June as well as differences from cod in these locations and cod from the South coast of Ireland during October±December. These researchers also studied seasonal changes in trypsin and chymotrypsin activity in viscera from cod, saithe, haddock, tusk and ling viscera from different locations (Sovik and Rustad, 2004) and found that enzyme activities differed among species. Krill proteases have been researched thoroughly and are well defined. Sjodahl et al. (2002) have identified three trypsin-like proteinases, and four carboxypeptidases. The trypsinlike proteinases were as much as 60 times as active as bovine trypsin. Yoshitomi (2005) found that crude krill digestive proteases were much more active in the Antarctic summer season (December to February) than the winter season and was correlated with phytoplankton abundance. Benjakul et al. (1996) recovered a major proteinase from Pacific whiting surimi wash water which was cathepsin L with molecular weight of 39.5 kDa. These researchers also characterized biochemical properties and stability of the proteinase recovered from Pacific whiting surimi wash water by ohmic heating, ultrafiltration, and freeze-drying (Benjakul et al., 1997) and found that they were stable at pH range of 3.0±9.0, having the highest activity at pH 4.0. 17.2.2 Collagenolytic enzymes Collagenolytic enzymes are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein (Kim et al., 2002). They can be divided into serine collagenases and metallocollagenases due to different physiological functions that they possess. Serine collagenases show their greatest activity in protein digestion, blood-clotting, fibrinolysis, complement activation and fertilization whereas metallocollagenases are involved in remodeling the extracellular matrix (Park et al., 2002). Collagenolytic enzymes have been extracted from shrimp (Brauer et al., 2003; Van Wormhoudt et al., 1992; Oh et al., 2000), cod (Kristjansson et al., 1995), filefish (Kim and Kim, 1991), yellow-tail pyloric ceca (Yoshinaka et al., 1972), crab (Roy et al., 1996; Grant et al., 1981, 1983; Klimova et al., 1990),

378

Maximising the value of marine by-products

winter flounder (Teruel and Simpson, 1995), and mackerel (Park et al., 2002). Related studies regarding its effect on autolysis of fish muscle tissues during storage period include cod (Hernandez-Herrero et al., 2003), and salmon (Hultmann and Rustad, 2004). These enzymes were most active in the pH range of 6.5±8.0 and are inactivated at pH < 6.0 (Haard and Simpson, 1994). Collagenase from internal organs of mackerel was purified and characterized, and its molecular weight was estimated to be 14.8 kDa, and the optimum pH and temperature were approximately pH 7.5 and 55ëC, respectively (Park et al., 2002). 17.2.3 Lipases Lipase is an enzyme produced primarily in the pancreas and hydrolyzes fatty acids from the glycerol backbone at the hydrophobic/hydrophilic interface of the lipid substrate. It is necessary for the absorption and digestion of lipid including mono-, di-, and triacylglycerols. Lipase has been purified and characterized from various aquatic organisms including cod (Gjellesvik et al., 1992), salmon (Gjellesvik et al., 1994), stomach, pyloric ceca, liver, and intestine of rohu, oil sardine, mullet, Indian mackerel (Nayak et al., 2003), red sea bream hepatopancreas (Iijima et al., 1998) and others. The optimum pH of lipase ranges from 6.0 to 9.0, and lipase is normally stable between 30 and 45ëC. These lipases could be suitable for various kinds of food applications because of their high activity at mild pH and temperature conditions. Phospholipases have also been extracted from pollock muscle (Audley et al., 1978), Atlantic cod muscle (Chawla and Ablett, 1987), gill membranes of red sea bream (Uchiyama and Nozaki, 2005), and hepatopancreas of red sea bream (Iijima et al., 1990, 1997). Recently, phospholipase A2 isozyme from starfish (Asterina pectinifera) was characterized (Kishimura and Hayashi, 2004) demonstrating that phospholipase A2 isozyme II had an optimum temperature of 50ëC and pH of 9.0. It did not show fatty acid specificity for hydrolysis of phosphatidylcholine; however, it had ten times the activity than that of commercially available porcine pancreatic phospholipase A2 and has been suggested as a potential source for phospholipase A2 for industrial production. Lipase from tilapia stomach and intestine were extracted and characterized by Taniguchi et al. (2001). The study showed that this lipase had a molecular weight of 54 kDa with optimum pH of 6.5, and optimum temperature of 40ëC. The lipases were stable at the pH range of 5.0 to 7.0 at 40ëC for 30 min while lipase from tilapia intestine showed its molecular weight of 46 kDa, optimum temperature of 35ëC, and was stable in the pH range of 6.5 to 8.5. The lipases were identified as non-specific in terms of position on the glycerol backbone but preferentially hydrolyzed triacylglycerol rather than di- and monoacylglycerol. The highest lipase activity was found when soybean oil was used for substrate showing 100% hydrolysis. A bile salt-activated lipase from hepatopancreas of red sea bream was extracted and characterized (Iijima et al., 1998). Molecular weight of this lipase

Marine enzymes from seafood by-products 379 was determined as approximately 64 kDa and had a pH optimum of 7.0 to 9.0. This lipase was homologous to mammalian bile salt-activated lipase which preferentially hydrolyzed ethyl esters of polyunsaturated fatty acids. 17.2.4 Chitinolytic enzymes Chitinolytic enzymes are widely distributed in crustaceans and play an important role in the degradation of chitin. Two chitinolytic enzymes are involved in crustaceans, which are endo-type (chitinase) and exo-type ( -N-acetylhexosaminidase), which are related to molting in several crustaceans (Kono et al., 1995). These enzymes have been purified mainly from processing by-products such as squid liver (Matsumiya, 2004), shrimp waste silage (Matsumoto et al., 2004), and shrimp by-products (Olsen et al., 1990). Chitinase was purified from the stomach of red sea bream (Pagrus major) and kinetic analysis (Karasuda et al., 2004) showed high enzyme activity at pH 2.5 and 9.0 toward glycolchitin, and at pH 2.5 and 5.0 toward N-acethylchitopentasaccharide. These multiple pH-peak activities are unique for chitinases. Prawn shell waste was used as a raw material for chitinase production by the marine fungus Beauveria bassiana BTMF S10 by solid state fermentation (Suresh and Chandrasekaran, 1998). Likewise, shrimp waste silage was used for production of -N-acetylhexosaminidase by Verticillium lecanii in submerged and solid state fermentations taking advantage of the abundance and composition of crustacean wastes (Matsumoto et al., 2004). They concluded that shrimp silage was an efficient inducer of the extracellular enzyme, compared with media supplemented with sucrose where enzymic activity was not detected. 17.2.5 Transglutaminases Transglutaminase is an aminoacyltransferase that catalyzes an acyl transfer reaction between -carboxyamide groups of glutamine residues in polypeptides and proteins. When the -amino group of lysine acts as an acyl acceptor, -( glutamyl) lysine crosslinks are formed in proteins which change protein texture and structure (Kumazawa, 2002). Protein properties, gelation capability, thermal stability, and water-holding capacity are uniquely affected by this crosslinkage (Kuraishi et al., 2001). A study showed that seafood products contained the highest level of -( -glutamyl) lysine crosslinks (43 moles/100 g protein) followed by meat products (38 moles/100 g protein) and soy product (15 moles/ 100 g protein) (Kuraishi et al., 2001). Transglutaminases have been purified and characterized from walleye pollock liver (Kumazawa et al., 1996), Japanese oyster (Kumazawa, 1997), red sea bream muscle (Yasueda et al., 1994; Noguchi et al., 2001), botan shrimp squid, carp, rainbow trout, atka mackerel (Nozawa et al., 1997), squid gill (Nozawa et al., 2001), scallop striated adductor muscle (Nozawa and Seki, 2001), and tilapia (Worratao and Yongsawatdigul, 2003, 2005). Two different kinds of transglutaminases were extracted from the Japanese oyster (Kumazawa, 1997) and had molecular weights of 84 and 90 kDa with

380

Maximising the value of marine by-products

optimum pH of 8.0 for both enzymes. The optimum temperature differed and showed 40 and 25ëC, respectively. Squid gill was used for transglutaminase extraction due to its high activity compared with other tissues (Nozawa et al., 2001). Its molecular weight was estimated to be 94 kDa and its activity showed an optimum pH and temperature at 8.0 and 20ëC, respectively with 10 mM CaCl2. Tissue transglutaminase from scallop striated adductor muscle was purified (Nozawa and Seki, 2001), and its molecular weight was estimated to be 95 kDa. Optimum pH and temperature were pH 8.0 and 35ëC, respectively. This enzyme was Ca2+ dependent and inactivated by -chloromercuribenzoic acid, Nethylmaleimide, Cu2+, and Zn2+ showing that it belongs to the thiol group of enzymes. 17.2.6 Polyphenoloxidases Polyphenoloxidase which is also known as tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and catecholase is widely distributed in nature (Chen et al., 1991). Polyphenoloxidase oxidizes diphenols to quinones, which undergo autoxidation and polymerization to form dark pigments in fruits, vegetables, and crustacean species (Bartolo and Birk, 1998). Of these, crustacean species can be a source for polyphenoloxidase from seafood by-products of the shellfish processing industry. Polyphenoloxidase can be found mainly in shellfish and by-products including shrimp (Nakagawa et al., 1992), prawns (Montero et al., 2001), lobster (Opoku-Gyamfua et al., 1992) and cuttlefish (Zhou et al., 2004). A polyphenoloxidase fraction was isolated and characterized from lobster (Opoku-Gyamfua et al., 1992) using the skin layer between the muscle and the exoskeleton (Homarus americanus) and compared with those of commercial tyrosinase. The lobster polyphenoloxidase fraction was activated by trypsin, and it showed a more heat labile nature as compared with commercial tyrosinase. The enzyme was most stable at pH 7.5, while tyrosinase exhibited a much broader pH stability ranging between 6.5 and 10.0. The thermotolerance of polyphenoloxidase activity could depend on its source and the environmental factors under which the species are grown. One of the significant environmental factors is water temperature although polyphenoloxidase from shrimp are usually stable between 30 and 50ëC. Polyphenoloxidase activity from Florida spiny lobster (Panulirus argus) and Western Australian lobster (Panuliruscygnus) was studied (Chen et al., 1991). Both enzymes showed similar characteristics that catalyzed oxidation of catechol and dl- -3,4dihydroxyphenylalanine and showed optimum pH stability at pH 7.0. However, they differed with respect to activation energy and thermal stability. Western Australian lobster polyphenoloxidase showed decreased activity when preincubated at temperatures greater than 30ëC whereas that of Florida spiny lobster showed greater stability at 35ëC. Chen et al. (1991) suggested that this is because Florida spiny lobsters live in warm water areas while Western Australian lobsters live in cold water areas. Authors stated that these differences in environmental

Marine enzymes from seafood by-products 381 conditions of their natural habitats may account for the difference in optimal thremostability between the enzymes.

17.3

Producing enzymes from seafood processing by-products

Enzyme extraction/solubilization, concentration, fractionation, and purification are the main steps of enzyme production. To produce enzymes at the industrial level from seafood by-products, there are several requirements including: 1) a large quantity of raw material is needed because the target enzymes are usually in very low concentration; 2) facilities are required to efficiently remove significant amounts of water to concentrate/purify enzyme; and 3) the removal of particulate matter (including cell and/or cell debris) from the final product is required. The following applications are examples achieving these objectives using a combination of several techniques. 17.3.1 Enzyme extraction/solubilization Extraction of enzymes largely depends on the localization of the target enzymes. For instance, if the starting material contains intracellular enzymes, cell membrane should be disrupted and homogenized in a buffer. The membrane disruption can be done by various methods; alkalization, addition of Ethylenediaminetetraacetic acid (EDTA), detergents or osmotic shock, or by physical methods, including sonication, alternating freezing and thawing phases, solid or liquid shear, or grinding or agitation with abrasives. Then, insoluble impurities and cellular waste should be removed by filtration or centrifugation (Barthomeuf, 1989). On the other hand, if the enzyme is located in the extracellular fluid, cells should be removed by centrifugation to eliminate contaminants. 17.3.2 Enzyme concentration As mentioned earlier, the target enzyme is normally of very low concentration in a large volume of raw material; therefore, the concentration of enzymes after extraction is necessary. To maintain enzyme activity, harsh conditions such as high temperature and strong mechanical stress should be avoided. The enzyme concentration process includes removal of water and other molecules or use of precipitation methods. The following methods are examples that can be applied with relatively mild condition throughout the process. Membrane technology The food industry started incorporating membrane technology with reverse osmosis technology for water purification as well as ultrafiltration technology for concentration of products (Cuperus and Nijhuis, 1993). Since then, membrane processes have become major tools in food processing and for extraction of bioactive ingredients.

382

Maximising the value of marine by-products

A membrane is a thin sheet of artificial or natural polymeric material with pores distributed throughout the material. Under certain pressure, the membrane rejects large particles, and allows species smaller than the pores to pass through (Morrissey et al., 2005). Membrane technology includes microfiltration, ultrafiltration, nanofiltration, reverse osmosis and electrodialysis. Ultrafiltration, where particles smaller than 0.2 m pass through the membrane, is the most widely used process for protein and enzyme recovery. Ultrafiltration has been used extensively in many industries including the pharmaceutical as well as the food and beverage industry. A good example of membrane technology and use in the dairy industry is to recover and separate out different whey proteins (Dsouza and Mawson, 2005). Ultrafiltration is a pressure-driven process and works by passing low-molecular-weight products through the membrane while retaining high-molecular-weight compounds such as protein. Cellulose acetate is one of the traditional materials for ultrafiltration membrane. Compared to others such as polyacrylnitrile and polyethersulphone, it is extremely hydrophilic. Having fewer fouling problems, it tends to have weak resistance against heat and chemicals. With the development of chemical and heat stable membrane such as polysulphone membrane, the feasibility of this technology has greatly increased. Protease recovery from surimi wash water containing various cathepsins was studied using ultrafiltration technology with various pretreatments (MirelesDewitt and Morrissey, 2002b). Preliminary tests showed that fish proteins will rapidly clog membrane pores actually changing the dynamics of the filtration (Huang and Morrissey, 1998). Acidification of wash water (pH 4.0) and mild heat treatment (60ëC) followed by centrifugation were necessary as pretreatments before ultrafiltration was applied. This helped to remove highmolecular-weight proteins that might interfere with ultrafiltration without reducing protease activity. The supernatant was then subjected to both highmolecular-weight ultramicrofiltration and low-molecular-weight ultrafiltration. The result showed that concentration of protease using 50 kDa ultrafiltration polysulphone membranes was successful in recovering approximately 80% of original protease activity (Mireles-Dewitt and Morrissey, 2002b). Enzyme concentration by precipitation An alternative and relatively traditional way to concentrate enzymes is to use salts for enzyme precipitation. This is due to its ability to change electrostatic forces which affect enzyme solubility. Enzymes are usually soluble in water/ buffer solutions because hydrophobic residues tend to locate the interior of the globular proteins (enzymes). At low salt concentration (0.5±1 M), protein solubility increases (salting in) as ions from salt decrease the electrostatic attraction between opposite charges of neighboring molecules. At high salt concentration (>1 M), protein solubility decreases resulting in protein precipitation. The protein can also be precipitated by adjusting pH to pI of proteins which is usually between 5.2 and 5.5. However, use of salt, especially ammonium sulphate (NH4)2SO4, is the most common way to precipitate and concentrate

Marine enzymes from seafood by-products 383 enzymes. Ammonium sulphate possesses a relatively strong `salting out' effect without causing significant levels of protein denaturation. It has been intensively used in research areas including seafood and seafood by-products as a first step to accomplish separation of crude enzymes. The advantages of using these tecniques include: 1) high solubility, 2) high commercial availability with inexpensive costs, 3) lack of toxicity, and 4) its stabilizing effect on precipitates. A disadvantage of using mineral salts is that the method requires another step such as dialysis or gel filtration (Barthomeuf, 1989). In industries where enzymes are purified on a large scale, the corrosion of stainless steel by ammonium sulphate is a disadvantage and possibly causes additional environmental concerns. Sodium sulphate may be more suitable from this point of view, but it is not as effective (Naz, 2002). Freeze-drying or lyophilization Freeze-drying, also known as lyophilization, has been widely used in the food industry for various protein products including enzymes. Freeze-drying removes water from a frozen sample by sublimation and desorption in a three-step process, which includes freezing, primary drying and secondary drying. Freezedrying technology is used in combination with other concentration/purification methods including ultrafiltration in seafood enzyme recovery research. Dry powder forms of enzymes are more stable than enzymes in aqueous solution, since water can facilitate enzyme denaturation. However, it is also known that conformational changes of enzymes by freeze-drying can result in lower enzyme activity. Alternative forms of enzyme preparations have been developed to increase enzyme stabilization, including immobilization with sol-gel methods, cross-linked enzyme crystals (Altus Biologics, Inc., Cambridge, MA, USA), soluble enzymes with polymers such as ethylene glycol, and surfactant-modified enzymes such as sorbitan monostearate-modified lipase (Roy and Gupta, 2004). 17.3.3 Fractionation/purification Concentration techniques will remove considerable extraneous material but only partially purify the enzyme. The degree of purity requires further downstream processing that often includes chromatography and electrophoresis. Since electrophoresis is mainly an analytical procedure, only chromatography will be discussed here. Chromatography Although there are limitations for industrial scale use of chromatography, it is frequently used in the laboratory to fractionate components including enzymes with high degrees of purity. Chromatography technology can be categorized into various methods based on protein size, charge, hydrophobicity and molecular recognition. These include size exclusion chromatography, ion exchange chromatography, and affinity chromatography. Enzymes can be fractionated depending on their hydrodynamic volume by size exclusion chromatography

384

Maximising the value of marine by-products

whereas affinity chromatography is based on specific recognition between two relevant biomolecules (An and Visessanguan, 2000). Affinity chromatography is commonly used for the separation of one enzyme from others. Fractionation of target enzyme by ion exchange chromatography is based on the electrostatistic interactions between the enzyme and charged groups on the exchangers. Cellulosic ion exchange chromatography has been one of the most common methods for protein separation (Naz, 2002). Separation of enzymes can be controlled by changing pH of the elution buffer. Gastricsin-like proteinase was purified from Atlantic cod viscera after lyophilization with a single step purification scheme on ion-exchange of Amberlite CG-50 with efficient recovery (Amiza and Owusu Apenten, 2002). Crude cod pepsinogen was dissolved in 10 ml of 0.2 M sodium citrate buffer (pH 2.1) and introduced to the column. The pH of elution buffer was changed to 3.8, 4.2, and 4.6 to fractionate proteinases followed by pooling the target peaks, dialyzing, and freeze-drying to produce purified proteinaes. Pepsin A was eluted at a lower pH (pH 4.0) while gastricsin appeared at higher pH (pH 4.4). The study demonstrated that final proteinase recovery was high which could be related to the salt activation by citrate buffer or removal of inhibitors. Chen et al. (1997) investigated the purification methods for shrimp polyphenoloxidase from frozen powder of white shrimp (Penaeus setiferus) and pink shrimp (P duorarum) and found that the use of butanol treatment followed by phenyl sepharose CL-4B chromatography was better than ammonium sulphate fractionation and then phenyl sepharose chromatography. They also found that different species exhibited different activity through the purification process which demonstrated that activity of white shrimp polyphenoloxidase was more susceptible than that of pink shrimp during the process. Taniguchi and coworkers (2001) extracted lipase from the stomach and intestines of tilapia. They extracted crude lipase by chromatofocusing and applying the extract on a polyexchanger column previously equilibrated with 25 mM imidazole-acetic acid buffer. The lipase was eluted with polybuffer 96acetic acid. Iijima et al. (1998) characterized a bile salt-activated lipase from hepatopancreas of red sea bream. A delipidated powder processed from red sea bream hepatopancreas was used as a raw material. Lipase was extracted by fractional precipitation with ammonium sulphate and sequential chromatography.

17.4

Marine by-product enzyme utilization

Despite extensive research in marine enzyme technology, there are only a few applications in the food processing sector. Limitations of marine by-product enzyme utilization are often due to the cost of enzyme recovery and competition with more mainstream enzyme sources. However, there are some commercial operations that use marine by-product enzymes such as the enzyme recovery process from cod viscera decribed in Fig. 17.1 (Gildberg, 2004). This figure shows a Norwegian multi-purpose processing plant manufacturing several

Marine enzymes from seafood by-products 385

Fig. 17.1

Flowchart of by-product processing and crude extract production using cod viscera (Gildberg 2004).

products including a protein hydrolysate and a crude pepsin extract as a fish processing aid for fish caviar and descaling operations. The crude extract will vary in pepsin concentration from 2 to 10%, depending on the quality and source of raw material and operating conditions. Crude extracts can undergo further seperation by chromatography or other methods to produce a purified enzyme. However, these are often expensive procedures and most commercial uses require only the crude extract form except in the medical/biotechnology field. Several of these uses, from traditional seafood products to advanced biochemical/medical applications, are described below. 17.4.1 Fermented fish products Fish fermentation products, such as fish sauces, are expanding in the marketplace and there have been increased efforts to better define their manufacturing parameters and control the fermentation process (Lopetcharat et al., 2001). More success has been found using enzymes to hasten the process and quality of fish sauce production. Researchers (Tungkawachara et al., 2003; Chaveesuk et al., 1993) have shown the efficiency of using specific enzymes for the production of fish sauce and producing an acceptable product. Maatjes is a fermented product using the natural viscera enzymes in herring and several efforts have been made to describe the enzymatic reaction and mimic the end result (Olsen and Skara, 1997; Nielsen and Borrensen, 1997; Nunes et al., 1997). 17.4.2 Protein hydrolysates There is an increasing amount of interest in protein hydrolysate production as this product has shown unique biological properties such as stimulating the immune system as well as having peptide fractions that stimulate growth. A good general review on hydrolysates can be found by Kristinsson and Rasco

386

Maximising the value of marine by-products

(2000). Recent research in the area of production hydrolysates from by-products include threadfin bream (Normah et al., 2005), cod by-products (Slizyte et al., 2004, 2005), gold carp (Sumaya-Martinez et al., 2005), salmon heads (Gbogouri et al., 2004), mackerel (Wu et al., 2003), and herring (Sathivel et al., 2003). Although enzymes from plant and microbial sources are commonly used for the production of fish protein hydrolysates, research with marine enzymes has shown comparable production rates (Benjakul et al., 1997). Shahidi and his coworkers have produced fish protein hydrolysates using mixtures of extracted and endogenous enzymes in both capelin and seal meat (Shahidi et al., 1995; Shahidi and Synowiecki, 1997). 17.4.3 Deskinning The enzymatic removal of fish skin from certain species has proven advantageous as an alternative method for mechanical deskinning (Tschersich and Choudhury, 1998; Kim et al., 1993) since mechanical methods tend to be harsh processes resulting in lower fillet recovery. The use of proteases for removal of squid skin is a standard process, although non-marine proteases are often used. Several researchers have shown that marine enzymes can be used and often leave a product that has several advantages over papain or ficin proteases (Wray, 1988). 17.4.4 Fish roe (caviar) production Caviar is the salt-cured and preserved eggs of aquatic animals that have been separated from the supporting connective tissue. The most widely recognized and valued caviar is made from sturgeon harvested from the Caspian Sea (Bledsoe et al., 2003). There are enzyme-based processes for removing the connective tissue that surrounds the eggs, which reduces human handling and increases caviar recovery. Commercially available fish enzymes such as RozymTM (Biotec-Mackzymal AS, Tromso, Norway) and DigestaseTM (Alaska Russia Salmon Caviar Co., Anchorage, AK, USA) have been used for salmon caviar. Enzymes from fish viscera such as pepsin can be also used for skein removal. Pepsins split the linkages that adhere the egg cells to the roe sack without affecting the eggs. Such application with marine derived pepsin has advantages over the enzymes from mammalian origin including higher optimum pH and higher activity at lower temperatures (Raa, 1996). Research has shown the efficacy of proteolytic enzymes for higher yields in fish caviar for salmon and lumpfish (Raa, 1997). 17.4.5 Gel formation of food Transglutaminase can be used in certain food products such as surimi, seafood and meat products that require improved gel formation and gel strength resulting in better texture. The advantages of transglutaminase addition in the surimi

Marine enzymes from seafood by-products 387 products include: 1) to increase gel strength of surimi resulting in higher quality, and 2) to lower production costs by increasing water content in surimi (Kumazawa, 2002). Currently, transglutaminase has also been used for meat products, noodles, soy protein products such as tofu, dairy and baked products. Commercially available transglutaminase, such as Activa TG-K has high potential for food applications which to date are only from microbial sources (Kuraishi et al., 2001). 17.4.6 Meat tenderization Currently, proteases from plant sources, such as papain and bromelain, are the principle enzymes used as meat tenderizers (Haard et al., 1994). However, these enzymes attack both connective and myofibrillar proteins which often lead to over-tenderization whereas marine collagenase could only hydrolyze connective tissue protein possibly resulting in optimum tenderization of meat products. Aoki et al. (2004) extracted collagenase from northern shrimp by products and suggested potential use for the meat industry in reducing toughness of the meat products cased by connective tissues claiming that it works better than currently available enzymes from plant sources. 17.4.7 Biotechnology/medical Although many of the marine enzyme technologies are in the initial phases, there have been notable successes in biochemistry/biotechnology field. The isolation and purification of shrimp alkaline phosphatase (SAP) from cold water shrimp (Pandalus borealis) has led to the use of this compound in gene splicing with plasmid and bacteriophage vectors in most biotechnology laboratories (Olsen et al., 1991; Sambrook and Russell, 2001). SAP completely dephosphorylates DNA and has the advantage over other alkaline phosphatases as it can be inactivated at lower temperatures (65ëC for 15 min) and thus not denature DNA materials. The enzyme is recovered and purified from the shrimp processing waste water and is a highly sought after chemical. This success has created other potential opportunities with by-product enzymes and Biotec, ASA in Norway also lists cod uracil-DNA glycosylase and other enzymes for use in biotechnology and food processing fields. Atlantic cod viscera is an abundant fishery by-product in Iceland and the purification and characterization of trypsin followed by chymotrypsin from the viscera has led to its utilization (Asgeirsson et al., 1989; Asgeirsson and Bjarnason, 1991). After ten years of research and collaboration between University of Virginia and University of Iceland, researchers developed a new product using both trypsin and chymotrypsin from cod viscera. The product, called Penzim, is used as a gel and lotion in the treatment of skin ailments including psoriasis and other skin conditions. Recently, researchers have reported the successful cloning and expression of trypsin I from cod in E. coli (Jonsdottir et al., 2004).

388

Maximising the value of marine by-products

There has also been active research in investigating krill proteolytic enzymes as an active ingredient in wound debridement or the removal of necrotic tissue in wounds (Mekkes et al., 1997, 1998). Research suggests that it is more active than several plant proteases and can help accelerate wound healing.

17.5

Future trends

Despite tremendous scientific strides in identification and purification of marine enzymes and technological advances in the recovery of specific compounds from fish by-products, the question remains concerning the economic feasibility of enzyme recovery from fish and shellfish by-products. Many of the specific enzymatic activities, e.g. proteases for fish hydrolysates, have been preempted by other enzymes from plant and microbial sources that often prove to be less expensive. In addition to scale-up limitations, there is also strong market competition for the by-product raw material. Many fish processing operations already use solid by-products for established-market products such as fish meal, fertilizers, silage and more recently hydrolysates. As aquaculture expands over the next decade, demand for the raw material for producing fish meal and other products for fish feed formulations will also increase (Kilpatrick, 2003). These products have relatively stable global markets in which there are known risks, capital investment needs and available technology which facilitates entry into the marketplace. Other markets for fish by-products have also developed over the last decade. Viscera by-products, e.g. stomachs from the Alaska pollock industry and other organs, are now being marketed into Asian niche markets (Morrissey et al., 2005). Extraction of specific enzymes from fish by-products requires considerable investment in technologies that often have high costs, varying efficiencies and require skilled technicians which can be problematic in remote areas. Some of the best examples of utilization of marine enzymes from seafood byproducts come from the biotechnology/medical field. Companies such as Biotec Pharmacon ASA in Tromso, Norway, which produces shrimp alkaline phosphatase for biotechnology laboratories and Zymetec in Iceland currently marketing a marine trypsin as a skin healing lotion, are examples of companies that have successfully taken extractive by-product research into the marketplace. Perhaps the future of marine enzymes utilization rests more in biotechnology/medical uses than in food processing; however, even this is a two-edged sword. Rapid advances in biotechnology have also revolutionized the field of enzyme production providing researchers and companies with the potential to produce specific enzymes more economically. Haard (1998) addressed the uniqueness of aquatic enzymes in their diverse environments in his review of `specialty enzymes'. It is unlikely that many of these marine enzymes could be produced in large enough quantities due to limitations in obtaining sufficient raw material. However, their unique properties may have industrial or medical applications that warrant production through biotechnological techniques. The advent of biotechnology and the production of specific compounds through gene transfer and use of microbial organisms for

Marine enzymes from seafood by-products 389 production are making inroads in the commercial production of enzymes. As this technology continues to develop over the next decade, marine enzymes with unique characteristics could be cloned and produced more economically through biotechnology than from by-product recovery operations. Even in the case of shrimp alkaline phosphatase and Penzim, there is active research to transfer these genes to bacteria which would be able to produce commercial quantities of this valuable enzyme. In many cases, the scientific information about biochemical properties of the marine enzymes themselves, might prove to be the most valuable component of the by-product itself. Although the science of seafood enzyme research remains an exciting one due, in part, to the uniqueness of its resources and the opportunities it may provide in the biotechnology/medicine fields, the question remains whether it will reach its true potential beyond the laboratory setting and become a viable force in the global marketplace.

17.6

References

2002. A single step purification of gastricsin-like proteinease from Atlantic cod (Gadus morhua). Online Journal of Biological Sciences 2(9): 591±5. AN H, VISESSANGUAN W. 2000. Recovery of enzymes from seafood-processing wastes. In: Haard NF, Simpson BK (eds), Seafood Enzymes. New York: Marcel Dekker, Inc., pp. 641±64. AN HA, SEYMOUR TA, WU J, MORRISSEY MT. 1994. Assay systems and characterization of Pacific whiting (Merluccus prodctus) protease. J Food Sci 59 (2): 277±81. AOKI H, AHSAN N, MATSUO K, HAGIWARA T, WATABE S. 2004. Partial purification of proteases that are generated by processing of the Northern shrimp Pandalus borealis and which can tenderize beef. Int J Food Sci and Technol 39(5): 471±80. AOKI T, YAMASHITA T, UENO R. 1995. Purification and characterization of a cathepsin Blike enzyme from mackerel white muscle. Fish Sci 61(1): 121±6. ASGEIRSSON B, BJARNASON JB. 1991. Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua) comparison with bovine chymotrypsin. Comp Biochem Physiol B 99(2): 327±35. ASGEIRSSON B, FOX JW, BJARNASON JB. 1989. Purification and characterization of trypsin from the poilikotherm Gadus morhua. Eur J Biochem 180: 85±94. ASHIE INA, SIMPSON BK. 1996. Application of high hydrostatic pressure to control enzyme related fresh seafood texture deterioration. Food Res Intl 29: 569±75. AUDLEY MA, SHETTY KJ, KINSELLA JE. 1978. Isolation and properties of phospholipase A from pollock muscle. J Food Sci 43(6): 1771±5. BARTHOMEUF C. 1989. Review Application of enzyme purification process to proteolytic enzymes. J Chromatography 473: 1±26. BARTOLO I, BIRK E. 1998. Some factors affecting Norway lobster (Nephrops norvegicus) cuticle polyphenol oxidase activity and blackspot development. Int J Food Sci and Technol 33: 329±36. BENJAKUL S, SEYMOUR TA, MORRISSEY MT, AN H. 1996. Proteinase in Pacific whiting surimi wash water: identification and characterization. J Food Sci 61(6): 1165±70. AMIZA MA, OWUSU APENTEN RK.

390

Maximising the value of marine by-products

1997. Characterization of proteinase recovered from Pacific whiting surimi wash water. J Food Biochem 22: 1±16. BLEDSOE GE, BLEDSOE CD, RASCO B. 2003. Caviars and fish roe products. Crit Rev Food Sci Nutr 43(3): 317±56. BRAUER JME, LEYVA JAS, ALVARADO LB, SANDEZ OR. 2003. Effect of dietary protein on muscle collagen, collagenase and shear force of farmed white shrimp (Litopenaeus vannamei). Eur Food Res Technol 217 (4): 277±80. CARDENAS-LOPEZ JC, HAARD NF. 2005. Cysteine proteinase activity in jumbo squid (Dosidicus gigas) hepatopancrease extracts. J Food Biochem 29(2): 1745±53. CAVICCHIOLI R, SIDDIQUI KS, ANDREWS D, SOWERS KR. 2002. Low-temperature extremophiles and their applications. Curr Opion Biotechnol 13: 253±61. CHAVEESUK R, SMITH JP, SIMPSON BK. 1993. Production of fish sauce and acceleration of sauce fermentation using proteolytic enzymes. J of Aquatic Food Product Technol 2(3): 59±77. CHAWLA P, ABLETT RF. 1987. Detection of microsomal phospholipase activity in myotomal tissue of Atlantic cod (Gadus morhua). J Food Sci 52(5): 1194±7. CHEN JS, ROLLE RS, MARSHALL MR, WEL CI. 1991. Comparison of phenoloxidase activity from Florida spiny lobster and western Australian lobster. J Food Sci 56(1): 154±7. CHEN JS, CHAREST DJ, MARSHALL MR, WEI CI. 1997. Comparison of two treatment methods on the purification of shrimp polyphenol oxidase. J of Sci of Food and Agric 75(1): 12±18. CHOI YJ, CHO Y-J, LANIER TC. 1999a. Purification and characterization of proteinase from Atlantic menhaden muscle. J Food Sci 64(5): 772±5. CHOI YJ, LANIER TC, LEE HG, CHO Y-J. 1999b. Purification and characterization of alkaline proteinase from Atlantic menhaden muscle. J Food Sci 64(5): 768±71. CRAWFORD A, RICHARDSON RN, MATHER P. 2005. A comparative study of cellulose and xylanase activity in freshwater crayfish and marine prawns. Aquaculture Research 36: 586±92. CUPERUS EP, NIJHUIS HH. 1993. Applications of membrane technology to food processing. Trends Food Sci Technol 4: 277±82. DSOUZA NM, MAWSON AJ. 2005. Membrane cleaning in the dairy industry: review. Crit Rev Food Sci Nutr 45: 125±34. EHARA T, TAMIYA T, TSUCHIYA T. 1992. Investigation of myosin heavy chain-degrading proteinase in decapoda muscle. Nippon Suisan Gakkaishi 58(12): 2379±82. EHARA T, TAMIYA T, TSUCHIYA T. 1994. Investigation of myosin heavy chain-degrading proteinase in squid muscle. Nippon Suisan Gakkaishi 60(4): 527±8. GBOGOURI G, LINDER M, FANNI J, PARMENTIER M. 2004. Influence of hydrolysis degree on the functional properties of salmon by-products hydrolysates. J Food Sci 69(8): C 615±22. BENJAKUL S, SEYMOUR TA, MORRISSEY MT, AN H.

GERDAY C, AITTALEB M, BENTAHIR M, CHESSA JP, CLAVERIE P, COLLINS T, D'AMICO S, DUMONT

2000. Cold-adapted enzymes: from fundamentals to biotechnology. Tibtech 18: 103±7. GILDBERG A. 2004. Enzymes and bioactive peptides from fish waste related to fish silage, fish feed and fish sauce production. J Aquatic Food Product Technol 13(2): 3±11. GILDBERG A, SIMPSON BK, HAARD NF. 2000. Use of enzymes from marine organisms. In: Haard NF, Simpson BK (eds), Seafood Enzymes. New York: Marcel Dekker, pp. 619±39. GJELLESVIK DR, LOMBARDO D, WALTHER BT. 1992. Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties. Biochem Biophys Acta J, GARSOUX G, GEORLETTE D, HOYOUX A, LONHIENNE T, MEUWIS MA, FELLER G.

Marine enzymes from seafood by-products 391 1124: 123±34.

1994. Pancreatic carboxylester lipase from Atlantic salmon (Salmo salar) cDNA sequence and computer-assisted modeling of tertiary structure. Eur J Biochem 226: 603±12. GRANT GA, EISEN AZ, BRADSHAW RA. 1981. Collagenolytic protease from fiddler crab (Uca pugilator). Methods Enzymol 80: 722±34. GRANT GA, SACCHETTINI JC, WELGUS HG. 1983. A collagenolytic serine protease with trypsin-like specificity from fiddler crab Uca pugilator. Biochem 22: 354±6. GUDMUNDSDOTTIR A, PASLSDOTTIR HM. 2005. Atlantic cod trypsin: from basic research to practical applications. Mar Biotechnol 7(2): 77±88. HAARD NF. 1998. Specialty enzymes from marine organisms. Food Technol 53(7): 64±7. HAARD NF, SIMPSON BK. 1994. Proteases from aquatic organisms and their uses in the seafood industry. In: Martin AM (ed.), Fisheries Processing Biotechnology Applications. London: Chapman and Hall. pp. 132±54. HAARD NF, SIMPSON BK. 2000. Seafood Enzymes. New York: Marcel Dekker. HARRD NF, SIMPSON BK, SIKORSKI ZE. 1994. Biotechnological applications of seafood proteins and other nitrogenous compounds. In: Sikorski ZE, Sun Pan B, Shahidi F (eds), Seafood Proteins. New York: Chapman and Hall, pp. 194±222. HERNANDEZ-HERRERO MM, DUFLOS G, MALLE P, BOUQUELET S. 2003. Collagenase activity and protein hydrolysis as related to spoilage of iced cod (Gadus morhua). Food Res Int 36(2): 141±7. HEU MS, KIM HR, PYEUN JH. 1995. Comparison of trypsin and chymotrypsin from the viscera of anchovy, Engraulis japonica. Comp Biochem Physiol 112B(3): 557±67. HONJO I, KIMURA S, NONAKA M. 1990. Purification and characterization of trypsin-like enzyme from shrimp Penaeus indicus. Nippon Suisan Gakkaishi 56(10): 1627±34. HUANG L, MORRISSEY MT. 1998. Fouling of membranes during microfiltration of surimi wash water: Roles of pore blocking and surface cake formation. J Membrane Sci 144: 113±23. HULTMANN L, RUSTAD T. 2004. Iced storage of Atlantic salmon (Salmo salar) ± effects on endogenous enzymes and their impact on muscle proteins and texture. Food Chem 87(1): 31±41. IIJIMA N, NAKAMURA M, UEMATSU K, KAYAMA M. 1990. Partial purification and characteriazation of phospholipase A2 from the hepatopancreas of red sea bream. Nippon Suisan Gakkaishi 56: 1331±9. IIJIMA N, CHOSA S, UEMATSU M, GOTO T, HOSHITA T, KAYAMA M. 1997. Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major. Fish Physiol Biochem 16: 487±98. IIJIMA N, TANAKA S, OTA Y. 1998. Purification and characterization of bile salt-activated lipase from the hepatopancreas of red sea bream, Pagrus major. Fish Physiol and Biochem 18: 59±69. JOHNSON HM. 2004. Annual Report on the United States Seafood Industry (11th edn). H.M. Johnson and Associates, Jacksonville, OR. JONSDOTTIR G, BJARNASON JB, GUDMUNSDOTTIR A. 2004. Recombinant cold-addapted trypsin I from Atlantic cod ± expression, purification and identification. Protein Expr Purif 33(1): 11±22. KARASUDA S, YAMAMOTO K, KONO M, SAKUDA S, KOGA D. 2004. Kinetic analysis of a chitinase from red sea bream, Pagrus major. Biosci Biotechnol Biochem 68(6): 1338±44. KILPATRICK JS. 2003. Fish processing waste: opportunity or liability. In: Advances in Seafood GJELLESVIK DR, LORENS JB, MALE R.

392

Maximising the value of marine by-products

Byproducts: 2002 Conference Proceedings, Alaska Sea Grant, Fairbanks, AK. 1993. Removal of skin from filefish using enzymes. Bulletin of the Korean Fisheries Society 26: 159±72. KIM SK, PARK PJ, KIM JB, SHAHIDI F. 2002. Purification and characterization of a collagenolytic protease from the filefish, Novoden modestrus. J of Biochem and Molecular Biol 35(2): 165±71. KIM YT, KIM SK. 1991. Purification and characterization of the collagenase from the tissue of filefish, Novoden modestrus. Korean Biochem J 24(4): 401±9. KISHIMURA H, HAYASHI K. 2004. Purification and properties of phospholipase A2 isozymes from pyloric ceca of the starfish (Asterina pectinifera). J Food Biochem 28(3): 181±94. KLIMOVA OA, BORUKHOV SI, SOLOVYEVA NI, BALAEVSKAYA TO, STRONGIN AY. 1990. The isolation and properties of collagenolytic proteases from crab hepatopancreas. Biochem Biophys Res Commun 166: 1411±20. KONO M, WILDER MN, MATSUI T, FURUKAWA K, KOGA D, AIDA K. 1995. Chitinolytic enzyme activities in the hepatopancreas, tail fan and hemolymph of kuruma prawn penaeus japonicus during the molt cycle. Fish Sci 61(4): 727±8. KRISTINSSON HG, RASCO BA. 2000. Fish protein hydrolysates: production, biochemical, and functional properties. Crit Rev Food Sci Nutr 40(1): 43±81. KRISTJANSSON MM, GUDMUNDSDOTTIR S, BJARNASON JB, FOX JW. 1995. Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua). Comp Biochem Physiol B 110 (4): 707±17. KUDA T, TSUDA N, YANO T. 2004. Thermal inactivation characteristics of acid and alkaline phosphatase in fish and shellfish. Food Chem 88: 543±8. KUMAZAWA Y. 2002. Development and application of tranglutaminase for seafood products. Nippon Suisan Gakkaishi 68(5): 633±6. KUMAZAWA Y, NAKANISHI K, YASUEDA H, MOTOKI M. 1996. Purification and characterization of transglutaminase from walleye pollack liver. Fish Sci 62(6): 959±64. KUMAZAWA Y, SANO K, SEGURO K, YASUEDA H, NIO N, MOTOKI M. 1997. Purification and characterization of transglutaminase from Japanese oyster (Crassostrea gigas). J Agric Food Chem 45: 604±10. KURAISHI C, YAMAZAKI K, SUSA Y. 2001. Transglutaminase: Its utilization in the food industry. Food Rev Intl 17: 221±46. LEE CM. 2000. Technology development for flavor production from seafood processing wastes. National Marine Fisheries Service, Northeast Region, One Blackburn Drive, Gloucester, Massachusetts. LOPETCHARAT K, CHOI YJ, PARK JW, DAESCHEK M. 2001. Fish sauce products and manufacturing: a review. Food Rev Intl 17(1): 65±88. MAKINODAN Y, TOKOYAMA Y, KINOSHITA M, TOYOHARA H. 1987. Characterization of an alkaline proteinase of fish muscle. Comp Biochem Physiol B 87 (4): 1041±6. MATSUMIYA M. 2004. Enzymatic production of N-Acetyl-D-Glucosamine using crude enzyme from the liver of squids. Food Sci Technol Res 10(3): 296±9. MATSUMOTO Y, SAUCEDO-CASTANEDA G, REVAH S, SHIRAI K. 2004. Production of -Nacehylhexosaminidase of Verticillium lecanii by solid state and submerged fermentations utilizing shrimp waste silage as substrate and inducer. Process Biochem 39: 665±71. MEKKES JR, LE POOLE IC, DAS PK, KAMMEYER A, WESTERHOF W. 1997. In vitro tissuedigesting properties of krill enzymes compared with fibrinolysin/DNAse, papain and placebo. Int J Biochem Cell Biol 29: 703±6. KIM SK, BYUN HG, CHOI KD, ROH HS, LEE WH, LEE EH.

Marine enzymes from seafood by-products 393 1998. Efficient debridement of necrotic wounds using proteolytic enzymes deived from Antarcic krill: a doubleblind, placebo-controlled study in a standardized animal wound model. Wound Repair Regen 6(1): 50±7. MIRELES-DEWITT CA, MORRISSEY MT. 2002a. Parameters for the recovery of proteases from surimi wash water. Bioresource Technol 81: 241±7. MIRELES-DEWITT CA, MORRISSEY MT. 2002b. Pilot plant recovery of catheptic proteases from surimi wash water. Bioresource Technol 82: 295±301. MONTERO P, AVALOS A, PEREZ-MATEOS M. 2001. Characterization of polyphenol oxidase from prawns (Penaeus japonicus). Alternatives to inhibition: additives and highpressure treatment. Food Chem 75: 317±24. MORRISSEY MT, LIN J, ISMOND A. 2005. Waste management and by-product utilization. In Park JW (ed.), Surimi and Surimi Seafood. New York: Taylor and Francis, pp. 279±323. NAKAGAWA T, MAKINODAN Y, HUJITA M. 1992. Distribution and properties of polyphenol oxidase from shrimp Penaeus spp. Kinki University Agriculture Department. 25: 27±32. NAKAGAWA T, ANDO M, MAKINODAN Y. 1996. Properties of glycogenolytic enzymes from fish muscle. Nipon Suisan Gakkaishi 62(3): 434±8. MEKKES JR, LE POOLE IC, DAS PK, BOS JD, WESTERHOF W.

NAKAI H, IIZUKA T, FUKUKAWA T, NISHIOKA K, TANIZAWA S, OKUYAMA M, MORI H, CHIBA S,

2005. Screening of enzymes having excellent functions from the digestive caecum of scallop. 21st COE program. Marine Bio-Manipulation Frontier for Food Production, The 2nd International Symposium. Japan. NATALIA Y, HASHIM R, ALI A, CHONG A. 2003. Characterization of digestive enzymes in a carnivorous ornamental fish, the Asian bony tongue Scleropages formosus (Osteoglossidae). Aquaculture 233: 305±20. NAYAK J, VISWANATHAN NAIR P, AMMU K, MATHEW S. 2003. Lipase activity in different tissues of four fish. J Sci and Food Agric 83(11): 1139±42. NAZ SHAHINA. 2002. Enzymes and Food. New York: Oxford University Press. NIELSEN HH, BORRENSEN T. 1997. The influence of intestinal proteinases on ripening of salted herring. In: Luten JB, Brresen T, Oehlenschlager J (eds), Seafood from Producer to Consumer, Integrated Approach to Quality. Amsterdam: Elsevier Science, pp. 293±303. NOGUCHI K, ISHIKAWA K, YOKOYAMA, KI OHTSUKA T, NIO N, SUZUKI EI. 2001. Crystal structure of red sea bream transglutaminase. J Biological Chem 276: 12055±9. NORMAH I, JAMILAH B, SAARI N, CHEMANYAAKOB B. 2005. Optimization of hydrolysis conditions for the production of threadfin bream (Nemipterus Japonicus) hydrolysate by alcalase. J Muscle Foods 16(2): 87±102. NOZAWA H, SEKI N. 2001. Purification of transglutaminase from scallop striated adductor muscle and NaCl-induced inactivation. Fish Sci 67: 493±9. NOZAWA H, MAMEGOSHI S, SEKI N. 1997. Partial purification and characterization of six transglutaminases from ordinary muscles of various fishes and marine invertebrates. Comp Biochem Physiol B Biochem Mol Biol 118(2): 313±17. NOZAWA H, CHO SY, SEKI N. 2001. Purification and characterization of transglutaminase from squid gill. Fish Sci 67: 912±19. NUNES ML, CAMPOS RM, BATISTA I. 1997. Sardine ripening: evolution of enzymatic, seasonal and biochemical aspects. In: Luten JB, Borresen T, Oehlenschlager J (eds), Seafood from Producer to Consumer, Integrated Approach to Quality. Amsterdam: Elsevier Science, pp. 319±29. KIMURA A.

394

Maximising the value of marine by-products

2000. Enzymatic properties of a protease from the hepatopancreas of shrimp, Penaeus orientalis. J Food Biochem 24: 251±64. OLSEN RL, JOHANSEN A, MYRNES B. 1990. Recovery of enzymes from shrimp waste. Process Biochem 25(2): 67±8. OLSEN R, OVERBO MYMES B. 1991. Alkaline phosphatase from the hepatopancreas of shrimp (Pandalus borealis), a dimmer enzyme with catalytically active subunits. Comp Biochem Physiol 99B: 755±61. OLSEN SO, SKARA T. 1997. Chemical changes during ripening of North Sea herring. In: Luten JB, Borresen T, Oehlenschlager J (eds), Seafood from Producer to Consumer, Integrated Approach to Quality. Amsterdam: Elsevier Science, pp. 305±17. OPOKU-GYAMFUA A, SIMPSON BK, SQUIRES EJ. 1992. Comparative studies on the polyphenoloxidase fraction from lobster and tyrosinase. J Agric Food Chem 40: 772±5. PANGKEY H, HARA K, TACHIBANA K, CAO MJ, OSATOMI K, ISHIHARA T. 2000. Purification and characterization of cathepsin S from hepatopancreas of carp Cyprinus carpio. Fish Sci 66: 1130±7. PARK PJ, LEE SH, BYUN HG, KIM SH, KIM SK. 2002. Purification and characterization of a collagenase from the mackerel, scomber japonicus. J Biochem and Molecular Biol 35(6): 576±82. RAA J. 1996. Biotechnology and aquaculture and the fish processing industry: A success story in Norway. Center of Aquaculture Research and the University of Tromso, PO Box 677 Brevika, N-9001, Tromso, Norway. RAA J. 1997. New commercial products from waste of the fish processing industry. In: Bremner A, Davis C, Austin B (eds), Making the Most of the Catch. Proceedings of the Seafood Symposium, AUSEAS, Brisbane, Australia, pp. 33±6. ROY I, GUPTA MN. 2004. Freeze-drying of proteins: some emerging concerns. Biotechnol Appl Biochem 39: 165±77. ROY P, COLAS B, DURAND P. 1996. Purification, kinetical and molecular characterizations of a serine collagenolytic protease from greenshore crab (Carcinus maenas) digestive gland. Comp Biochem Physiol 115B: 87±95. SABROWSKI R, SHALING G, NAVARETTE DEL TORO MA, WALTER I, GARCIA-CARRENO FL. 2004. Stability and effects of organic solvents on endopeptidases from the gastric fluid of the marine crab Cancer pagurus. J Molecular Catalysis B: Enzymatic 30: 109±18 SAMBROOK J, RUSSELL DW. 2001. Molecular cloning: A Laboratory Manual, 3rd edn. New York: Laboratory Press, Cold Springs Harbor. SATHIVEL S, BABBITT J, SMILEY S, CRAPO C, REPPOND KD, PRINYAWIWATKUL W. 2003. Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates. J Food Sci 68(7): 2196±200. SHAHIDI F, KAMIL YVA. 2001. Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends in Food Sci and Technol 12: 435±64. SHAHIDI F, SYNOWIECKI J. 1997. Protein hydrolysate from seal meat as phosphate alternative in food processing application. Food Chem 60: 29±32. SHAHIDI F, HAN XQ, SYNOWIECKI J. 1995. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem 53: 285±93. SJODAHL J, EMMER A, VINCENT J, ROERAADE J. 2002. Charcterization of proteinases from Antarctic krill (Euphausia superba). Protein Expr Purif 26(1): 153±61. SKEA LG, MOUNTFORT OD, CLEMENTS DK. 2005. Gut carbohydrases from the New Zealand marine herbivorous fishes Kyphosus sydneyanus (Kyphosidae), Aplodactylus arctidens (Aplodactylidae) and Odax pullus (Labridae). Comp Biochem Physiol OH ES, KIM DS, KIM JH, KIM HR.

Marine enzymes from seafood by-products 395 Part B 140: 259±69.

2004. Hydrolysis of cod (Gadus morhua) byproducts: influence of initial heat inactivation, concentration and separation conditions. J Aquat Food Prod Technol 13(2): 31±48. SLIZYTE R, DAUKSAS E, FALCH E, STORRO I. 2005. Characteristics of protein fractions generated from hydrolysed cod (Gadus morhua) by-products. Process Biochem 40(6): 2021±33. SOVIK SL, RUSTAD T. 2004. Seasonal changes in trypsin and chymotrypsin activity in viscera from cod species. J Aquatic Food Product Technol 13(2): 13±30. SOVIK SL, RUSTAD T. 2005. Proteolytic activity in byproducts from cod species caught at three different fishing grounds. J Agric Food Chem 53: 452±8. SLIZYTE R, VAN NGUYEN J, RUSTAD T.

SUMAYA-MARTINEZ T, CASTILLO-MORALES A, FAVELA-TORRES E, HUERTA-OCHOA S, PRADO-

2005. Fish protein hydrolysates from gold carp (Carassius auratus). J Sci Food Agric 85(1): 98±104. SUPPASITH K, SOOTAWAT B, WONNOP V. 2004. Comparative studies on proteolytic activity of splenic extract from three tuna species commonly used in Thailand. J Food Biochem 28(5): 355±72. SURESH PV, CHANDRASEKARAN M. 1998. Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation. World J of Micro and Biochem 14: 655±60. TANIGUCHI A, TAKANO K. 2001. Purification and properties of carboxypeptidase from tilapia intestine-digestive enzyme of tilapia VIII. Nippon Suisan Gakkaishi 67(6): 1096±102. TANIGUCHI A, TAKANO K. 2002. Purification of properties of aminopeptidase from Tilapia intestine digestive enzyme of tilapia-IX-. Nippon Suisan Gakkaishi 68(3): 382±8. TANIGUCHI A, TAKANO K, KAMOI I. 2001. Purification and properties of lipase from tilapia stomach ± digestive enzyme of tilapia-VII. Nippon Suisan Gakkaishi 67(1): 96±101. TERUEL SRL, SIMPSON BK. 1995. Characterization of the collagenolytic enzyme fraction from winter flounder (Pseudopleuronectes americanus). Comp Biochem and Physiol- Part B Biochem and Molecular Biol 112(1): 131±6. TSAO CY, HSU YH, CHAO LM, JIANG ST. 2004. Purification and characterization of three amylases from viscera of hard clam Meretrix lusoria. Fish Sci 70: 174±82. TSCHERSICH P, CHOUDHURY GS. 1998. Arrowtooth flounder (Atheresthes stomias) protease as a processing aid. J Aquatic Food Product Technol 7: 77±89. TUNGKAWACHARA S, PARK JW, CHOI YJ. 2003. Biochemical properties and consumer acceptance of Pacific whiting fish sauce. J Food Chem 68: 855±60. UCHIYAMA S, I NOZAKI. 2005. Partial purification and characterization of prophospholipase A2 activating proteases from gill membranes of the red sea bream, Chrysophrys major. Comp Biochem Physiol B 141B(1): 121±7. VAN WORMHOUDT A, LE CHEVALIER P, SELLOS D. 1992. Purification, biochemical characterization and N-terminal sequence of a serine-protease with chymotrypsic and collagenolytic activities in a tropical shrimp, Penaeus vannamei (Crustacea, Decapoda). Comp Biochem Physiol 103B: 675±80. VANNUCCINI S. 2004. Overview of fish production, utilization, consumption and trade based on 2002 data fishery information, data and statistics unit Food and Agriculture Organization of United Nations. VECCHI SD, COPPES Z. 1996. Marine fish digestive proteases ± relevance to food industry and the south-west Atlantic region ± a review. J Food Biochem 20: 193±214. WILSON DD, CHOUDHURY GS. 2004. Modulation and inhibition of arrowtooth flounder BARRAGAN LA.

396

Maximising the value of marine by-products

(Atheresthes stomias) protease. J Aquat Food Prod Technol 13(1): 24±45. 2003. Cross-linking of actomyosin by crude tilapia (Oreochromis niloticus) transglutaminase. J Food Biochem 27: 35±51. WORRATAO A, YONGSAWATDIGUL J. 2005. Purification and characterization of transglutaminase from tropical tilapia (Oreochromis niloticus). Food Chem 93 (4): 651±8. WRAY T. 1988. Fish processing: new uses for enzymes. Food Manufac 63: 64±5. WU HC, SHIAU CY, CHEN HM. 2003. Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Res Int 36(9): 949±57. XU RA, WONG RJ, ROGERS ML, FLETCHER GC. 1996. Purification and characterization of acidic proteases from the stomach of the deepwater finfish orange roughy (Hoplostethus Atlanticus). J Food Biochem 20: 31±48. YAMASHITA M, KONAGAYA S. 1995. Purification and characterization of cathepsin L from the white muscle of chum salmon, Oncorhynchus keta. Comp Biochem Physiol B 111(4): 587±96. YASUEDA H, KUMAZAWA Y, MOTOKI M. 1994. Purification and characterization of a tissuetype transglutaminase from red sea bream (Pagrus major). Biochimica Biophysica. 58: 2041±5. YOSHINAKA R, SATO M, IKEDA S. 1972. Studies on collagenase of fish-I. Existence of collagenolytic enzyme in pyloric caeca of seriola quinqueradiata. Bulletin of the Japanese Society of Scientific Fisheries 39 (3): 275±81. YOSHITOMI BUNJI. 2005. Seasonal variation of crude digestive protease activity in Antarctic krill Euphausia Superba. Fish Sci 71: 12±19. ZHOU P, QI X, ZHENG X. 2004. Purification and some properties of cuttlefish ink polyphenol oxidase. In: Sakaguchi M (ed.), More Efficient Utilization of Fish and Fisheries Products. New York: Elsevier, pp. 223±41. WORRATAO A, YONGSAWATDIGUL J.

18 Antioxidants from marine by-products F. Shahidi and Y. Zhong, Memorial University of Newfoundland, Canada

18.1

Introduction

Lipid oxidation, which involves the generation of reactive oxygen species (ROS) such as superoxide anion and hydroxyl radicals, is one of the major reasons for food quality deterioration during processing and storage with concurrent decrease in nutritional value, safety and appearance of products. In living organisms, oxidation is associated with aging, membrane damage, heart disease, stroke, emphysema and cancer through free radical-mediated modification of DNA, proteins, lipids and small cellular molecules (Marx, 1987). Antioxidants in foods can retard lipid oxidation and thus extend the shelf-life of products. Furthermore, intake of antioxidants can protect the body against oxidative stress and lead to disease risk reduction. Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG) are commonly used in food products. However, these synthetic antioxidants are suspected to cause potential health hazards, and their use in foods has been discouraged. Therefore, the search for effective and safe antioxidants from natural sources is of great interest to researchers, producers and consumers alike. Higher plants and their constituents provide for a rich source of natural antioxidants such as tocopherols and polyphenols. Spices and herbs and hulls from seeds as well as their extracts are known to exert antioxidant activity, albeit to different degrees. More recently, marine organisms have attracted special interest for their potential use in drugs and value-added food production, thus their antioxidant properties have been investigated. Antioxidants from marine sources may be used as substitutes for plant antioxidants such as those from rosemary and sage.

398

Maximising the value of marine by-products

Most of the research on marine antioxidants has focused on potency of crude extracts with effective antioxidant compounds remaining unisolated and unidentified. Only few studies have been carried out aiming at purification and characterization of antioxidant compounds from marine resources. The previously characterized marine antioxidants are mainly substances that are structurally related to plant-derived antioxidants (Takamatsu et al., 2003). These include pigments such as chlorophylls and carotenoids as well as tocopherol derivatives, related isoprenoids and certain phenolic compounds and UVabsorbing mycosporine-like amino acids (Takamatsu et al., 2003). Naturally occurring antioxidants can be found in a variety of marine organisms including marine algae, invertebrates, fish, shellfish and marine bacteria. For instance, polyphenols and carotenoids are present in marine algae and micro-algae; some seafoods such as oyster and eel are well known to contain high levels of tocopherols; antioxidants produced by marine bacteria isolates were also reported. In addition, protein hydrolysates from marine animals or their processing by-products as well as chitinous material exhibit antioxidant activity. This chapter provides a cursory overview of antioxidants from marine sources and their by-products.

18.2

Antioxidants from marine algae

Marine algae are well known as a rich source of polyunsaturated fatty acids (PUFA), especially omega 3 PUFA. However, in spite of their high content of highly unsaturated fatty acids (HUFA) which are very susceptible to oxidation, their quality is not changed during storage (Sakata, 1997). It is believed that marine algae are protected against oxidative deterioration by certain antioxidant systems. While marine algae are primarily used for production of single-cell oil rich in docosahexaenoic acid (DHA, 22:6n-3) and other omega 3 PUFA (Kyle, 2001; Zeller et al., 2001), the leftover material after processing may contain a variety of antioxidative substances, including phenolics, and can potentially be utilized as a source of natural antioxidants. A number of studies have been conducted to verify and evaluate the antioxidant activity of marine algae. Mori et al. (2003) found that methanol extract of a marine brown alga Sargassum micracanthum inhibited oxidation in rat liver homogenates (Table 18.1). A red alga Grateloupia filicina was reported to contain compounds with high antioxidant efficacy equal to or better than that of commercial antioxidants such as BHA, BHT and -tocopherol, thus its use as an antioxidant in food formulations was suggested (Athukorala et al., 2003a,b, 2005). Mediterranean marine algae of genus Cystoseira were found to possess antioxidant activity comparable to that of -tocopherol (Table 18.2) (Foti et al., 1994; Ruberto et al., 2001). Furthermore, water, methanol and ethanol extracts of an edible seaweed Hizikia fusiformis showed significant ROS scavenging activity, indicating that this alga might be a valuable source of both water- and fat-soluble antioxidants (Siriwardhana et al., 2003). In addition, Park et al. (2004b) demonstrated that

Antioxidants from marine by-products 399 Table 18.1 Effects of methanol extract of Sargassum micracanthum (SM) and tocopherol acetate on CCl4 induced liver injury in rats1 Groups SM extract SM extract SM extract

Dose (mg/kg)

Inhibition of formation of malondialdehyde in rat liver (%)

120 400 1200

4.4 11.1 14.7

1 Percent inhibition of malondialdehyde formation for tocopheryl acetate at 400 mg/kg dose was 17.5. Adapted from Mori et al. (2003).

enzymatic hydrolysates of an edible seaweed Sargassum horneri exhibited strong radical scavenging activity on hydroxyl and alkyl radicals. Enzymatic extracts from various brown algae were reported to exert a positive effect in reducing oxidative damage to DNA (Heo et al., 2005a,b; Park et al., 2005). Antioxidant activity of marine algae may arise from pigments such as chlorophylls and carotenoids, vitamins and vitamin precursors including tocopherol, -carotene, niacin, thiamin and ascorbic acid, phenols such as polyphenolics and hydroquinones, phospholipids particularly phosphatidylcholine, terpenoids, peptides, and other antioxidant substances. These compounds directly or indirectly contribute to inhibition or suppression of free radical generation. Although chlorophyll-related compounds are photosensitizers under the light, they are potent antioxidants in the dark. Chlorophyll a, chlorophyllonolacetone a, chlorophyllonic acid a methyl ester and pyropheophorbide a produced by microalgae showed higher antioxidant activity at certain concentrations in linoleic acid than -tocopherol and exerted about the same level of potency as BHT (Sakata, 1997). Carotenoids, another important group of pigments in nature, can also act as effective antioxidants. While most other well-known Table 18.2 Effects of Cystoseira extracts on linoleic acid micellar suspension measured as conjugated dienes Algae species -tocopherol Cystoseira amentacea var. stricta Cystoseira amentacea var.amentacea Cystoseira algeriensis Cystoseira elegans Cystoseira elegans x C. algeriensis Cystoseira jabukae Cystoseira barbata Cystoseira crinita Adapted from Ruberto et al. (2001).

Relative antioxidant activity 100 83 57 54 62 31 37 58 43

400

Maximising the value of marine by-products

carotenoids such as -carotene, lutein and lycopene are derived from plant sources, astaxanthin originates from marine sources, primarily from microalgae. Astaxanthin is both water- and fat-soluble, and is considered 80 times more potent than vitamin E and 10 times more potent than -carotene as an antioxidant (Express press release, 2005). It is thought to have great potential in improving the health of the eyes and the skin (Express press release, 2005). Astaxanthin from microalgae is commercially available. Phenolics, in many cases, are claimed to be the major active constituents that account for the antioxidant activity of marine algae. Duan et al. (2006) have demonstrated that antioxidant potency of crude extract from a red alga (Polysiphonia urceolata) correlated well with its total phenolic content. Strong correlation also existed between the polyphenol content and DPPH radical scavenging activity of a seaweed (Hizikia fusiformis) extract (Siriwardhana et al., 2003). Phenolic antioxidants act as free radical scavengers, reducing agents and metal chelators, and thus effectively inhibit lipid oxidation. Phenolic compounds, especially polyphenols, are widely distributed throughout the plant kingdom. Some important polyphenols in higher plants, such as catechin, epicatechin, epigallocatechin, catechin gallate, epicatechin gallate and epigallocatechin gallate, are also present in marine algae such as Halimada algae (Yoshie et al., 2002). The content and profile of phenolic substances in marine algae varies with the species (Table 18.3). In marine brown algae, a group of phloroglucine polymers called phlorotannins comprises the major phenolic compounds (Chkhikvishvili and Ramazanov, 2000). Brown algae contain various phlorotannins such as fucolls, phlorethols, fucophlorethols, fuhalols and halogenated and sulphited phlorotannins (Chkhikvishvili and Ramazanov, 2000); structures of these phlorotannins are similar to those of condensed tannins (Fig. 18.1). PhloroTable 18.3 Content of phenolic compounds in selected brown algae Alga

Phenolic compounds (% of dry weight)

Cystoseira compressa Cystoseira foeniculaceae Dictyota sp.1 Dictyota sp.2 Dictyota ciliolate Dictyopteris membranacea Focus spiralis Halopteris scoparia Lobophora variegate Padina pavonica Sargassum desfontrainessi Sargassum furcatum Stypopodium zonale Zonaria tonznefortii Adapted from: Chkhikvishvili and Ramazanov (2000).

4.83 2.16 0.03 0.001 0.08 0.09 2.17 0.16 1.20 0.69 1.68 2.97 1.22 1.06

Antioxidants from marine by-products 401

Fig. 18.1

Chemical structures of monomeric units of phlorotannins.

tannins are known to possess a number of biological activity properties, including antiplasmin inhibition (Nakayama et al., 1989), detoxification of heavy metals (Eide et al., 1980), antibacterial effects (Nagayama et al., 2002), UV protection (Swanson and Druehl, 2002) and chemoprevention against vascular risk factors (Kang et al., 2003). It has also been reported that phlorotannins extended the induction period in oxidation of methyl -linolenate (Nakamura et al., 1996) and ROS generation (Kang et al., 2004). These findings suggest that phlorotannins, the natural antioxidant compounds found in edible brown algae, can protect food products against oxidative degradation as well as preventing and/or treating free radical-related diseases. In addition to phlorotannins, bromophenols also play an important role as antioxidants in marine algae, especially in red algae. Fugimoto et al. (1985) isolated four bromophenols from a red alga Polysiphonia ulceolata. Takamatsu et al. (2003) showed that bromophenols (Fig. 18.2) isolated from several marine algae not only exhibited activity in antioxidant assays, but also were taken up by living cells and maintained their activity. Tetraprenyltoluquinols, known phenolic compounds formed by coupling of a hydroquinone ring and a diterpenoidic chain, are characteristically synthesized and accumulated in algal species Cystoseira (Ruberto et al., 2001). Species Cystoseira are the most widely-spread marine flora along the Mediterranean coasts. The extracts of Cystoseira showed significant antioxidant activities comparable to that of -tocopherol in a micellar model system (Ruberto et al., 2001). The antioxidant activities were found to be proportional to the tetraprenyltoluquinol contents in the extracts, and were ascribed to the presence of these compounds (Ruberto et al., 2001). Tetraprenyltoluquinols are tocopherol-like compounds with their diterpenoid chain being very simple, linear and little functionalized in some cases, and complex, largely cyclized and functionalized in others (Fig. 18.3).

402

Maximising the value of marine by-products

Fig. 18.2 Chemical structures of selected bromophenols.

Tocopherols, the most important natural antioxidants, are also tetraprenyltoluquinols. Considering the high content of tetraprenyltoluquinols and their potential antioxidant efficacy, genus Cystoseira can provide for an alternative source of natural antioxidant for food and cosmetic industries (Ruberto et al., 2001).

Fig. 18.3

Chemical structures of chemical tetraprenyltoluquinols.

Antioxidants from marine by-products 403 Polysaccharides are another group that account for the antioxidant activity of marine algae. Polysaccharides are present in the cell walls of marine algae, generally in the form of alginates, fucans, lamininarans, cellulose and sulphated galactans such as agar and carrageenans (Ruperez et al., 2002). Cell walls of marine algae characteristically contain sulphated polysaccharides, which are not found in land plants and which are believed to possess specific functions (Ruperez et al., 2002). Zhang et al. (2003) found that water extracted polysaccharides from Porphyra haitanesis exhibited antioxidant activity. Antioxidant effects of polysaccharides from Fucus vesiculosus (Ruperez et al., 2002) and Laminaria japonica (Xue et al., 2001) have also been reported. Sulphated polysaccharides are by-products in the preparation of alginates from edible brown seaweeds and could be used as a good source of natural antioxidants with potential application in the food industry (Ruperez et al., 2002). The hydrolysis products of polysaccharides, mainly oligosaccharides also showed antioxidant activity, which is thought to be associated with their radical scavenging and metal chelation capacity. Agar oligosaccharides produced by marine bacterial agarase were found to be effective in inhibiting lipid oxidation and scavenging superoxide anion and hydroxyl radicals (Wang et al., 2004). The antioxidant activity of oligosaccharides was structure-dependent; the activity increased with increasing molecular mass and sulphate content (Wang et al., 2004). Some unsaturated fatty acids have been reported to play an effective role in antioxidant activity. The lipophilic extracts from 16 species of seaweeds showed potential antioxidant activities proportional to the content of unsaturated fatty acids (Huang and Wang, 2004). In addition to the groups of substances mentioned above, other compounds also make contribution to the antioxidant efficacy of marine algae. Among them are indoles and dimethylsulphoniopropionate (DMSP). Indole compounds isolated from marine algae have proven to exert inhibitory effect on lipid oxidation (Takahashi et al., 1998). Investigations on DMSP have recently revealed that this compound from marine algal species could serve as an effective antioxidant (Athukorala et al., 2005). Besides, some unknown compounds present in marine algae may also act as active constituents in inhibiting lipid oxidation. Various extraction methods have been used to release these identified and unidentified antioxidant substances from marine algae. Solvent extraction methods employ different solvent systems depending on the solubility of the desired bioactive materials in certain solvents. More recently, enzyme-assisted extraction has been proposed to prepare potential natural water-soluble antioxidants from marine algae. Enzymes such as carbohydrases and proteinases are used to macerate the tissues of the algae, break down the cell walls and complex interior storage materials of algae, such as laminarians, to release interior compounds (Heo et al., 2005c). In the meantime, the breakdown/releasing of high-molecular-weight polysaccharides and proteins themselves may contribute to the antioxidant activity of the extracts (Heo et al., 2005c). Strong dose-dependent radical scavenging capacities were found in the proteolytic hydrolysates of a brown marine alga (Ecklonia cava) (Heo et al., 2005a).

404

18.3

Maximising the value of marine by-products

Antioxidants from marine animals and their by-products

Marine animals including fish, shellfish and mammals have received considerable attention for their application in food and pharmaceutical industries. The processing of marine animals for food production yields a large amount of byproducts, which have been recognized to have special values as natural materials. Issues have been addressed on the utilization of marine discards and by-products. Heu et al. (2003) investigated the components and nutritional quality of shrimp processing by-products. Onodenalore (1998) showed that enzymatic extract of shrimp heads displayed antioxidant activity in a meat model system. Crude and purified extracts from shrimp shell waste have been found to inhibit lipid oxidation and improve colour stability of red rockfish (Li et al., 1998). A study on hag fish and eel skin extracts revealed that they were rich with heat-stable antioxidants with strong radical scavenging activities (Ekanayake et al., 2004, 2005). Compounds responsible for the antioxidant properties of marine animals and/or their processing by-products have been isolated and characterized. Protein hydrolysates and chitosan are among those most frequently studied. Marine animals and their processing by-products are rich in protein. Hydrolysis of protein leads to the production of protein hydrolysates, which have been shown to exert inhibitory effects on lipid oxidation. Protein hydrolysates from many animal and plant sources, individual peptides and amino acids have been tested as antioxidants in a number of studies. Large quantities of yeast and soybean protein hydrolysates were shown to inhibit the oxidation of tocopherol-free corn oil (Benshov and Henick, 1972, 1975). Some amino acids showed strong antioxidant activity in linoleic acid and methyl linoleate model systems (Marcuse, 1962). A polar fraction from krill extract containing a mixture of numerous amino acids possessed strong antioxidant activity (Seher and LoÈschner, 1985). A combination of tryptophan and lysine was effective in inhibiting the oxidation of butterfat (Merzametov and Gadzhieva, 1976). Furthermore, antioxidant properties of proline, methionine, histidine and thronine in fish and vegetable oil or oil emulsion model systems have been reported (Revankar, 1974; Sims and Fioriti, 1977; Riison et al., 1980). Taurine, hypotaurine, carnosine and anserine were found to exert antioxidant effects in vivo (Aruoma et al., 1988). On the other hand, some amino acids such as cysteine may act as prooxidants (Marcuse, 1962; Kanner, 1979). Amino acids which show marked antioxidant activity at low concentrations may become prooxidants at high concentrations (Marcuse, 1962). Protein hydrolysates from marine animal sources and their antioxidant activity have been investigated. Shahidi et al. (1995) reported that capelin protein hydrolysates at a level of 0.5±3.0% inhibited the formation of thiobarbituric acid reactive substances (TBARS) by 17.7±60.4% (Table 18.4). By combining membrane filtration, separation and chromatography techniques, the antioxidant fraction could be partly purified (Guerard et al., 2005). Peptides fractions from protein hydrolysates showed different antioxidant effectiveness.

Antioxidants from marine by-products 405 Table 18.4 Inhibition of TBARS formation by capelin protein hydrolysates (CPH) in cooked meats stored at 4ëC CPH (%)

0.5 1.0 2.0 3.0

% inhibition (days) 0

1

3

5

Mean

29.4 36.0 62.3 76.4

15.9 16.5 62.3 76.4

7.3 14.6 34.6 48.2

18.3 19.9 41.2 58.1

17.7 21.8 44.4 60.4

Adapted from Shahidi et al. (1995).

According to Amarowicz and Shahidi (1997), among the four peptide fractions isolated from capelin protein hydrolysates, one fraction possessed a notable antioxidant activity and another two had a weak efficacy while the fourth fraction exerted prooxidant effect in a -carotene-linoleate model system. He et al. (2006) demonstrated that protein hydrolysates prepared from shrimp Acetes chinensis by a crude protease inhibited hydroxyl radical generation by about 42%. The inhibition by their ultrafiltrate which contained 41% of oligopeptides with a molecular mass of lower than 3 kDa, however, was nearly 68% (He et al., 2006). Kim et al. (2001) isolated two peptides composed of 13 and 16 amino acid residues, respectively from Alaska Pollack skin, both of which contained a glycine residue at the C-terminus and the repeating motif Gly-Pro-Hyp (Table 18.5). The peptide with sequence of Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-HypGly-Pro-Hyp-Gly was more effective in inhibiting the formation of TBARS in linoleic acid compared to another peptide fraction whose sequence was Gly-GluHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly. The difference in antioxidant activity between the two peptide isolates was thought to be Table 18.5 Antioxidative peptides from gelatin hydrolysate of Alaska pollack skin in comparison with that of soy 75 protein Peptide

Amino acid sequence

Alaska pollack skin P1 Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly P2 Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly Soy 75 protein P1 Val-Asn-Pro-His-Asp-His-Glu-Asn P2 Leu-Val-Asn-Pro-His-Asp-His-Glu-Asn P3 Leu-Leu-Pro-His-His P4 Leu-Leu-Pro-His-His-Ala-Asp-Ala-Asp-Tyr P5 Val-Ile-Pro-Ala-Gly-Tyr-Pro P6 Leu-Glu-Ser-Gly-Asp-Ala-Leu-Arg-Val-Pro-Ser-Gly-Thr-Tyr-Tyr Adapted from Shahidi (2003).

406

Maximising the value of marine by-products

attributed to the additional three amino acid residues (Gly-Glu-Hyp) at Nterminus of the latter peptide (Kim et al., 2001). Antioxidant properties of peptides isolated from protein hydrolysates of fish by-products such as skin and frame has also been reported (Jun et al., 2004; Mendis et al., 2005; Je et al., 2005). Proteins can be recovered from marine organisms and their processing byproducts by base extraction. An alkali-assisted extraction process was employed to obtain protein hydrolysates from meat and bone residues of heap seal (Shahidi and Synowiecki, 1996). Besides, enzyme technology has been applied in converting marine by-products and under-utilized species into protein hydrolysates. Use of protein hydrolysates obtained from marine animals, especially from their processing waste, as a source of natural antioxidants has been discussed (Guerard et al., 2005). In addition to their antioxidant effectiveness, protein hydrolysates were found to be useful in improving water-binding capacity of meat products as phosphate alternatives (Shahidi and Synowiecki, 1997). Shellfish processing by-products are also a rich source of chitin (poly-Nacetyl-D-glucosamine). Deactylation of chitin affords chitosan. Depending on the duration of the deacetylation process, the chitosan produced may assume different viscosities and molecular weights. Chitosan is a linear polysaccharide composed mainly of -(1-4)-linked D-glucosamine and is one of the most common polymers found in nature. The most practical source for chitosan is processing discards of shellfish such as shrimp, crab, lobster and crayfish. Chitosan possesses multiple functional properties and has been used in the food, pharmaceutical, cosmetic, paint and textile industries. These include the use of chitosan in medical area as a wound-healing agent, a coating agent for prosthetic articles, a dietary supplement for reducing body weight, an antihypercholesterolemic agent, and an antitumor and antiulcer agent (Shahidi and Abuzaytoun, 2005). The food applications of chitosan include its role as an antimicrobial agent, fruit preservation agent, acidity adjusting and antioxidant agent (Shahidi and Abuzaytoun, 2005). The potential antioxidant activity of chitosan has been investigated. Chitosans of different viscosity were found effective in controlling lipid oxidation in cooked comminuted fish, and the inhibition of oxidation was concentration- and viscosity-dependent (Shahidi et al., 2002; Kamil et al., 2002; Jeon et al., 2002). The use of chitosan as an edible invisible film for quality preservation of fish fillet has been proposed. The content of propanal, an indicator of oxidation of omega 3 fatty acids, was decreased when chitosan was used as an edible invisible film in herring (Table 18.6). Deactylated chitosan showed strong free radical scavenging activity, which positively correlated with the degree of deactylation (Park et al., 2004a). Kanatt et al. (2004) reported that antioxidant activity of chitosan was increased six-fold by gamma irradiation at 25 kGy dose. In addition, chitosan act as an elicitor that induces phytochemicals, mainly phenolic compounds, in plants and hence enhancing the antioxidant activity of the plant, as observed in sweet basil (Kim et al., 2005). Chitosan derivatives may also be produced in order to obtain more effective products for certain applications. For instance, N, O-carboxymethylchitosan (NOCC) and its

Antioxidants from marine by-products 407 Table 18.6 Content of propanal (mg/kg dried fish) in headspace of chitosan-coated herring samples stored at 4ëC Chitosan

Storage period (days)

Uncoated 14 cps 57 cps 360 cps

0

2

4

6

8

10

12.6 13.8 12.6 14.2

23.7 18.3 15.5 15.7

29.9 24.6 19.7 17.6

34.3 30.9 24.9 20.2

44.1 33.0 22.8 18.3

46.3 39.7 24.2 22.7

Adapted from Shahidi (2003).

lactate, acetate and pyrrolidine carboxylate salts were able to inhibit lipid oxidation and off-flavor development in cooked meat stored for nine days in a refrigerator (Shahidi et al., 1999). Chitosan is water-insoluble and highly viscous in dilute acidic solutions, which may restrict its use in physiological functional foods. The oligomers of chitosan, however, are not only water-soluble with low viscosity values, but may also be absorbed in the human intestine, suggesting that they may have much physiological functionality in the in vivo systems (Jeon et al., 2000). Chitosan oligomers with strong physiological activities can be prepared by chemical and enzymatic hydrolyses (Jeon et al., 2000).

18.4

Antioxidants from other marine sources

Marine invertebrates and bacteria have also been explored for their potential applications in biomedicine, food processing as well as in cosmetics and related products. Marine invertebrates, especially tropical marine invertebrates which are chronically exposed to high levels of solar UV radiation, suffer from oxidative stress. Furthermore, unicellular algae residing in symbiosis within their tissues continuously release photosynthetic oxygen that far exceeds the respiratory demand of the invertebrate (Dunlap et al., 2003). For example, coral tissues are hyperoxic (>250% air saturation) during daylight exposure (Dunlap et al., 2003). This, in combination with the high levels of light intensity, can cause photooxidative toxicity to the invertebrate via photodynamic production of cytotoxic ROS (Dunlap et al., 2003). However, marine invertebrates are protected against deleterious ROS, possibly by endogenous antioxidants in their tissues or metabolites and/or the `UV-extremophilic' bacteria inhabiting their tissues. This sheds light on discovery of structurally novel and biologically active antioxidants rich in biodiversity. Novel sunscreening agents derived from tropical marine organisms continue to be developed (Dunlap et al., 1999). Investigations on marine invertebrate metabolites have revealed that metabolites consisting of benzenoid (phenol or quinoid) and terpenoid parts are among the most active antioxidant substances in marine invertebrates

408

Maximising the value of marine by-products

Table 18.7 DPPH scavenging capacity (at 45 min) and inhibitory effect of marine sponge metabolites on Fe2+/ascorbate-induced oxidation of rat brain homogenate Compound

DPPH trapping (%)

Oxidation inhibition (%)

13.4 15.8 70.9 90.7 68.4

56.3 27.2 72.8 67.0 55.0

22.1 24.5 36.6 93.3

59.3 77.0 58.1 ±

ilimaquinone isospongiaquinone puupenone 15-methoxypuupenol 2-methyl-2-pentaprenyl-6hydroxychromene (+)-curcuphenol (+)-curcudiol BHT -tocopherol Adapted from Utkina et al. (2004).

(Utkina et al., 2004). A number of terpenoid phenols and sesquiterpenequinones were isolated from marine sponges. These compounds exhibit various degrees of activity in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and in inhibiting Fe2+/ascorbate-induced lipid oxidation in a rat brain homogenate (Table 18.7) (Utkina et al., 2004). Nevertheless, some of these compounds such as curcuphenol and curcudiol, although active in chemical assays, had no significant activity inside living cells, as reported by Takamatsu et al. (2003). It is suggested that these compounds did not enter the cells due to their poor cellular uptake or low solubility, or perhaps their antioxidant capacity was suppressed in the cellular environment. Marine bacteria have been found to produce compounds with antioxidant activity, such as the sunscreen pigment scytonemin from Scytonema spp. (Takamatsu et al., 2003), astaxanthin and 4-ketozeaxanthin from Agrobacterium auranticam (Yokoyama et al., 1994), as well as 3,4-dimethoxyphenol and indole from Ruditapes phillipinarum bacterial isolate (Sakata, 1997). Sakata (1997) obtained 112 bacterial isolates from 16 fish and shellfish species that possessed antioxidant activity.

18.5

References

and SHAHIDI, F. (1997). Antioxidant activity of peptide fractions of capelin protein hydrolysates. Food Chem., 58, 355±359. ARUOMA, O.I., HALLIWELL, B., HOEY, B.M. and BUTLER, J. (1988). The antioxidant action of taurine, hypotaurine and their metabolic precursors. Biochem. J., 256, 251±255. ATHUKORALA, Y., LEE, K.W., SHAHIDI, F., HEU, M.S., KIM, H.T., LEE, J.S. and JEON, Y.J. (2003a). Antioxidant efficacy of extracts of an edible red alga (Grateloupia filicina) in linoleic acid and fish oil. J. Food Lipids, 10, 313±327. ATHUKORALA, Y., LEE, K.W., CHOONBOK, S., CHANG, B.A., TAI, S.S., YONG, J.C., SHAHIDI, F. and AMAROWICZ, R.

Antioxidants from marine by-products 409 (2003b). Potential antioxidant activity of marine red algae Grateloupia filicina extracts. J. Food Lipids, 10, 251±265. ATHUKORALA, Y., LEE, K.W., PARK, E.J., HEO, M.S., YEO, I.K., LEE, Y.D. and JEON, Y.J. (2005). Reduction of lipid peroxidation and H2O2-mediated DNA damage by a red alga (Grateloupia filicina) methonolic extract. J. Sci. Food Agric., 85, 2341±2348. BENSHOV, S.J. and HENICK, A.S. (1972). Antioxidant effct of protein hydrolysates in a freeze-dried model system. J. Food Sci., 37, 873±875. BENSHOV, S.J. and HENICK, A.S. (1975). Antioxidant effect of protein hydrolysates in a freeze-dried model system. Synergistic action with a series of phenolic antioxidans. J. Food Sci., 40, 345±348. CHKHIKVISHVILI, I.D. and RAMAZANOV, Z.M. (2000). Phenolic substances of brown algae and their antioxidant activity. Appl. Biochem. Microbiol., 36, 289±291. DUAN, X.J., ZHANG, W.W., LI, X.M. and WANG, B.G. (2006). Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem., 95, 37±43. DUNLAP, W.C., MALCOLM SHICK, J. and YAMAMOTO, Y. (1999). Sunscreens, oxidative stress and antioxidant functions in marine organisms of the Great Barrier Reef. Redox Report, 4, 301±306. DUNLAP, W., LLEWELLYN, L., DOYLE, J. and YAMAMOTO, Y. (2003). A microtiter plate assay for screening antioxidant activity in extracts of marine organisms. Mar. Biotechnol., 5, 294±301. EIDE, I., MYKLESTAD, S. and MELSON, S. (1980). Long-term uptake and release of heavy metals by Ascophyllum nodosum (L). Le Jol. (Phaeophyceae) in situ. Environ. Pollut., 23, 19±28. EKANAYAKE, P., LEE, Y.D. and LEE, J. (2004). Antioxidant activity of flesh and skin of Eptatretus burgeri (hag fish) and Enedrias nebulosus (white spotted eel). Food Sci. Tech. Int., 10, 171±177. EKANAYAKE, P.M., PARK, G.T., LEE, Y.D., KIM, S.J., JEONG, S.C. and LEE, J. (2005). Antioxidant potential of eel (Anguilla japonica and Conger myriaster) flesh and skin. J. Food Lipids, 12, 34±47. EXPRESS PRESS RELEASE. (2005). New marine based antioxidant blows others out of the water. http://www.express-press-release.com FOTI, M., PIATTELLI, M., AMICO, V. and RUBERTO, G. (1994). Antioxidant activity of phenolic meroditerpenoids from marine algae. J. Photochem. Photobiol. B: Biology, 26, 159±164. FUGIMOTO, K., OHMURA, H. and KANEDA, T. (1985). Screening for antioxygenic compounds in marine algae and bromophenols are effective principles in red alga Polysiphonia ulceolate. J. Jap. Soc. Sci. Fish., 51, 1139±1143. GUERARD, F., SUMAYA-MARTINEZ, M.T., LINARD, B. and DUFOSSE, L. (2005). Marine protein hydrolysates with antioxidant properties. Agro. Food Ind. Hi. Tech., 16, 16±18. HE, H.L., CHEN, X.L., SUN, C.Y., ZHANG, Y.Z. and GAO, P.J. (2006). Preparation and functional evaluation of oligopeptide-enriched hydrolysate from shrimp (Acetes chinensis) treated with crude protease from Bacillus sp. SM98011. Bioresource Technol., 97, 385±390. HEO, S.J., PARK, P.J., PARK, E.J., CHO, S.K., KIM, S.K. and JEON, Y.J. (2005a). Antioxidant effect of proteolytic hydrolysates from Ecklonia cava on radical scavenging using ESR and H2O2-induced DNA damage. Food Sci. Biotech., 14, 614±620. HEO, S.J., PARK, E.J., LEE, K.W. and JEON, Y.J. (2005b). Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresource Technol., 96, 1613±1623. JEON, Y.J.

410

Maximising the value of marine by-products

and JEON, Y.J. (2005c). Antioxidant activity of enzymatic extracts from a brown seaweed Ecklonia cava by electron spin resonance spectrometry and comet assay. Eur. Food Res. Technol., 221, 41±47. HEU, M.S., KIM, J.S. and SHAHIDI, F. (2003). Components and nutritional quality of shrimp processing by-products. Food Chem., 82, 235±242. HUANG, H.L. and WANG, B.G. (2004). Antioxidant capacity and lipophilic content of seaweeds collected from the Qingdao Coastline. J. Agric. Food Chem., 52, 4993± 4997. JE, J.Y., PARK, P.J. and KIM, S.K. (2005). Antioxidant activity of a peptide isolated from Alaska Pollack (Theregra chalcogramma) frame protein hydrolysates. Food Res. Int., 38, 45±50. JEON, Y.J., SHAHIDI, F. and KIM, S.K. (2000). Preparation of chitin and chitosan oligomers and their applications in physiological functional foods. Food Rev. Int., 16, 159±176. JEON, Y.J., KAMIL, J.Y.V.A. and SHAHIDI, F. (2002). Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod. J. Agric. Food Chem., 50, 5167± 5178. JUN, S.Y., PARK, P.J., JUNG, W.K. and KIM, S.K. (2004). Purification and characterization of an antioxidative peptide from enzymatic hydrolysate of yellowfin sole (Limanda aspera) frame protein. Eur. Food Res. Technol., 219, 20-26. KAMIL, J.Y.V.A., JEON, Y.J. and SHAHIDI, F. (2002). Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus). Food Chem., 79, 69±77. KANATT, S.R., CHANDER, R.C. and SHARMA, A. (2004). Effect of irradiated chitosan on the rancidity of radiation-processed lamb meat. Int. J. Food Sci. Technol., 39, 997± 1003. KANG, H.S., CHUNG, H.Y., KIM, J.Y., SON, B.W., JUNG, H.A. and CHOI, J.S. (2004). Inhibitory phlorotannins from the edible brown alga Ecklonia stolonifera on total reactive oxygen species (ROS) generation. Arch. Pharm. Res., 27, 194±198. KANG, K., PARK, Y., HWANG, H.J., KIM, S.H., LEE, J.G. and SHIN, H.C. (2003). Antioxidative properties of brown algae polyphenolics and their perspectives as chemopreventive agent against vascular risk factors. Arch. Pharm. Res., 26, 286±293. KANNER, J. (1979). S-Nitrosocysteine (RSNO) an effective antioxidant in cured meat. J. Am. Oil Chem. Soc., 56, 74±76. KIM, S.K., KIM, Y.T., BYUN, H.G., NAM, K.S., JOO, D.S. and SHAHIDI, F. (2001). Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska Pollack skin. J. Agric. Food Chem., 49, 1984±1989. KIM, J.H., CHEN, F., WANG, X. and RAJAPAKSE, N.C. (2005). Effect of chitosan on the biological properties of sweet basil (Ocimum basilicum L.). J. Agric. Food Chem., 53, 3696±3701. KYLE, D.J. (2001). The large-scale production and use of a single-cell oil highly enriched in docosahexaenoic acid. In: Omega-3 fatty acids: chemistry, nutrition, and health effects. Shahidi, F. and Finley, J.W. eds., ACS Symposium Series 788. American Chemical Society, Washington, DC, pp. 92±107. LI, S.J., SEYMOUR, T.A., KING, A.J. and MORRISSEY, M.T. (1998). Color stability and lipid oxidation of rockfish as affected by antioxidant from shrimp shell waste. J. Food Sci., 63, 438±441. MARCUSE, R. (1962). The effect of some amino acids on oxidation of linoleic acid and its methyl esters. J. Am. Oil Chem. Soc., 39, 97±103. MARX, J.L. (1987). Oxygen free radicals linked to many diseases. Science, 234, 529±531. HEO, S.J., PARK, P.J., PARK, E.J., KIM, S.K.

Antioxidants from marine by-products 411 and KIM, S.K. (2005). Antioxidant properties of a radicalscavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. J. Agric. Food Chem., 53, 581±587. MERZAMETOV, M.M. and GADZHIEVA, L.I. (1976). Certain amino acids as antioxidants in butter fat. Izv. Ucheb. Zaved. Pischew. Technol., 115, 21±27. MORI, J., MASTUNAGA, T., TAKAHASHI, S., HASEGAWA, C. and SAITO, H. (2003). Inhibitory activity on lipid peroxidation of extracts from marine brown alga. Phytother. Res., 17, 549±551. NAGAYAMA, K., IWAMURA, Y., SHIBATA, T., HIRAYAMA, I. and NAKAMURA, T. (2002). Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome. J. Antimicro. Chemother., 50, 889±893. NAKAMURA, T., NAGAYAMA, K., UCHIDA, K. and TANAKA, R. (1996). Antioxidant acitivity of phlorotannins isolated from the brown alga Eisenia bicyclis. Fish. Sci., 62, 923± 926. NAKAYAMA, Y., TAKAHASHI, M., FUKUYAMA, Y. and KINZYO, Z. (1989). An anti-plasmin inhibitor, echol, isolated from the brown alga Ecklonia kurome OKAMURA. Agric. Biol. Chem., 63, 3025±3030. ONODENALORE, A.C. (1998). Value-added functional protein products and endogenous antioxidants from aquatic species. PhD Thesis, Memorial University of Newfoundland, St. John's, NL, Canada. PARK, P.J., JE, J.Y. and KIM, S.K. (2004a). Free radical scavenging activities of differently deactylated chitosans using an ESR spectrometer. Carbohydrate Polymers, 55, 17± 22. PARK, P.J., SHAHIDI, F. and JEON, Y.J. (2004b). Antioxidant activities of enzymatic extracts from an edible seaweed Sargassum horner using ESR spectrometry. J. Food Lipids, 11, 15±28. PARK, P.J., HEO, S.J., PARK, E.J., KIM, S.K., BYUN, H.G., JEON, B.T. and JEON, Y.J. (2005). Reactive oxygen scavenging effect of enzymatic extracts from Sargassum thunbergii. J. Agric. Food Chem., 53, 6666±6672. REVANKAR, G.D. (1974). Proline as an antioxidant in fish oil. J. Food Sci. Technol. Mysore, 11, 10±11. RIISON, T., SIMS, R.J. and FIORITI, J.A. (1980). Efect of amino acids on the autoxidation of safflower oil in emulsions. J. Am. Oil Chem. Soc., 57, 354±359. RUBERTO, G., BARATTA, M.T., BIONDI, D.M. and AMICO, V. (2001). Antioxidant activity of extracts of the marine algal genus Cystoseira in a micellar model system. J. Appl. Phycol., 13, 403±407. RUPEREZ, P., AHRAZEM, O. and LEAL, J.A. (2002). Potential antioxidant capacity of sulfated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. J. Agric. Food Chem., 50, 840±845. SAKATA, K. (1997). Antioxidative compounds from marine organisms. In: Food and Free Radicals. Hiramatsu, M., Yoshikawa, T. and Inoue, M. eds., Plenum Press, New York, pp. 85±100. È SCHNER, D. (1985). Naturliche Antioxidantien. V: Andioxidantien und SEHER, A. and LO Synergisten aus antarktischen Krill. Fette-Seifen-Anstrichmittel, 87, 454±457. SHAHIDI, F. (2003). Nutraceuticals and bioactives from seafood by-products. In: Advances in seafood byproducts: 2002 Conference proceedings. Bechtel, P.J. ed., Alaska Sea Grant College Program, University of Alaska Fairbanks, Fairbanks, Alaska, pp. 247±263. SHAHIDI, F. and ABUZAYTOUN, R. (2005). Chitin, chitosan, and co-products: chemistry, MENDIS, E., RAJAPAKSE, N.

412

Maximising the value of marine by-products

production, applications, and health effects. In: Advances in food and nutrition research, volnume 49. Taylor, S. ed., Academic Press, San Diego, CA, pp. 93±137. SHAHIDI, F. and SYNOWIECKI, J. (1996). Alkalie-assisted extraction of proteins from meat and bone residues of harp seal (Phoca groenlandica). Food Chem., 57, 317±321. SHAHIDI, F. and SYNOWIECKI, J. (1997). Protein hydrolyzates from seal meat as phosphate alternatives in food processing applications. Food Chem., 60, 29±32. SHAHIDI, F., HAN, X.Q. and SYNOWIECKI, J. (1995). Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem., 53, 285±293. SHAHIDI, F., ARACHCHI, J.K.V. and JEON, Y.J. (1999). Food application of chitin and chitosan. Trends Food Sci. Technol., 10, 37±51. SHAHIDI, F., KANIL, J., JEON, Y.J. and KIM, S.K. (2002). Antioxidant roleof chitosan in a cooked cod (Gadus morhua) model system. J. Food Lipids, 9, 57±64. SIMS, R.J. and FIORITI, J.A. (1977). Methional as an antioxidant for vegetable oils. J. Am. Oil Chem. Soc., 54, 4±7. SIRIWARDHANA, N., LEE, K.W., KIM, S.H., HA, J.W. and JEON, Y.J. (2003). Antioxidant activity of Hizikia fusiformis on reactive oxygen species scavenging and lipid peroxidation inhibition. Food Sci. Technol. Int., 9, 339±346. SWANSON, A.K. and DRUEHL, L.D. (2002). Induction, exudation and the UV protective role of kelp phlorotennins. Aquatic Botany, 73, 241±253. TAKAHASHI, S., MATSUNAGA, T. and HASEGAWA, C. (1998). Martefragin A, a novel indole alkaloid isolated from red alga inhibits lipid peroxidation. Chem. Pharm. Bull., 46, 1527±1529. TAKAMATSU, S., HODGES, T.W., RAJBHANDARI, I., GERWICK, W.H., HAMANN, M.T. and NAGLE, D.G. (2003). Marine natural products as novel antioxidant prototypes. J. Nat. Prod., 66, 605±608. UTKINA, N.K., MAKARCHENKO, A.E., SHCHELOKOVA, O.V. and VIROVAYA, M.V. (2004). Antioxidant activity of phenolic metabolites from marine sponges. Chem. Nat. Comp., 40, 373±377. WANG, J.X., JIANG, X.L., MOU, H.J. and GUAN, H.S. (2004). Anti-oxidation of agar oligosaccharides produced by agarases from a marine bacterium. J. Appl. Phycol., 16, 333±340. XUE, C., FANG, Y., LIN, H., CHEN, L., LI, Z., DENG, D. and LU, C. (2001). Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica. J. Appl. Phycol., 13, 67±70. YOKOYAMA, A., IZUMIDA, H. and MIKI, W. (1994). Production of astaxanthin and 4-ketozeaxanthin by the marine bacterium Agrobacterium aurantiacum. Biosci. Biotech. Biochem., 58, 1842±1844. YOSHIE, Y., WANG, W., HSIEH, Y.P. and SUZUKI, T. (2002). Compositional differences of phenolic compounds between two seaweeds Halimeda spp.. J. Tokyo Univ. Fish., 88, 21±24. ZELLER, S., BARCLAY, W. and ABRIL, R. (2001). Production of docosahexaenoic acid from microalgae. In: Omega-3 fatty acids: chemistry, nutrition, and health effects. Shahidi, F. and Finley, J.W. eds., ACS Symposium Series 788. American Chemical Society, Washington, DC, pp 108±124. ZHANG, Q., YU, P., LI, Z., ZHANG, H., XU, Z. and LI, P. (2003). Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis. J. Appl. Phycol., 15, 305±310.

19 Pigments from by-products of seafood processing B. K. Simpson, Department of Food Science and Agricultural Chemistry, Canada

19.1

Introduction

The term `pigments from aquatic species' is used here to encompass all those compounds that are naturally present in aquatic animals, plants and algae, and impart a plethora of colors to these species. These colors may be bright yellow, red or orange as found in the flesh of salmon and trout, or in the exoskeletons of raw and/or cooked shrimp, crab, krill and lobster; they may be brown as found in the marine algae collectively known as the bryophytes; or they may range from brown to black ± as occurs in the eyes, skins and other tissues of some of these species. There are yet others that are either intensely red in color as found in the red algae that inhabit greater depths of the ocean where hydrostatic pressure is high; or they may be intensely green as in the green algae or seaweeds. At first glance, it may be tempting for one to simply perceive these compounds as nature's way of adding appeal, variety and delight to the (aquatic) environment, which undoubtedly would have been monotonous, drab and dreary otherwise. However, as will be shown in the following pages, these compounds serve several very fundamental and crucial functions within the species they occur in. For instance, some of them serve as mating signals in certain species; some provide camouflage and concealment for prey from their predators; some others potentiate crucial biological processes like photosynthesis whereby complex biomolecules (carbohydrates) are fabricated from simpler molecules (CO2 and H2O); while others function as antioxidants; and yet others participate in vision or protect skin and membranes against the ravaging effects of sunlight and radiation.

414

Maximising the value of marine by-products

From time immemorial, humans have derived benefit and delight from these naturally occurring pigments present in aquatic species, and this tendency does not show any signs of abating any time soon. One is rather inclined to predict that our reliance on these compounds for good health and well-being will continue to increase in light of new discoveries about their health and related benefits, as well as the general preferences by consumers for all things `natural' versus their synthetic or artificial counterparts. It is not possible to fully and extensively cover all the different pigments in a single chapter of this book. Thus, the focus will be placed on carotenoid pigments from crustacean processing discards, due to current interest in these biological molecules for commercial use in health, food/feed and related applications, as well as the relative abundance of the source material that currently represents an environmental pollution problem ± in great need to be put to more profitable use.

19.2

Pigment types and sources

There are several classes or types of pigments known at the present time. These include the carotenoids ± responsible for the bright red, orange and yellow coloration of the flesh and skins of species like salmonids (e.g., salmon and trout), as well as the exoskeletons of crustacea (e.g., shrimp, lobster, krill, crayfish and crab); melanins ± the brown to black pigments formed by enzyme catalyzed oxidation of phenolic compounds that are found in the skins, eyes and peritoneal lining of certain species; the green chlorophyll pigments that occur in all photosynthetic organisms to enable these species to function as primary producers in the food chain. The chlorophylls are invariably found together with carotenoid pigments including fucoxanthin (the brown and dominant pigment in brown seaweeds or brown algae) and accessory pigments like phycocyanin and phycoerythrin. As indicated earlier, this chapter will cover carotenoid pigments more extensively, while the other pigments will be briefly mentioned to highlight their significance or potential.

19.3

Carotenoid pigments

The carotenoids are synthesized in plants, bacteria and microalgae (RodriquezConcepcion and Boronat, 2002) from the simple precursor molecules, pyruvate and acetyl CoA, via a 5-carbon intermediate compound known as isopentenyl pyrophosphate (C5H9O7P2) or IPP (Fig. 19.1) for short, via the mevalonic acid (MVA) and the deoxyxylulose (DOXP) pathways (Kasahara et al., 2002). The IPP formed from pyruvate and acetyl CoA, is converted by a series of enzyme mediated steps to a 40-carbon polyunsaturated hydrocarbon compound with nine double bonds known as phytoene (Fig. 19.2). It (phytoene) has a molecular formula of C40H64, and after it is formed, it undergoes four desaturation or

Pigments from by-products of seafood processing

415

Fig. 19.1 Isopentenyl pyrophosphate.

Fig. 19.2 Phytoene.

Fig. 19.3 Lycopene.

dehydrogenation reactions catalyzed by the enzyme phytoene desaturase to form lycopene, C40H56 (Fig. 19.3), a hydrocarbon carotenoid compound with thirteen double bonds. Lycopene may then undergo isomerization and cyclization by carotene isomerase and lycopene cyclase, respectively, to form other hydrocarbon carotenoids like -carotene, -carotene and -carotene (Fig. 19.4). These cyclic hydrocarbon carotenoids may then be hydroxylated by hydroxylase enzymes (carotene hydrolases) to form oxygenated carotenoids like crypto-

416

Maximising the value of marine by-products

Fig. 19.4 (a) -carotene, (b) -carotene and (c) -carotene.

xanthin, lutein and zeaxanthin (Fig. 19.5). Other oxygenated carotenoids such as astaxanthin and canthaxanthin (Fig. 19.6) may subsequently be formed from the carotenes or hydroxylated carotenoids by epoxidation and de-epoxidation reactions. Unlike plants and microorganisms, animals are incapable of de novo synthesis of these carotenoid compounds. Rather, animals must obtain these molecules preformed in their diets; and they have the capacity to modify dietary carotenoids for storage in their tissues for various physiological functions. This capacity by animals to modify carotenoids from the diets is exemplified by the conversion of -carotene into canthaxanthin in species like the Artemia (Tanaka et al., 1976).

Pigments from by-products of seafood processing

417

Fig. 19.5 (a) Cryptoxanthin, (b) lutein and (c) zeaxanthin.

19.3.1 Properties and functions Carotenoid pigments serve several functions besides imparting beautiful bright red, orange and yellow colors to these species. For instance, carotenoids have antioxidant properties by virtue of their highly unsaturated nature, which enable them to lend themselves to (sacrificial) oxidation instead of other molecules. Carotenoids like -carotene, -carotene, zeaxanthin and -cryptoxanthin are cleaved by dioxygenase in the gastrointestinal tract to release, at least a molecule of vitamin A ± thus these carotenoids are said to have provitamin A activity. Carotenoids also participate in energy transfer reactions during photosynthesis and in singlet oxygen quenching. As a result of these properties, carotenoids play crucial roles in vision and are effective in: curtailing agerelated eye disorders such as cataracts; preventing the oxidation of low density

418

Maximising the value of marine by-products

Fig. 19.6 (a) Astaxanthin and (b) canthaxanthin.

lipoprotein (LDL) that culminates in platelet formation and aggregation leading to cardiovascular diseases (Hadley et al., 2003); prevention of various cancers via free radical scavenging; as well as protection of tissues against damage from exposure to light. Carotenoids also enhance/regulate the immune system in several ways, including increasing the activities of lymphocytes, as well as protecting macrophages and the immune system against oxidative damage from exposure to ultraviolet light and X-rays. Fucoxanthin, shown in Fig. 19.7, is an oxygenated carotenoid pigment found in brown algae. It is yellowish-brown in color and has a molecular formula of C40H60O6. Fucoxanthin is mentioned here because of its demonstrated health benefits. Various studies have shown it to induce apoptosis and enhance antiproliferative effects on certain cancer cells (Kotake-Nara et al., 2005; Hosokawa et al., 2004). An example of economically important brown algae with high fucoxanthin content is the kelp that is consumed as food by humans.

Fig. 19.7 Fucoxanthin.

Pigments from by-products of seafood processing

419

19.3.2 Preparation and stabilization Crustacean processing waste is a rich source of carotenoid pigments. However, the use of this abundant resource as source material for carotenoid pigments in aquaculture feeds and similar products is not widespread and also not very practical because the material deteriorates rapidly in the raw form. Other disadvantages with direct incorporation of the offal in feeds include bulkiness, variable pigment levels and high content of calcium and chitin. Various methods have been devised to either extract the carotenoid pigments from crustacean waste, or modify the offal into semi-stable or stable forms. These include the organic solvent extraction process described by Bligh (1978). The organic solvent extraction process involves soaking the waste in an acetone-petroleum ether-water mixture (75:15:10, v/v/v) and then filtering out the de-pigmented residue. The pigment trapped in the acetone-petroleum ether-water mixture is then transferred to petroleum ether, and then dried with anhydrous sodium sulphate. Crustacean offal has also been processed into meals and used in feeds (Ruthledge, 1971), although this approach invariably requires decalcification of the meals prior to use as feed ingredient. The acid ensilage method developed in Norway for producing fish protein hydrolysates from processing discards and underutilized by-catch (Torrissen et al., 1981) has also been adapted for use with crustacean waste. The acid ensilage process involves treatment of comminuted heat processed crustacean waste with formic acid, and the process successfully recovers a product that is high in carotenoid pigments, low in calcium and chitin, and is also stable at ambient temperature. However, the drawback with the acid ensilage method is the high acidity of the product and the need to adjust the pH prior to use (Meyers and Chen, 1982a). Vegetable (soy) oil has also been used to strip carotenoid pigments from crustacean offal into the oil. This involves blending the vegetable oil with comminuted heat-processed crustacean waste, and heating the concoction with continuous stirring at a temperature ranging between 40 and 50ëC in the dark. The process achieves high yields of the pigment that may be stabilized by the addition of suitable antioxidants such as ethoxyquin, -tocopherol and butylated hydroxytoluene (BHT) (Shahidi and Synowiecki, 1991; Meyers and Chen, 1982b); however, this approach recovers only the free pigment, and excludes valuable protein nutrients from the offal. Another process is based on the treatment of the raw offal with proteolytic enzymes and chelating agents to co-extract the carotenoid pigments with proteins (Cano-Lopez et al., 1987; Simpson and Haard, 1985); this last approach recovers valuable protein nutrient with the pigment. The advantages with the latter approach include the fact that chelating agents used in the process together with the proteins coextracted with the pigments help to stabilize the pigments. As well, the carotenoprotein product is depleted in ash and chitin, and also achieves substantial reduction in the bulk of the offal to ease storage, transportation and distribution.

420

Maximising the value of marine by-products

19.3.3 Uses in food and feed products The global market for carotenoid pigments is estimated at about US$935 million (Fraser and Bramley, 2004). Carotenoid pigments are used as colorants for food, drugs and cosmetics, and as animal feed and nutritional supplements. In the food industry, carotenoid pigments are used to impart color to confectionery, as well as in bakery and dairy products (Britton, 1996). They are also used as colorant for butter, egg yolk, salmon and lobster. There is no gainsaying the fact that aquaculture feeds represent the leading user sector of natural carotenoid pigments from crustacean waste. This fact may hold true for the foreseeable future, given the steady increase in salmon and trout farming in the leading producer countries like Norway and Chile and elsewhere in the world. Salmonids, crustaceans and other animals all have a demanding requirement for carotenoid pigments for various functions including provitamin A activity, antioxidant effects, as hormone precursors, in immune response, and in growth, maturation and reproduction (Lorenz and Cysewski, 2000). However, because animals are incapable of de novo synthesis of carotenoid pigments (Fraser and Bramley, 2004), they must have these compounds in their diets for the various functions listed above. As mentioned above, microorganisms and plants first synthesize carotenoid compounds such as lycopene, -carotene and canthaxanthin from pyruvate and acetyl Co A via the isoprenoid pathway. The carotenoids from these primary sources are subsequently ingested by animals with their diet and converted enzymatically to other carotenoids like astaxanthin. In some of these animals, deficiencies in carotenoids lead to undesirable defects like the blue color syndrome in shrimp. The strategies that have been used to obviate such defects and/or impart the desirable bright orange or red pigmentation in these animals reared in captivity include supplementation of the diets with synthetic colorants like NatuRoseTM astaxanthin (Lorenz, 1998) and CarophyllÕ pink, or carotenoid pigments derived from crustacean meals. These materials have also been incorporated in poultry feed to impart desirable coloration to egg yolks of various poultry animals. 19.3.4 Comparison with synthetic colorants Carotenoid pigments are available for commercial use in both `natural' and synthetic forms. The natural source materials for carotenoid pigments include krill oil (White et al., 2003), shrimp waste (Saito and Regier, 1971), crayfish oil extract (Peterson et al., 1966), crab meals and oil extracts (Spinelli et al., 1974), Spirulina and paprika (Meyers, 1994), the microalga Haematococcus sp. (Johnson and An, 1991), and the yeast Phaffia rhodozyma (Parajo et al., 1998). The commercially available synthetic forms include canthaxanthin ( , carotene-4,40 -dione), astaxanthin (3,30 -dihydroxy- -carotene-4-40 -dione) and astaxanthin dipalmitate (Storebakken et al., 1987). Synthetic astaxanthin and synthetic canthaxanthin are also known as CarophyllÕ pink and CarophyllÕ red, respectively. Both have been approved for food use by the US Food and Drug Administration.

Pigments from by-products of seafood processing

421

Carotenoids from different sources may occur either in the free or the esterified form. For example krill oil astaxanthin is found predominantly in the diester form (Yamaguchi et al., 1983); astaxanthin from Haematococcus sp. occurs predominantly as monoesters (Harker et al., 1996; Breithaupt, 2004); while Phaffia rhodozyma astaxanthin occurs in the free non-esterified form (Johnson and An, 1991; Breithaupt, 2004). The form in which astaxanthin occurs is believed to influence the uptake of the pigment from the diet and its subsequent incorporation into the flesh of the animal (White et al., 2003). The view is that the free and the monoester forms of the pigment are taken up more rapidly and more extensively than the diester forms, thus lending credence to the notion that astaxanthin is deposited in the flesh of the animal in the free unesterified form, so that the esterified forms need first to be hydrolyzed in the guts before they can be absorbed and assimilated (Choubert and Heinrich, 1993; White et al., 2003). The differences in the degree of esterification of the carotenoids in feed supplements from various sources is believed to be one of the main reasons for the observed differences in the extent of deposition and coloration of the flesh of cultured salmonids and crustaceans. Other studies have shown that astaxanthin in the free form is also found to color the flesh of salmonids more extensively than canthaxanthin (Negre-Sadargues et al., 1993). Furthermore, fish flesh color achieved with canthaxanthin tends to be more yellowish, while astaxanthin imparts a more pinkish/orange color (Bjerkeng, 2000). There have been a number of studies that have compared coloration achieved by feeding astaxanthin from `natural sources' with their synthetic counterparts. Examples of those studies include the feeding of rainbow trout with diets supplemented with CarophyllÕ Pink (unesterified synthetic astaxanthin), astaxanthin monoesters and astaxanthin diesters extracted from the microalga H. pluvialis. This study suggested an influence of the degree of astaxanthin esterification on pigment uptake and deposition in the fish flesh (White et al., 2003). In another study, rainbow trout (Oncorhynchus mykiss) were fed diets supplemented with shrimp carotenoproteins or CarophyllÕ Pink and compared with control fish samples that were maintained on the non-pigmented ration (Nguyen et al., 2003). The overall mean fish size at the start of the feeding trials was 166.6  31.2 g. The final average mass values for each treatment were: fish fed carotenoprotein supplemented ration, 375.1 g; fish fed CarophyllÕ Pink supplemented ration, 390.4 g; and the control fish fed the non-pigmented ration, 391.7 g. The fish fed CarophyllÕ Pink and control diets showed slightly higher specific growth rate (SGR) than those fed the carotenoprotein diet. The carotenoprotein fed fish showed a slight aversion to the carotenoprotein diet at the beginning of the trial, but rapidly overcame this aversion, as evidenced by the very minor differences in fish size at the end of the trial. The flesh from the fish samples that were fed carotenoprotein or CarophyllÕ Pink diets had similar intense orange color compared with the control fish samples. Thus, shrimp carotenoprotein was as effective in coloring the rainbow trout flesh as the synthetic product (CarophyllÕ Pink) and did not appear to adversely affect the overall growth of the animal.

422

Maximising the value of marine by-products

In general, these studies have shown that carotenoid pigments from `natural' or synthetic origins were both effective in achieving flesh coloration, and there were no significant differences in the absorption and deposition of the pigments from these two sources. However, considerably larger quantities of the `natural' source material were required to achieve the same effect that is achievable with much smaller amounts of the synthetic pigment, because the pigment levels in the natural source materials tend to be quite low. Similar observations were made when the diets used in the studies by Nguyen et al. (2003) were used to feed brook trout, Salvelinus fontinalis (Finn, 2004). Other limitations with some of the `natural' source materials (such as crustacean offal/meals, and Phaffia) include high contents of moisture, ash and/or chitin. 19.3.5 Methods for measuring carotenoid pigments Carotenoid pigments (as astaxanthin) may be quantitated spectrophotometrically, by measuring the absorbance at 485 nm (Saito and Regier, 1971). Other methods that have been used to study the molecular properties of carotenoids include field desorption mass spectrometry (Takaichi et al., 2003), thin layer chromatography (TLC) separation followed by transmethylation and analysis by gas chromatography (GC) (Renstrom and Liaaen-Jensen, 1981). High pressure liquid chromatography (HPLC) has also been used for the separation and identification of astaxanthin esters and chlorophylls in the Japanese fresh water algae, Haematococcus lacustris (Yuan et al., 1996). More recently, detection and measurement of carotenoid pigments was accomplished by the negative ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (negative ion LC-[APC]-MS) method described by Breithaupt (2004) for studying astaxanthin esters in shrimp and algae. In practice, the most commonly used instrumental methods for measuring fish flesh color are the Hunter L, a, b, and the Commission Internationale de l'Eclairage/International Commission on Illumination (CIE) L*, a*, b* systems.

19.4

Other pigments

Apart from carotenoids, there are other natural pigments also found in various species that inhabit the aquatic environment. Examples of these other natural pigments include the green chlorophyll pigments, the blue and red chromoproteins (phycocyanin and phycoerythrin) from in the blue-green algae and cyanobacteria, and melanins. There are also other minor pigments such as flavins, pterins, quinones and porphyrins. 19.4.1 Chlorophylls The chlorophylls are the green pigments whose primary function is photosynthesis ± whereby they permit the species that harbor these pigments to

Pigments from by-products of seafood processing

423

Fig. 19.8 Chlorophylls.

synthesize complex biomolecules (carbohydrates) from simpler sources (carbon dioxide and water). Chlorophylls occur abundantly in green algae, also known as chlorophytes. They are predominantly freshwater, with only a small percentage inhabiting the marine environment. Chlorophylls are tetrapyrolle compounds ± i.e., four pyrolle groups are linked together by a central Mg++ ion to form a porphyrin ring, which together with phytol (a 20-carbon hydrocarbon chain) makes the chlorophyll molecule (Fig. 19.8). The chlorophyll pigments are best known as the basis of all plant life because of its functions as light trapping pigment and electron donor in photosynthesis. Perhaps, a lesser mentioned property of chlorophylls is their demonstrated benefits to human health. For example, as far back as 1936, Patek demonstrated that chlorophyll (as wheatgrass) rebuilds the bloodstream (Patek, 1936), and studies using various animals showed that dispensation of chlorophyll restored red blood cell counts to normal levels within 4 to 5 days, even in extremely anemic animals. Chlorophyll is nontoxic even in large doses when administered intravenously, intramuscularly, or orally and as a colon implant to animals and humans without toxic side effects. It is anti-bacterial and can be used inside and outside the body as a healer (Bowers, 1947). Furthermore, the high magnesium content in chlorophyll is thought to enhance fertility by building up the enzymes that modulate the sex hormones. Chlorophyll is believed to cleanse drug deposits from the body, neutralize toxins in the body, purify the liver, and alleviate blood sugar problems (Colio and Babb, 1948). Other uses for chlorophyll include clearing up foul smelling odors, neutralizing streptococcus infections in the buccal cavity, promoting wound healing, hastening skin grafting, reducing varicose veins, healing rectal sores, and reducing typhoid fever (Offenkrantz, 1950). Excellent sources of chlorophylls (in commercial quantities) include the green algae chlorella, the blue-green algae spirulina, the string lettuce (Entero-

424

Maximising the value of marine by-products

morpha) and the sea lettuce (Ulva). Spirulina has both chlorophyll (green) and phycocyanin (blue) pigments in its cellular structure. All four major sources of chlorophyll listed above are consumed as food by humans for their known health benefits (Ayehunie et al., 1996). They are used in salads and soups. Also because of their intense green color, chlorophylls are used in many applications in coloring soaps, oils, creams and body lotions, oral hygiene products as well as confectionery. 19.4.2 Phycocyanin and phycoerythrin The blue pigment, phycocyanin is found in blue-green algae and cyanobacteria. The red pigment, phycoerythrin is found in the red algae commonly referred to as the rhodophytes. Phycocyanin and phycoerythrin pigments are conjugated chromoproteins and are composed of a number of subunits, each having a protein backbone covalently attached to open chain tetrapyrrole groups. Their molecular weights range from 44 000 daltons (monomers) to 260 000 daltons (hexamers) (Boussiba and Richmond, 1979). They function as light-absorbing substances together with chlorophyll during photosynthesis. An example of red algae of commercial relevance is the nori or Porphyra. It is consumed as food and is used as wraps for sushi and in several other Japanese dishes. The two pigments are produced commercially from the blue-green algae Spirulina platensis. Spirulina grows well in warmer climates and warm alkaline waters. There are several Spirulina species, although Spirulina platensis and Spirulina maxima are the best known. The former is cultivated in California while S. maxima are cultivated in Mexico. Phycocyanin and related compounds found in Spirulina are believed to have antiviral and anticancer properties as well as the ability to stimulate the immune system, and also promote formation and development of red blood cells, and thereby curtail the incidence of anemia (Jensen and Ginsberg, 2000; Jensen et al., 2001; Mani et al., 2000; Mathew et al., 1995; Samuels et al., 2002; Shih et al., 2003). They are also thought to promote the development of healthy skin. Thus, it is used to treat skin disorders like eczema and psoriasis. Phycocyanin and phycoerythrin are found together with carotenoids and chlorophylls in the blue-green and red algae. The red pigment, phycoerythrin, in particular, is believed to facilitate red seaweed subsistence at greater depths in the ocean where hydrostatic pressures are high, unlike the other seaweed species (e.g., brown and green algae) that can thrive only in shallow waters. Studies with various experimental animals have demonstrated the potent antioxidant, free radical scavenging and anti-inflammatory properties of phycocyanin, as well as antiviral activity of the pigment against herpes simplex and anti-arthritic effects (Bhat and Madyastha, 2000). Some other studies have also revealed protective effects by phycocyanin against neuronal damage and oxidative damage to DNA (Bhat and Madyastha, 2001; PinÄero Estrada et al., 2001). Phycocyanin is used as a natural coloring agent in several food products including dairy products (yoghurts, milk shakes and ice creams), alcoholic and

Pigments from by-products of seafood processing

425

non-alcoholic beverages, desserts, and also in cosmetic products. The phycoerythrin pigment is one of the brightest dyes and is commonly used as fluorescent dyes for FACS analysis (Hardy, 1986). Phycocyanin is used in medicine to facilitate selective destruction of atherosclerotic plaques or cancer cells by radiation with little or no damage to the surrounding cells or tissue (Morcos and Henry, 1989). 19.4.3 Melanins Melanins are partly responsible for the dark brown and black colorations found in aquatic species. Melanins are formed from the amino acid, tyrosine, and phenolic compounds by enzymatic oxidation through a series of intermediates like dihydroxyphenolics and quinones, followed by polymerization to form the large molecular weight melanins. Melanins occur in the eyes, skins and peritoneal lining of these species, and they play the role of protecting tissues against light/UV radiation, among others. In particular, species living in clear, shallow waters are exposed to the damaging effects of ultraviolet radiation from the Sun, and the melanins these species are endowed with help to curtail such damaging UV effects. Damage from UV radiation may come about in various ways, such as thymine dimerization in DNA, or via the effects of reactive oxygen species such as singlet oxygen and superoxide radical (Jagger, 1985; Tyrrell, 1991). The capacity of aquatic species to subsist in these habitats suggests that they have the mechanism(s) to protect themselves against the adverse effects of the radiation from the Sun. This protective ability derives from naturally present melanins or melanin-like substances present in these species. Simple life forms such as algae and cyanobacteria make these melanin-type compounds as part of their normal metabolism, and these compounds subsequently pass along the food chain to provide similar protective effects in higher forms of aquatic life. The enzymes responsible for the oxidation steps are the phenolases or phenoloxidases. In crustacean species such as shrimp, prawn, crab and lobster, the formation of melanins is referred to as melanosis or `blackspot' formation. Although these `blackspots' formed on the animals are not toxic, consumers nevertheless find them unappealing. The `blackspot' phenomenon in crustaceans has been extensively studied by various researchers with the goal to curtail this undesirable effect of polyphenoloxidases in these animals (Benjakul et al., 2005; Chen et al., 1993; Ferrer et al., 1989; Ogawa et al., 1984; Yan et al., 1989). Nevertheless, these same melanins may be recovered and put to very good use. For instance, in humans melanins protect against skin damage from UV radiation from sunlight and also minimize glare within the eyes. This property of melanin has been exploited in making superior quality sun lenses that better filter colors to reduce their damaging effects, and thus mitigate the risks of macular degeneration and cataracts. A company in the US, PhotoProtective Technologies, produces melanins for incorporation in a myriad of lenses including sunglasses, reading glasses, computer glasses, pilot

426

Maximising the value of marine by-products

glasses and other special purpose glasses (Anon, 2005). Melanin has also been incorporated in a formula known as Melancor-NH for reducing gray hair in men and women (www.911healthshop.com/melancornh.html).

19.5

Economic, environmental, and safety considerations

Agricultural harvesting and processing lead to the production of large quantities of residual materials that are either underutilized or not utilized at all. Invariably such unutilized material ends up as waste, polluting the environment. For instance, most commercial shrimp processing operations entail semimechanized peeling operations to remove the heads, viscera, carapace and legs. These parts account for between 70 and 80% of the whole animal and are commonly not consumed in certain cultures or communities, and end up as a major waste disposal problem. Although there are efforts by several companies to make products like chitin, chitosan, glucosamine, etc., from crustacean waste, there is still a large bulk of it that is dumped as waste. Because of growing concerns for environmental health and safety, stringent environment regulations have been promulgated aimed at curtailing the dumping of such processing discards back into the ocean or in landfills. So in some countries like Canada, disposal of agricultural harvesting and processing waste is becoming quite costly. This high waste disposal cost is making processors more amenable to the idea of converting processing discards into profitable by-products. Fortunately, these processing discards have high levels of useful nutrients and other bioingredients that may be recovered by relatively simple procedures to increase profits. As alluded to elsewhere in this chapter, animals such as salmonids and crustaceans have a demanding requirement for carotenoids for several functions, yet are incapable of making these compounds on their own from scratch. Apart from the normal metabolic functions of these pigments in the live animal, these compounds also impart colors to foodstuffs ± for example, the reddish orange colors associated with salmonids are due to the carotenoid pigments derived from the diet (Fox, 1957). Consumers have come to associate these animals with these colors, and in some products (like salmon, trout, crab, lobster and shrimp), these colors are used (rightly or wrongly) as one of the measures of quality and acceptability. For instance, the market value of prawn is determined largely on the visual appeal of its body color (Lorenz, 1998). For some time now, the wild salmon harvest has been in steady decline. The annual wild salmon harvest worldwide is estimated at less than a million tonnes, while the world production of farmed salmon has been increasing for the same period. The 1999 harvest of farmed salmon was greater than 750 000 metric tonnes, and this figure is projected to reach about 1.3 million tonnes for 2005 (Lorenz and Cysewski, 2000). For cultured salmonids, carotenoid pigments in either the `natural' or synthetic forms, are incorporated in the diet to impart the desired flesh color. According to Lorenz and Cysewski (2000), more than 95%

Pigments from by-products of seafood processing

427

of the carotenoid pigments used to supplement aquaculture diets is synthetic. Because of consumer aversion to the use of synthetic products in foodstuffs, fish farmers desire to use carotenoid pigments from `natural' sources. Furthermore, the synthetic colorants currently available for use as feed supplements are quite expensive and constitute from 40 to 60% of the total operating costs in intensive aquaculture operations (Meyers, 1994). Synthetic astaxanthin, for example, retails for about US$2,500 per kilogram, and the global market for astaxanthin was estimated in the year 2000 at US$200 million (Lorenz and Cysewski, 2000). Thus, there is great interest in crustacean processing discards and microorganisms (e.g. Phaffia yeast and the Haematococcus microalgae) as cheaper sources of carotenoid pigments for food, feed and health use; and for various reasons such as their being perceived as more `natural', putting discards from the crustacean harvest to profitable use, as well as reducing environmental pollution. Some of the factors that augur well for crustacean waste versus microorganisms as `natural' source of carotenoid pigments include: · the pressure on the fishing industry by environmentalists and regulatory agencies to convert the discards into high value products instead of simply dumping them into the environment to aggravate the pollution problem · the added profits that could accrue to the fishing industry from recovering useful bioingredients from these sources for use in human food, nutraceuticals and animal feed · the relatively high production cost in producing the pigments from microalgae (e.g., Haematococcus pluvialis) and yeast (Phaffia rhodozyma) that is estimated at about US$5 to US$20 per kg dry weight (Lorenz and Cysewski, 2000) · the presence of tough cell walls in microalgae and yeast that could limit the bioavailability of carotenoid pigments from these sources (Burcyk, 1987). Crustacean offal has been used in both the fresh and frozen forms in aquaculture diets (Saito and Regier, 1971). Other forms in which crustacean offal have been used include its processing into meals to reduce bulkiness prior to incorporation in aquaculture feeds (Spinelli et al., 1974); differential screening of the crustacean meal to exclude chitin and calcium before feeding it to farmed animals (Rutledge, 1971); acid ensilage of the offal (Torrissen et al., 1981); stripping the carotenoid pigments from the crustacean offal with soy oil for incorporation in aquaculture rations (Meyers and Chen, 1982a); or by coextraction of the carotenoids as pigment-protein (carotenoprotein) complexes (Cano-Lopez et al., 1987; Simpson and Haard, 1985).

19.6

Future trends

This discussion has mentioned various sources of natural pigments. Carotenoid pigments from crustacean offal were treated at some length compared with the

428

Maximising the value of marine by-products

other natural pigment sources because of their importance as antioxidants, and as colorants for food and farmed fish. Various algae such as chlorophyll, fucoxanthin, phycocyanin and phycoerythrin were mentioned as sources of pigments. So far, the foremost use of algae or seaweeds is as source material for (food) hydrocolloids such as agar, alginates and carrageenan for food and cosmetic uses. The recovery of these ingredients is carried out without much regard for the pigments present with the result that the bulk of these pigments are wasted. This neglect is bound to change with the growing awareness of the health benefits that could be derived from these pigments, as well as their uses to color food, cosmetic and related products. This is especially so given that pigments from `natural' sources are gaining in importance over their synthetic counterparts, as the former are perceived to be relatively non-toxic and noncarcinogenic. The literature is also replete with conflicting reports on the effects of degree of esterification of carotenoid pigments and their uptake by fish flesh. More studies aimed at clarifying this situation would help to improve feed supplementation/formulation to assure better bioavailability of the pigments. Several companies based on bioingredients or by-products from marine algae are sprouting all over the world and are all touting the spectacular health benefits of these products. When it comes to foods or ingredients that we ingest through our mouths into our bodies, most consumers see `natural' products as better and/ or safer. Nevertheless, it is known that excessive intake of iodine from highiodine-content macro algae can upset thyroid function (Teas et al., 2004). Some consumers are wary about heavy metal levels in their foods, and these toxicants may either be absorbed by (or accumulated in) some of these seaweeds, or coextracted with useful bioingredients from the seaweeds (Ethus, 2003). Furthermore, with the continued disposal of waste (including sewage) into oceans and landfills (that may seep into rivers, streams and oceans), and the incidence of nuclear accidents, there are justifiable concerns about the possible contamination of these `natural' bioingredients or by-products with environmental toxicants and hazardous radioactive fallout materials (Barnaby and Boeker, 1999). Thus far, food and health regulatory agencies such as the US Food and Drug Administration and Health and Welfare Canada do not stringently regulate seaweeds and similar herbaceous products used as dietary supplements. Thus, aspects such as levels of incorporation, purity and safety, as well as the interactive effects of these products with various prescription drugs need to be verified by more research.

19.7

Sources of further information and advice

(1981), Carotenoids as Colorant and Vitamin A Precursors ± Technological and Nutritional Applications, New York, Academic Press. HENDRY G A F and HOUGHTON J D (1996), Natural Food Colorants, London, Blackie Academic & Professional. BAUERNFEIND J C

Pigments from by-products of seafood processing

429

(1998), Carotenoids, Carey, NC, IARC. JOHNSON I and WILLIAMSON G (2003), Phytochemical Functional Foods, Boca Raton, CRC Press. MESKIN M S, BIDLACK W R, DAVIES, A J and OMAYE S T (2002), Phytochemicals in Nutrition and Health, Boca Raton, CRC Press. IARC WORKING GROUP ON THE EVALUATION OF CANCER-PREVENTIVE AGENTS

19.8

References

(2005), 1996±2005, MelaninProducts.com ± PhotoProtective Technologies, Inc. and RUPRECHT R (1996), 7th IAAA Conference, Knysna, South Africa April 17, Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis). BARNABY F and BOEKER E (1999), `Is technetium-99 (Tc-99) radiologically significant?' Med Confl Surviv. 15, 57±70. BENJAKUL S, VISESSANGUAN W and TANAKA M (2005), `Properties of phenoloxidase isolated from the cephalothorax of kuruma prawn (Penaeus japonicus)', J. Food Biochem, 29, 470±485. BHAT V B and MADYASTHA K M (2000), `C-Phycocyanin: A potent peroxyl radical scavenger in vivo and in vitro', Biochem Biophys Res Comm, 275, 20±25. BHAT V B and MADYASTHA K M (2001), `Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: protection against oxidative damage to DNA', Biochem Biophys Res Commun, 286, 262±266. BJERKENG B (2000), `Carotenoid pigmentation of salmonid fishes ± recent progress'. In: Avances en NutricioÂn AcuõÂcola V. Memorias del V Simposium Internacional de NutricioÂn AcuõÂcola. Cruz -SuaÂrez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A. y Civera-Cerecedo, R. (eds). 19±22 Nov, 2000. MeÂrida, YucataÂn. BLIGH D (1978), `Separation, identification, and biochemical degradation of the carotenoid pigments of Louisiana crawfish processing waste', M.Sc. thesis, Louisiana State University, Baton Rouge, LA. BOUSSIBA S and RICHMOND A E (1979), `Isolation and characterisation of phycocyanins from the blue-green alga Spirulina platensis', Arch Microbiol, 120, 155±159. BOWERS W F (1947), `Chlorophyll in wound healing and suppurative disease', Am J Surg 71, 37±50. BREITHAUPT D E (2004), `Identification and quantification of astaxanthin esters in shrimp (Pandalus borealis) and in a Microalga (Haematococcos pluvialis) by liquid chromatography-mass spectrometry using negative ion atmospheric pressure chemical ionization', J Agric Food Chem, 52, 3870±3875. BRITTON G (1996), `Carotenoids', in Hendry G A F and Houghton J D, Natural Food Colorants, 2nd edition, London, Blackie Academic & Professional, 197±243. BURCYZK J (1987), `Biogenetic relationships between ketocarotenoids and sporopollenins in green algae', Phytochemistry, 26, 113±119. CANO-LOPEZ A, SIMPSON B K and HAARD N F (1987), `Extraction of carotenoprotein from shrimp process waste with the aid of trypsin from Atlantic cod', J Food Sci, 52, 503±506. CHEN J S, BALABAN M O, WEI C I, GLEESON R A and MARSHALL M R (1993), `Effect of carbon ANON

AYEHUNIE S, BELAY A, HU Y, BABA T

430

Maximising the value of marine by-products

dioxide on the inactivation of Florida spiny lobster polyphenol oxidase', J Sci Food & Agric, 61, 253±259. CHOUBERT G and HEINRICH O (1993), `Carotenoid pigments of the green alga Haematococcus pluvialis: assay on rainbow trout, Oncorhynchus mykiss, pigmentation in comparison with synthetic astaxanthin and canthaxanthin', Aquaculture, 112, 217± 226. COLIO L G and BABB V (1948), `Study of a new stimulatory growth factor', J Biol Chem, 174, 405±409. ETHUS E (2003), `Arsenic essentiality: A role affecting methionine metabolism', J Trace Elem Exp Med, 16, 345±355. FERRER O J, KOBURGER J A, SIMPSON B K, GLEESON R A and MARSHALL M R (1989), `Phenoloxidase levels in Florida spiny lobster (Panulirus argus): relationship to season and molting stage', Comp Biochem Physiol, 93B, 595±599. FINN E (2004), `Shrimp carotenoprotein as a pigment source for brook trout (Salvelinus fontinalis)'. Final report. Advance Diploma in Sustainable Aquaculture, Marine Institute, Memorial University of Newfoundland, St. John's, NL, Canada FOX D L (1957), `The pigments of fishes', in Brown M E, Physiology of Fishes, New York, Academic Press, 2, 367±385. FRASER P D and BRAMLEY P M (2004), `The biosynthesis and nutritional uses of carotenoids', Prog Lipid Res, 43, 228±265. HADLEY C W, CLINTON S K and SCHWARTZ S J (2003), `The consumption of processed tomato products enhances plasma lycopene concentrations in association with a reduced lipoprotein sensitivity to oxidative damage', J Nutr, 133, 727±732. HARDY R R (1986), `Purification and coupling of fluorescent proteins for use in flow cytometry'. In: Handbook of Experimental Immunology, 4th edn. D M Weir, C Blackwell and L A Herzenberg, eds. Blackwell Scientific Publications, Boston, pp. 31.1±31.12. HARKER M, TSAVALOS A J and YOUNG A J (1996), `Factors responsible for astaxanthin formation in the chlorophyte Haematococcus pluvialis', Biores Technol, 55, 207± 214. HOSOKAWA M, KUDO M, MAEDA H, KOHNO H, TANAKA T and MIYASHITA K (2004), `Fucoxanthin induces apoptosis and enhances the antiproliferative effect of the PPAR ligand, troglitazone, on colon cancer cells', Biochim Biophys Acta 1675, 113±119. JAGGER J (1985), Solar UV Actions on Living Cells. New York, Praeger. JENSEN G S and GINSBERG D I (2000), `Consumption of Aphanizomenon flos aquae has rapid effects on the circulation and function of immune cells in humans', J Amer Nutraceut Assoc, 2, 50±58. JENSEN G S, GINSBERG D I and DRAPEAU C (2001), `Blue-green algae as an immunoenhancer and biomodulator', J Amer Nutraceut Assoc 3, 24±30. JOHNSON E A and AN G H (1991), `Astaxanthin from microbial sources', Crit Rev Biotechnol, 11, 297±326. KASAHARA H, HANADA A, KUZUYAMA T, TAKAGI M, KAMIYA Y and YAMAGUCHI S (2002), `Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis', J Biol Chem, 277, 45188±45199. KOTAKE-NARA E, TERASAKI M and NAGAO A (2005), `Characterization of apoptosis induced by fucoxanthin in human promyelocytic leukemia cells', Biosci Biotechnol Biochem, 69, 224±227. LORENZ R T (1998), `A review of carotenoid, astaxanthin, as a pigment and vitamin source for cultured Penaeus prawn', NatuRoseTM Technical Bulletin #051. Cyanotech

Pigments from by-products of seafood processing

431

Corporation. November 24. and CYSEWSKI G R (2000), `Commercial potential for Haematococcus microalgae as a as a natural source of astaxanthin', TIBTECH, 18, 160±167. MANI UV, DESAI S and IYER U (2000), `Studies on the long-term effect of spirulina supplementation on serum lipid profile and glycated proteins in NIDDM patients', J Nutraceut, 2, 25±32. MATHEW B, SANKARANARAYANAN R, NAIR PP, et al. (1995), `Evaluation of chemoprevention of oral cancer with Spirulina fusiformis', Nutr Cancer, 24,197±202. MEYERS S P (1994), `Developments in world aquaculture, feed formulations, and the role of carotenoids', Pure & Appl Chem, 66, 1069±1076. MEYERS S P and CHEN H-M (1982a), `Extraction of astaxanthin pigment from crawfish waste using a soy oil process', J Food Sci, 47, 892±896, 900. MEYERS S P and CHEN H-M (1982b), `Effect of antioxidants on stability of astaxanthin pigment from crawfish waste', J Agric Food Chem, 30, 469±473. MORCOS N C and HENRY W L (1989), Medical uses of phycocyanin, US Patent 4886831. LORENZ R T

NEGRE-SADARGUES G, CASTILLO R, PETIT H, SANCE S, MARTINEZ R G, MILICUA J, CHOUBERT G

and TRILLES J (1993), `Utilization of synthetic carotenoids by the prawn Penaeus japonicus reared under laboratory conditions', Aquaculture, 110, 151±159. NGUYEN T T D, SIMPSON B K, RIDEOUT K and ALLEN N (2003), `Feeding shrimp carotenoprotein to rainbow trout', Ann Meeting of the Pacific Fish Technol, February 2003, Astoria, OR. OFFENKRANTZ W (1950), `Water-soluble chlorophyll in ulcers of long duration'. Review of Gastroenterology, 17, 359±367. OGAWA M, PERDIGAÄO N B, SANTIAGO M E and KOZIMA T T (1984), `On physiological aspects of blackspot appearance in shrimp', Nip Sui Gakkai, 50, 1763±1769. PARAJO J C, SANTOS V and VAZQUEZ M (1998), `Production of carotenoids by Phaffia rhodozyma growing in media made from hemicellulosic hydrolysates of Eucalyptus globulus wood', Biotechnol Bioeng, 59, 501±506. PATEK A (1936), `Chlorophyll and regeneration of blood', Arch Int Med, 57, 73±84. PETERSON D H, JAGER H K and SAVAGE G M (1966), `Natural coloration of salmonids using xanthophylls', Trans Am Fish Soc, 35, 408±415. Â S P and VILLAR DEL FRESNO A M (2001), `Antioxidant Ä ERO ESTRADA J E, BERMEJO BESCO PIN activity of different fractions of Spirulina platensis protean extract Il', Farmaco. 56, 5±7 & 497±500. RENSTROM B and LIAAEN-JENSEN S (1981), `Fatty acid composition of some esterified carotenoids', Comp Biochem Physiol, 69B, 625±627. Â N M and BORONAT A (2002), `Elucidation of the methylerythritol RODRIÂQUEZ-CONCEPCIO phosphate pathway for isoprenoid biosynthesis in bacteria and plastids, Plant Physiol, 130, 1079±1089. RUTHLEDGE J E (1971), `Decalcification of crustacean meals', J Agric Food Chem, 31, 236±237. SAITO A and REGIER L W (1971), `Pigmentation of brook trout (Salvelinus fontinalis) by feeding dried crustacean waste', J Fish Res Bd Canada, 28, 509±512. SAMUELS R, MANI U V, IYER U M and NAYAK U S (2002), `Hypocholesterolemic effect of spirulina in patients with hyperlipidemic nephrotic syndrome', J Med Food, 5, 91± 96. SHAHIDI F and SYNOWIECKI J (1991), `Isolation and characterization of nutrients and valueadded products from snow crab (Chinoecetes opilio) and shrimp (Pandalus borealis) processing discards', J Agric Food Chem, 39, 1527±1532.

432

Maximising the value of marine by-products

et al. (2003), `Inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis', J Med Virol 70, 119±125. SIMPSON B K and HAARD N F (1985), `The use of proteolytic enzymes to extract carotenoproteins from shrimp waste', J Applied Biochem, 7, 212±222. SPINELLI J, LEHMAN L and WIEG D (1974), `Composition, processing and utilisation of red crab (Pleuroncodes planipes) as an aquaculture feed ingredient', J Fish Res Bd Canada, 31, 1025±1029. STOREBAKKEN T, FOSS P, SCHEIDT K, AUSTRENG E, JENSEN S-L and MANZ U (1987), `Carotenoids in the diets for salmonids IV. Pigmentation of Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin', Aquaculture, 65, 279± 292. TAKAICHI S, MATSUI K, NAKAMURA M, MURAMATSU M and HANADA S (2003), `Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry', Comp Biochem Physiol, 136B, 317±322. TANAKA Y, MATSUGUCHI H, KATAYAMA T, SIMPSON K L and CHICHESTER C O (1976), `The biosynthesis of astaxanthin ± XVI. The carotenoids in crustacea', Comp Biochem Physiol, 54B, 391±393. TEAS J, PINO S, CRITCHLEY A and BRAVERMAN L E (2004), `Variability of iodine content in common commercially available edible seaweeds', Thyroid, 14, 836±841. TORRISSEN O, TIDEMANN E , HANSEN F and RAA J (1981), `Ensiling in acid ± a method to stabilize astaxanthin in shrimp processing and improve uptake of this pigment by rainbow trout (Salmo gairdneri)', Aquaculture, 26, 77±83. TYRRELL R M (1991), UVA (320±380 nm) as an oxidative stress. In Oxidative Stress: Oxidants and Antioxidants, ed. Sies H, pp. 57±83, San Diego, Academic. WHITE D A, MOODY A J, SERWARA R D, BOWEN J, SOUTAR C, YOUNG A J and DAVIES S J (2003), `The degree of carotenoid esterification influences the absorption of astaxanthin in rainbow trout, Oncorhynchus mykiss (Walbaum)', Aquaculture Nutr, 9, 247±251. www.911healthshop.com/melancornh.html. Melancor NH Hair Color Restorer & Rejuvenator. YAMAGUCHI K, MIKI W, TORIU N, KONDO Y, MURAKAMI M, KONSU S, SATAKE M and FUJITA T (1983), `The composition carotenoid pigments in the Antarctic Krill Euphasia superba', Nip Suis Gakkai, 49, 1411±1415. YAN X, TAYLOR K DA and HANSON S W (1989). `Studies on the mechanism of blackspot development in Norway lobster (Nephrops norvegicus)', Food Chem, 34, 273±283. YUAN J-P, GONG X-D and CHEN F (1996), `Separation and identification of astaxanthin esters and chlorophylls in Haematococcus lacustris by HPLC', Biotech Tech, 10, 655± 660. SHIH S R, TSAI K N, LI Y S,

Part III Non-food uses of marine by-products

20 By-products from seafood processing for aquaculture and animal feeds P. J. Bechtel, University of Alaska Fairbanks, USA

20.1

Introduction

There is a rich history associated with fish by-product utilization both abroad and in the United States of America. The classic reference in this area is the 1981 book Introduction to Fishery By-products by Windsor and Barlow (1981). In the USA, research in this area was conducted by the National Marine Fisheries Service at their Charleston, South Carolina Laboratory and focused on products from the menhaden fishery until the program area was phased out. The interesting history of Alaska marine by-products production and utilization from 1882 through 1989 was compiled by Meehan et al. (1990). Currently in Alaska there are a number of on-shore fish meal plants located close to large fish processing operations. These plants produce fish meals and oils from byproducts of fish processed for human consumption. Fish processing by-products refer to tissues that remain after much of the fish muscle has been removed and include heads, frames, viscera, and skin, among others. Edible parts such as heads, milt, and stomachs are on occasion collected and sold and some fish skin is made into gelatin or fish leather. By-products can be used to make fertilizer and other products; however, most of the fish byproducts produced in large shore-side fish processing operations are used to make fish meal and fish oil. Primary uses of fish meals and oils are as aquaculture feed ingredients for fish and shrimp, and as livestock and poultry feed ingredients. This chapter deals with making aquaculture and animal feed ingredients from fish processing by-products and focuses on by-products currently produced from marine finfish. The first section deals with the rationale for

436

Maximising the value of marine by-products

making feed ingredients from processing by-products. The second section deals with different by-product components obtained from the processing of finfish. The third and fourth sections briefly describe the process used to produce protein meals, solubles and oils from fish processing by-products and the production of hydrolysates and silages. This is followed by a discussion of the nutritional value and issues associated with feed ingredients made from fish processing by-products.

20.2

Driving forces for utilization of by-products

Ocean fish are used for human consumption as well as the production of feed ingredients for aquaculture and livestock. The world fish harvest from the oceans has been stagnant or in decline for a number of years. There is concern over the availability of high quality fish meal and oil for the world aquaculture industry. Hardy and Tacon (2002) have stated that the annual fish meal production has been more or less constant over the past 15 years at approximately 6.2 million metric tons (MMT). However, the proportion of global fish meal production utilized in feeds for farmed aquatic species has increased dramatically, because of the worldwide growth of aquaculture. Aquaculture is predicted to expand two to three fold over the next decades and create a large need for aquaculture feed ingredients (Hardy and Tacon, 2002). Most fish meal and fish oil are produced from the harvest of whole fish with high oil contents such as anchovy, capelin, and menhaden and less than 10% comes from white fish offal (Pike and Barlow, 1999). An additional supply of fish meal and oil can be recovered from seafood processing by-products. New (1996) has estimated that if seafood processing by-products and by-catch were converted into fish meal, the supply would be equivalent to a significant portion of the current global fish meal production. There can be practical problems in utilizing seafood processing by-products to make meals and oils and these can include seasonal availability, large volumes of by-products during a limited number of processing days, and remote fish processing locations. Also, because seafood by-products differ from whole fish in composition, due to the removal of much of the muscle flesh, modified production methods are required to make products that can compete with traditional fish meal made from whole fish. If aquaculture growth continues and supplies of fish meal and oil remain constant, fish meal and fish oil in aquaculture feeds will shift from being primary sources of protein and energy to being specialty products (Hardy, 2003). This trend is already underway for many aquaculture species. Federal and state regulations such as the Magnuson-Stevens Fishery and Conservation Act of 1996, The American Fisheries Act of 1998, and the permits required for discharging waste of on-shore processing plants, are encouraging the utilization of fish processing by-products. Producing fish meal is often the most economical solution for handling fish wastes produced from on-shore

By-products from seafood processing for aquaculture and animal feeds 437 processors. Without the development of effective means in dealing with fish byproducts, the fish processing industry will face increased costs associated with running their businesses. As an example of the potential to utilize fish processing by-products to make feed ingredients, Crapo and Bechtel (2003) estimated the total amount of Alaska by-products produced on a dry matter basis in 2000 was 208 599 MMT and that about 40% of the solids were reported to be recovered as fish meal and fish oil. On-shore processors recovered over 60% of the solids from their by-products, while catcher-processors recovered less. Increased utilization of fish wastes would allow for the production of more aquaculture fish feeds without increasing the harvest from the oceans. Other benefits include a cleaner environment and greater resource utilization.

20.3

By-product components

Substantial amounts of seafood by-products are available for processing into feed ingredients and for other products. The largest amount of these byproducts are from marine finfish and include viscera, frames, heads, skin and fins, among others. In addition, there are by-products available from squid, shrimp, crab, and other seafood industries that are used to make feed ingredients that have unique properties. Given the proper economic incentive it could be desirable to collect selected fish processing by-product components (e.g., viscera, heads, milt, liver) to make high quality feed ingredients that would command a premium price. Obtaining precise numbers for the amount of waste and by-products produced is difficult due to the proprietary nature and competitive aspects of the industry. The volume of processing by-products varies significantly by species, type of processing, and many other variables such as the time of year. Examples of variation in the percentage of by-product produced are 90% from fish harvested only for their roe to 30% for fish that are simply eviscerated-headed and frozen for the markets. As a broad generality the overall amount and quality of available fish processing by-products is being reduced as improved fish processing technology is developed, which removes more of the muscle tissue and leaves an increasing percentage of bone behind (Babbit and Stevens, 1996; Kelleher and Hultin, 2000, Gildberg et al., 2002). In addition, new technologies are being developed that increase the utilization of fish such as the Arrowtooth flounder and other species (Crapo et al., 1999). A broad guideline for recoveries and yields of common pacific fish and shellfish has been compiled (Crapo et al., 1993). There are good estimates of the harvest from different fisheries; however, there is less data on what happens to the fish wastes after processing. When fish by-products from food processing lines are made into fish meals and oils, much of the raw material is initially of the higher quality, e.g. human food grade. Advantages of seafood by-product derived from processing fish for human consumption include:

438 1. 2.

Maximising the value of marine by-products fresh food grade seafood by-products are available at the time of processing, because seafood quality must be maintained from the time of harvest through processing to meet edible food standards separate components can be obtained from the mechanical processing of these fish (e.g., heads removed first followed by viscera, frames, and skin during processing of boneless fillets).

Rather than mix by-products together and make a protein meal from all the byproducts, an active research area is the development of specialty feed ingredients and products from variety parts including skin, trim, frames, heads, viscera, liver, and milt, among others. There are chemical and biochemical differences in whole fish composition between fish species (Stansby, 1976; Krzynowek et al., 1989). This translates into species differences in fish waste composition and by-products made from wastes. There has been little evaluation of fish processing by-products such as heads, frames, viscera and skins beyond proximate analysis. The chemical composition of parts such as heads, viscera, frames and skin from a number of species has been determined (Gunasekera et al., 2002; Dong et al., 1993; Freeman and Hoogland, 1956; Kizevetter, 1971; Krzynowek et al., 1989; Montero et al., 1991; Nagai and Suzuki, 2000; Stansby, 1976; Olley et al., 1968; Bechtel, 2003; Bechtel and Johnson, 2004; Oliveira and Bechtel, 2005).

20.4 Overview of different products produced from fish by-products 20.4.1 Fish meal In the production of fish meal from the by-products of seafood processing, most large plants employ a wet reduction process (Hardy, 1992; Babbitt et al., 1994). This process includes cooking, pressing, and drying operations. The first step in the process of making fish meal involves heating the fish and/or fish waste to 95± 100ëC, which denatures the protein and facilitates the separation of oil and liquid from the solids. The cooked material is then passed through a press to produce both a press cake and a liquid fraction. The press cake is dried to less than 10% water and the resultant fish meal is milled to the desired particle size and usually stored and transported in bulk containers. The liquid, which contains the aqueous phase and associated water soluble proteins plus the fish oil, is usually passed through a centrifuge to separate the fish oil from the aqueous phase (stick water). Stick water contains substantial amounts of soluble protein, which is concentrated and added back to the press cake. Proteolytic enzymes are added during the concentration step to decrease viscosity and improve evaporator efficiency. Concentrated stick water can be added back to the press cake before drying or dried separately and sold as fish solubles. Marine fish lipids have a high degree of unsaturation and in the presence of oxygen will oxidize and reduce the quality and palatability of fish meal. An antioxidant such as ethoxyquin is added to many meals at levels of 130±150 ppm in the finished product (Hardy, 1998).

By-products from seafood processing for aquaculture and animal feeds 439 Most fish meal processing technologies have been developed for use with high-oil whole fish from the large industrial fisheries. Adaptations that have been devised for making white fish by-product meals include the potential for eliminating oil recovery (depending on the percentage lipid of the specific fish by-product) and the removal of bone fragments to decrease the ash content in the final product. In order to make fish meals with 65% protein and less than 20% ash from fish processing wastes, it has often been necessary to remove some of the bone from the dried meal. This is usually accomplished by using a gyrating sieve separator that removes bone and other fragments in the meal. This method has been successfully employed in a number of plants in Alaska. Another method of reducing ash content in the final product is to remove bone from the raw fish waste using a mechanical bone remover (Rathbone et al., 2001). Although the general operations for meal manufacture have changed little over the past 30 years, there have been improvements in the process conditions and equipment resulting in increased efficiencies and better quality products (see review by Tarr, 1982). Improved meal quality resulted from replacing direct drying of fish meal with indirect steam drying. Additional quality improvements have been made when low temperature (LT) drying had been employed, which results in products with higher digestibility than steam dried meals. Other advancements have been made in the development of more efficient evaporators for concentration of stick water. An alternative to using the press to separate solid and liquid fractions after the initial cooking step is to substitutes the use of a decanter type centrifuge for the separation (Windsor and Barlow, 1981). Over the years, fish meal quality has continued to improve due to a number of factors including an emphasis on raw material quality, and low-temperature drying. Recognized premium quality fish meals with high protein and low ash are now produced that have both manufacturing and product specifications. One example is the Norwegian reference meal (Norse-LT 94), which is approximately 74% protein, 11±13% fat, 10% ash and 7% moisture. Products that increase feed efficiency and have higher protein content generally have a higher market value. The importance of raw material freshness and low temperature processing have been documented in growth trials with fast-growing salmon, halibut, sea bream and Penaeus shrimp. Freshness in the raw material can be monitored using total volatile nitrogen, content of the main biogenic amines (histamine, cadaverine, putrescine and tyramine) and free fatty acid content. Koning (1999) and Hardy and Dong (1995) provided insight into the measurement of quality in fish meals and guidelines for the consistent production of high quality feed ingredients starting from the quality and composition of the raw materials to the storage properties of the feed ingredient. 20.4.2 Fish oil and bone From a historical perspective fish oil has been an important commodity with food, feed, fuel, and industrial applications. Processing of fish oils has been

440

Maximising the value of marine by-products

described by Bimbo (1990) and a host of others. Global fish oil production in 2000 exceeded 1.3 MMT and around half the production was projected for use in aquaculture diets. The requirements for the long chain omega-3 fatty acids in marine and freshwater aquaculture diets have been reviewed by Sargent and Tacon (1999). In spite of the importance of fish oil in aquaculture diets the price of fish oil remains relatively low. Refined fish oils from a variety of fish species including salmon are currently available in world markets. To make many fish meals produced from by-products competitive in the market place it has been necessary to remove bone in order to reduce the ash content below 20%. Removing the bone results in a bone by-product that is useful as a fertilizer soil amendment, and as a supplement in animal feeds; however, fish bone meal has a relatively low value. The profitability of a marginally-valued commodity can be a problem when transportation from remote areas is involved. A potential solution lies in the development of new, high-value uses for bonederived products. If organic certification can be obtained there may be additional markets for the product. The composition of fish bone meal depends on the species and the process used to clean the bone. However, the ash content of dried fish bone is high, often in the range of 40 to 55%. 20.4.3 Solubles The stick water fraction is usually 50±70% of the initial weight of the raw material and contains approximately 5±10% solids, most of which is protein. A limited number of studies have focused on concentration of stick water (Gaude, 1994; Valle, 1990). When stick water is concentrated and added to the fish meal press cake during drying, it can account for 20% or more of the solids in the meal (Pedersen L D et al., 2003; Soares et al., 1973; Ammu et al., 1986; Zarkadas et al., 1986). When stick water is concentrated and sold separately it is referred to as fish solubles. The price of fish solubles has been significantly below that of fish meal; therefore, it has been economically advantageous to sell the fish solubles as fish meal. This is often done by concentrating the fish solubles to 30±45% solids and then adding the concentrated solubles to press cake and drying to the desired endpoint. For some feed applications it is desirable to have a defined soluble protein content, which can be achieved with the inclusion of concentrated stick water in fish meal. A major problem is that the concentration of stick water traditionally requires a lot of energy-expensive evaporation systems. There are few studies on the biochemical characterization of stick water, although this soluble fraction should contain a rich assortment of biomolecules. Bechtel (2005) has reported that dried stick water contains substantial amounts of hydroxyproline, an amino acid abundant in connective tissue. 20.4.4 Protein powders Most protein powders and specialized protein ingredients will be produced for human use; however, there are some products such as insoluble protein fractions

By-products from seafood processing for aquaculture and animal feeds 441 that can be used as aquaculture, farm animal and pet animal feed ingredients. Protein powders have been made from fish by-products by Sathivel et al. (2004), in a process that involves grinding, heating to denature enzymes and release lipid, sieving to remove bone and large tissue fragments, centrifugation to remove lipid, and drying of both the soluble (supernatant) and insoluble protein fractions (pellet). Another method of extracting and concentrating protein from by-products (e.g., heads, frames, etc.) involves using pH extraction and isoelectric precipitation (Underland et al., 2002; Kristinsson and Demir, 2003; Choi and Park, 2002). In this process the ground tissue is mixed with water and the pH adjusted to an alkaline or acid pH to solubilize the protein. Bone and other unsolubilized materials are removed by centrifugation and then the pH is adjusted to 5.5, to precipitate the protein, which is collected by centrifugation.

20.5

Methods of producing hydrolysates and silage

An alternative to making fishmeal from seafood processing wastes is to make fish hydrolysates (Kristinsson and Rasco, 2000; Hardy, 1992; Raa and Gildberg, 1982; Sathivel et al., 2003; Shahidi, 1994). Fish hydrolysates are made by proteolytic digestion of the fish wastes. After proteolytic digestion, bones, undigested solids and often oil are separated from the hydrolysate. The liquid hydrolysates are usually concentrated and sometimes dried. Hydrolysates can be acidified to reduce microbial growth and oxidation retarded by adding an antioxidant. During the concentration process, protein hydrolysates tend to clump and become viscous. One solution has been to mix hydrolysates with dry ingredients (often plant materials) and then co-dry the mixture. There are three basic methods of producing protein hydrolysates from fish wastes: addition of acids or bases (the product is often referred to as silage), addition of proteolytic enzymes, and the use of microbial fermentations. 20.5.1 pH Producing silage (hydrolysate) using acidification can be accomplished by first decreasing fish waste particle size and then reducing the pH to approximately 3.5 with either organic or mineral acids (Espe et al., 1992; Skrede and Kjos, 1995). The low pH inhibits growth of microorganisms but allows proteolytic enzymes from the fish wastes to digest the wastes. Often the temperature is elevated to increase the enzymatic activity. Digestion is stopped by heating the hydrolysates to denature the proteolytic enzymes. Most microbes will not grow in the hydrolysate due to the low pH; however, chemical reactions still proceed and an antioxidant, such as ethoxyquine, reduces oxidation problems. The liquid material can be stored for later use, concentrated, or mixed with other dry materials. The advantages of the acidification procedure are low cost, minimal equipment requirements and low technical manpower requirements. The disadvantages are a lack of product uniformity and protein quality, increased levels

442

Maximising the value of marine by-products

of free fatty acids and ammonia in the product, increased length of time required for the process (days), large holding tank capacity required for multiple day production, and the handling of acids. 20.5.2 Commercial enzymes Hydrolysis can proceed faster by adding commercial proteolytic enzymes or adding by-products with high concentrations of proteolytic enzymes (Rebeca et al., 1991; Diniz and Martin, 1997; Ferreira and Hultin, 1994; Benjakul and Morrissey, 1997; Liceaga-Gesualdo and Li-Chan, 1999; Shahidi et al., 1995; Onodenalore and Shahidi, 1996). In this process the fish waste is ground and mixed with a defined amount of proteolytic enzyme and the time, temperature and other enzymatic reaction conditions are controlled. After the desired degree of hydrolysis has occurred, the reaction is stopped by heating to denature the proteolytic enzymes and an antioxidant added to reduce lipid oxidation. The hydrolysate can be acidified and stored, or concentrated immediately. Disadvantages include additional cost of the enzymes and the greater process control required to regulate the enzymatic activity. Advantages are fast processing (hours), uniform product, and ability to customize hydroysates for specific uses. Enzymatic hydrolysis of fish processing by-products offers a potentially less expensive technological solution to utilizing by-products than the relatively capital intensive process used to make fish meal. There are a number of variables that need to be controlled including the species and composition of fish used, the enzyme system used for the hydrolysis reaction, determining whether endogenous proteolytic enzymes should be inactivated prior to addition of the commercial enzymes, and determining the optimal degree of hydrolysis needed to obtain the desired product (Mackie, 1982; Rebeca et al., 1991). 20.5.3 Microbial fermentation The use of microbial fermentations to hydrolyze fish wastes is the third method used for producing hydrolysates. This method introduces and maintains a microbial culture to obtain hydrolysis of the ground waste material (Dapkevicius, 1998; Faid et al., 1997). There are two general methods: 1. 2.

use of microorganisms that produce an acid and thus lower the pH and allow the endogenous proteolytic enzymes to hydrolyze the wastes, and use of microbes that have additional proteolytic enzymatic activity to enhance by-product hydrolysis.

In either case conditions must be employed that support the microorganism population (microbial feed source, pH, temperature, etc.). After reaching an appropriate degree of hydrolysis, a heating step is used to destroy the microbes and denature the proteolytic enzymes. Hydrolysates can be preserved with the addition of acid to a pH below 4.5 and the addition of mold inhibitors such as sodium benzoate or formic acid.

By-products from seafood processing for aquaculture and animal feeds 443

20.6 Nutritional benefits and other properties of fish and animal feeds made from seafood processing wastes Soybean meal and corn gluten meal dominate the protein feed ingredient business and global production of soybean meal exceeds 130 MMT. Animal proteins derived from the rendering industry and those derived from capture fishing, e.g. fish meal and solubles, constitute only a small portion of the world's total protein meal production. Total world production of fish meal and solubles is approximately 6±7 MMT depending on the production from the large industrial fisheries of Peru and Chile (Barlow, 2003). To put the amount of fish meal produced from fish processing by-products in perspective, Alaska harvests over 2 MMT of fish for human consumption; however, the fish meal produced in Alaska accounts for only 1 to 2% of the total world production of fish meal (Crapo and Bechtel, 2003). Fish meals are used in many types of aquaculture feeds (Hardy and Masumoto, 1990; Pike and Hardy, 1997; Li et al., 2004; Hardy et al., 2005). Fish meal is a good aquaculture feed ingredient because it is a high quality protein, and compliments most vegetable proteins in feed formulations. In addition, fish meals usually have a high content of the long chain omega-3 fatty acids and minerals, and have good palatability characteristics. Fish meal quality has improved due to a number of factors including an emphasis on raw material freshness, and low-temperature drying. Babbitt et al. (1994) and Rathbone et al. (2001) evaluated the nutritional characteristics of meals made from white fish by-products. The digestibility of meals from fish processing by-products and whole fish have been reported in numerous studies including trout and salmon (Sugiura et al., 1998). The compositions of thirteen commercially available fish meals made from fish processing by-products compared well with other high quality fish meals (Smiley et al., 2003; Forster et al., 2004). The addition of stick water to fish meal from white fish processing by-products was evaluated in shrimp diets by Forster et al. (2003). Hydrolysates are often of lower nutritional value when used as feed ingredients than equivalent whole protein fish meal products (Stone and Hardy, 1986, 1989). However, the high level of digestibility of protein hydrolysates can be of importance in diet formulation for very young animals with immature digestive systems. Hydrolysates are potentially valuable aquaculture feed ingredients as feed binding agents and for their attractant and palatability properties (Lieske and Konrad, 1994). In addition, hydrolysates have been reported to stimulate an immune response in fry (Gildberg and Mikkelsen, 1998). Protein hydrolysates are commercially produced for use in animal milk replacers, and as animal feed and pet food ingredients that have unique palatability and functional properties. Hydrolysates made from seafood by-products are commercially available. Fish oils from cold water species are especially rich in long chain omega-3 fatty acids (Gruger et al., 1964) that are required as essential nutrients in marine and freshwater aquaculture diets (Sargent and Tacon, 1999). There are large

444

Maximising the value of marine by-products

seasonal changes in the lipid content of livers from number of species, and Aidos et al. (2002) reported large seasonal changes in the amount and composition of lipid in by-products. Oils extracted from fish by-products are utilized in aquaculture diets. The addition of a smaller volume of omega-3 rich fish oils to less costly vegetable oils can lower diet costs while providing other benefits associated with omega-3 fatty acids. Another strategy used to increase the omega-3 fatty acid content in aquaculture fish fillets is to feed diets rich in omega-3 fatty acids during the finishing phase of the production cycle. Freeman and Hoogland (1956), Olley et al. (1968), Dong et al. (1993) have reported nutritional values for fish viscera, and Ferreira and Hultin (1994) for liquefied cod frames. Recently, Gunasekera et al. (2002) reported the nutritional evaluation of selected by-products from three species of fish including carp offal, fish frames and trout offal. The chemical and nutritional properties of the individual pollock, cod, and salmon by-products have been determined (Bechtel, 2003; Bechtel and Johnson 2004; Oliveira and Bechtel, 2005). Small basic proteins associated with chromatin have been extracted from fish processing byproducts and reported to have anti-viral properties (Pedersen G M et al., 2003, 2004). It has been reported that high dietary nucleotide levels can have a prophylactic effect on salmonids when challenged by viral disease (Burrells et al., 2001a,b). Although yeast nucleotides were used, there is potential for using material derived from fish by-product. Fish meals are often used in the diets of young pigs, and feed ingredients developed from hydrolysates have been used in aquaculture and in the diets of young pigs and calves. Early weaned pigs require special dietary ingredients until their digestive system is fully developed. It has been suggested that cheaper specialty fish meals can replace expensive spray-dried animal plasma (Dijk et al., 2001) that is currently being used as a minor dietary ingredient during the early weaning period (BergstroÈm et al., 1997). Studies have suggested that inclusion of marine fish oils in diets for pregnant sows improves fetal survival rate (Rigau et al., 1995). In addition to uses as feed ingredients for livestock, fish by-products have been used in pet foods as sources of protein and oil and there is interest in using products made from fish by-products to enhance the health of pets.

20.7

Future trends

There are many exciting areas that are being explored in the field of by-product utilization for making feed ingredients; other uses include the following: 1.

Continue to provide ingredients that will reduce the overall aquaculture feed costs by increasing the use of cheaper plant-derived protein and oil ingredients. Although fish meal and oil are ideally suited for use as aquaculture feed ingredients, efficiencies can be gained by using cheaper sources of protein and oil during part of the production cycle and using fish

By-products from seafood processing for aquaculture and animal feeds 445

2.

3. 4.

meals and oils to retain palatability and attractant properties and improve the nutritional profile. Develop new products from fish by-products and separated components of the by-product stream such as separation of high valued viscera components for human use, higher valued protein and oil aquaculture feed components, unique nutritional ingredients for different segments of the life cycle for aquaculture, farm animal and pets, and mineral, protein and oil supplements. Extract interesting biomolecules and fractions from fish processing byproduct and hydrolysates that have unique applications for animal health and well-being. Develop economically viable processes and methods for utilizing and enhancing the value of fish by-products from small volume processors, seasonal processors, and processors in remote locations that cannot support traditional by-product processing operations.

20.8

References

and BOOM R (2002), `Seasonal changes in crude and lipid composition of herring fillets, byproducts, and respective produced oils', J. Agric. Food Chem., 50, 4589±4599. AMMU K, STEPHEN J and ANTONY P D (1986), `Nutritional evaluation of fish solubles', Fishery Tech., 23, 18±23. BABBITT J K and STEVENS W A (1996), `Evaluation of a new process for producing high quality fishmeal', Final Report of ASTF Grant No. 94-2-085S. Alaska Science and Technology Foundation, Anchorage, Alaska, 74. BABBITT J K, HARDY R W, REPOND K D and SCOTT T M (1994), `Processes for improving the quality of whitefish meal', J. Aquatic Food Product Tech., 3, 59±68. BARLOW S (2003), `World Market Overview of Fish Meal and Fish Oil', in Bechtel P J, Advances in seafood by-products: 2002 conference proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, 11±25. BECHTEL P J (2003), `Properties of different fish processing by-products from pollock, cod and salmon', J. Food Proc. and Pres., 27, 101±116. BECHTEL P J (2005), `Properties of stick water from fish processing byproducts', J. Aquatic Food Techn., 14(2), 25±38. BECHTEL P J and JOHNSON R (2004), `Nutritional properties of pollock, cod and salmon processing by-products', J. Aquatic Food Tech., 13(2), 125±142. BENJAKUL S and MORRISSEY M T (1997), `Protein hydrolysates from Pacific whiting solid by-products', J. Agric. Food Chem., 45, 3424±3430. BERGSTROÈM J R, NELSSEN J L, TOKACH, M D, GOODBAND R D, DRITZ S S, OWEN K Q and NESSMITH W B JR (1997), `Evaluation of spray-dried animal plasma and select menhaden fish meal in transition diets of pigs weaned at 12 to 14 days of age and reared in different production systems', J. Anim. Sci., 75, 3004±3009. BIMBO AP (1990), `Processing of fish oils', in Stansby M E, Fish Oils in Nutrition, Avi Book, Van Nostrand Reinhold, New York, 181±225. BURRELLS C, WILLIAMS P D and FORNO P F (2001a), `Dietary nucleotides: a novel AIDOS I, PADT A, LUTEN J B

446

Maximising the value of marine by-products

supplement in fish feeds. 2. Effects on vaccination, salt water transfer, growth rates and physiology of Atlantic salmon (Salmo salar)', Aquaculture, 199, 177±184. BURRELLS C, WILLIAMS P D, SOUTHGATE P J and WADSWORTH S L (2001b), `Dietary nucleotides: a novel supplement in fish feeds 1. Effects on resistance to disease in salmonids', Aquaculture, 199, 159±169. CHOI Y J and PARK J W (2002), Acid-aided protein recovery from enzyme-rich Pacific whiting', J. Food Sci., 67, 2962±2967. CRAPO C and BECHTEL P J (2003), `Utilization of Alaska's seafood processing by-product', in Bechtel P J, Advances in seafood by-products: 2002 Conference proceedings. Alaska Sea Grant College Program, University of Alaska Fairbanks, 105±119. CRAPO C, PAUST B and BABBITT J (1993), `Recoveries and yields from pacific fish and shellfish', Alaska Sea Grant College Program, Marine Advisory Bulletin. No. 37. CRAPO C, HIMELBLOOM B, PFUTZENREUTER R and CHONG L (1999), `Causes of soft flesh in Giant Grenadier (Albatrossia pectoralis) fillets', J. Aquatic Food. Prod. Tech., 8(3), 55±68. DAPKEVICIUS M (1998), `Lipid and protein changes during the ensilage of blue whiting by acid and biological methods', Food Chem., 63, 97±102. DIJK A J, EVERTS H, NABUURS M J A, MARGRY R J C F and BEYNEN A C (2001), `Growth performance of weanling pigs fed spray-dried animal plasma: a review', Livst. Prod. Sci., 68, 263±274. DINIZ F M and MARTIN AM (1997), `Optimization of dogfish (Squalus acanthias) protein. Composition of the hydrolysates', Int. J. Food Sci. Nutr., 48, 191±200. DONG, F M, FAIRGRIEVE W T, SKONBERG D I and RASCO B A (1993), `Preparation and nutrient analysis of lactic acid bacterial ensiled salmon viscera', Aquaculture, 109, 351± 366. ESPE M, HAALAAND H and NJAA L R (1992), `Autolysed fish silage as a feed ingredient for Atlantic salmon (Salmo salar)', Comparative Biochem. Physiol. Part A, 103A(2), 369±374. FAID M, ZOUITEN A, ELMARRAKCHI A and ACHKARI-BEGDOUI A (1997), `Biotransformation of fish waste into a stable feed ingredient', Food. Chem., 60, 13±18. FERREIRA N G and HULTIN H O (1994), `Liquifying cod frames under acidic conditions with a fungal enzyme', J. Food Proc. Pres., 18, 87±101. FORSTER I, BABBITT J K and SMILEY S (2003), `Nutritional quality of Alaska white fish meals made with different levels of hydrolyzed stickwater for Pacific threadfin (Polydactylus sexfilis)', in Bechtel P J, Advances in seafood by-products: 2002 conference proceedings, Alaska Sea Grant College Program, University of Alaska Fairbanks, 169±174. FORSTER I, BABBITT J and SMILEY S (2004), `Nutritional quality of fish meals made from by-products of the Alaska fishing industry in diets for Pacific white shrimp (Litopenaeus vannamei)', J. Aquatic Food Product Tech., 13, 115±123. FREEMAN H C and HOOGLAND P (1956), `Processing of cod and haddock viscera: 1. Laboratory experiments', J. Fish Res. Bd. Canada, 13, 869±877. GAUDE R (1994), `Menhaden condensed solubles', Feed Mgmnt, 45(8), 31. GILDBERG A and MIKKELSEN H (1998), `Effect of supplementing the feed to Atlantic cod (Gadus morhua) fry with lactic acid bacteria and immuno-stimulating peptides during a challenge trial with Vibrio anguillarum', Aquaculture, 167, 103±113. GILDBERG A, ARNESEN J A and CARLHOG M (2002), `Utilization of cod backbone by biochemical fractionation', Process Biochem., 38, 475±480. GRUGER E H, NELSON R W and STANSBY W E (1964), `Fatty acid composition from 21

By-products from seafood processing for aquaculture and animal feeds 447 species of marine fish, freshwater fish and shellfish', J. Amer. Oil Chem. Soc., 41, 662±667. GUNASEKERA R M, TUROCZY N J, DE SILVA S S and GOOLEY G J (2002), `An evaluation of the suitability of selected waste products in feeds for three fish species', J. Aquatic Food Prod. Tech., 11(1), 57±78. HARDY R W (1992), `Fish Processing By-products and their Reclamation', in Pearson A M, Advances in Meat Research, Vol. 8, Elsevier, Essex, England, 199±216. HARDY R W (1998), `Prevention and detection of fish oil oxidation', Aquaculture, 24(5), 93±98. HARDY R W (2003), `Marine Byproducts for Aquaculture Use', in Bechtel P J, Advances in seafood by-products: 2002 conference proceedings, Alaska Sea Grant College Program, University of Alaska Fairbanks, 105±119. HARDY R W and DONG F M (1995), `Aquatic Feed Ingredients: Quality Standards and Methods of Analysis', Proceedings: Feed Ingredients Asia, 95. Singapore. HARDY R W and MASUMOTO T (1990), `Specifications for marine by-products for aquaculture', in Bechtel P J, Advances in seafood by-products: 2002 conference proceedings, Alaska Sea Grant College Program, University of Alaska Fairbanks, 109±120. HARDY R W and TACON A G J (2002), `Fish meal ± historical uses, production trends and future outlook for sustainable supplies', in Stickney R R and McVey J P, Responsible Marine Aquaculture, CABI Publishing Co., Oxford, 311±325. HARDY R W, SEALY W M and GATLIN D M III (2005) `Fisheries by-catch and by-product meals as protein sources for rainbow trout Oncorhynchus mykiss', J. World Aquaculture Soc., 36, 393±400. KELLEHER S D and HULTIN H O (2000), `Functional chicken muscle protein isolates prepared using low ionic strength, acid solubilization/precipitation', in 53rd Annual Reciprocal Meat Conference, American Meat Science Association, Savoy, Ill, 76±81. KIZEVETTER I V (1971), Chemistry and technology of Pacific fish, (Translated in 1973 by Israel Program for Scientific Translations Ltd.). US Department of Commerce. Springfield, VA. KONING A J (1999), `Quantitative quality tests for South African fish meal: An investigation into the validity of a number of quality indices', Int. J. Food Prop., 2(1), 79±92. KRISTINSSON K G and DEMIR N (2003), `Functional fish protein ingredients from fish species of warm and temperate waters: comparison of acid- and alkali-aided processing vs. conventional surimi processing', in Bechtel P J, Advances in seafood by-products: 2002 conference proceedings, Alaska Sea Grant College Program, University of Alaska Fairbanks, 277±295. KRISTINSSON H G and RASCO B A (2000), `Fish protein hydrolysates: production, biochemical and functional properties', CRC Crit. Rev. Food Sci. Nutr., 40(1), 43±81. KRZYNOWEK J, MURPHY J, MANEY R S and PANUNZIO L J (1989), `Proximate composition and fatty acid and cholesterol content of 22 Species of northwest Atlantic finfish', NOAA Technical Report NMFS, 74. LI P, WANG X, HARDY R and GATLIN D M III (2004), `Nutritional value of fisheries by-catch and by-product meals in the diet of red drum (Sciaenops ocellatus)', Aquaculture, 236, 485±496. LICEAGA-GESUALDO A M and LI-CHAN L C (1999), `Functional properties of fish protein hydrolysate from herring (Clupea harengus)', J. Food. Sci., 64, 1000±1004.

448

Maximising the value of marine by-products

and KONRAD G (1994), `Protein hydrolysis-the key to meat flavoring systems', Food Rev. Int., 10, 287±312. MACKIE I M (1982), Fish protein hydrolysates', Process Biochemistry, Jan./Feb. 1982. MEEHAN M J, HUSBY F M, ROSIER C and KING R L (1990), `Historic and potential production and utilization of Alaskan marine by-products', in Keller S, Proceedings of the international conference on fish by-products, Alaska Sea Grant College Program, Fairbanks, Alaska, 31±38. MONTERO P, JIMENEZ-COLMENERO F and BORDERIAS J (1991), `Effect of pH and the presence of NaCl on some hydration properties of collagenous material from trout (Salmo irideus) muscle and skin', J. Sci. Food Agric., 54, 137±146. NAGAI T and SUZUKI N (2000), `Isolation of collagen from waste material-skin, bone and fins', Food Chem., 68, 277±281. NEW M B (1996), `Global aquaculture: Current trends and challenges for the 21st century', World Aquaculture Magazine, 8±13, 63±79. OLIVEIRA A C M and BECHTEL P J (2005), `Lipid composition of Alaskan pink salmon (Oncorhynchus gorbuscha) and Alaska walleye pollock (Theragra chalcogramma) by-products', J. Aquatic Food Tech., 14(1), 73±91. OLLEY J, FORD J E and WILLIAMS A P (1968), `Nutritional value of fish viscera meals', J. Sci. Fd. Agric., 19, 282±285. ONODENALORE A C and SHAHIDI F (1996), `Protein dispersions and hydrolysates from shark (Isurus oxyrinchus)', J. Aquatic Food Prod. Tech, 5(4), 43±59. PEDERSEN G M, GILDBERG A, STEIRO K and OLSEN R L (2003), 'Histone-like proteins from Atlantic cod milt: stimulatory effect on Atlantic salmon leucocytes in vivo and in vitro', Comp. Biochem. Physiol. Part B, 134, 407±416. PEDERSEN G M, GILDBERG A and OLSEN R L (2004), `Effects of including cationic proteins from cod milt in feed to Atlantic cod (Gadus morhua) fry during a challenge trial with Vibrio anguillarum', Aquaculture, 233, 31±43. PEDERSEN L D, CRAPO C, BABBITT J and SMILEY S. (2003), `Membrane filtration of stickwater', in Bechtel P J, Advances in seafood by-products: 2002 conference proceedings, Alaska Sea Grant College Program, University of Alaska Fairbanks, 359±369. PIKE I H and BARLOW S W (1999), `Fish meal and oil to the year 2010 ± supplies for aquaculture', Presentation copy; World Aquaculture 99, Sydney, Australia, 1±8. PIKE I H and HARDY R W (1997), `Standards for assessing quality of feed ingredients in crustacean nutrition', in Diagram L R, Conklin D E and Akiyama D M, Advances in World Aquaculture, World Aquaculture Society, Baton Rouge, Louisiana, 473± 492. RAA J and GILDBERG A (1982), `Fish silage: A review', CRC Crit. Rev. Food Sci. and Nutr., 16(4), 383±419. RATHBONE C K, BABBITT J K, DONG F M and HARDY R W (2001), `Performance of juvenile Coho Salmon Oncorhynchus kisutch fed diets containing meals from fish byproducts, deboned fish by-products, or skin-and-bone by-products as the protein ingredient', J. World Aquaculture Soc., 32, 21±29. Ä A-YERA M T and DõÂAZ-CASTAN Ä EDA M (1991), `Production of fish protein REBECA B, PEN hydrolysates with bacterial proteases; yield and nutritional value', J. Food Sci., 56, 309±314. RIGAU A P, LINDERMANN M D, KORNEGAY E T, HARPER A F and WATKINS B A (1995), `Role of dietary lipids on fetal tissue fatty acid composition and fetal survival in swine at 42 days of gestation', J. Anim. Sci., 73, 1372±1380. LIESKE B

By-products from seafood processing for aquaculture and animal feeds 449 and TACON A G J (1999), `Development of farmed fish: a nutritionally necessary alternative to meat', Proc. Nutr. Soc., 58, 377±383. SATHIVEL S, BECHTEL P J, BABBITT J, SMILEY S, CRAPO C, REPPOND K D and PRINYAWIWATKU, W (2003), `Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates', J. Food Sci., 68, 2196±2200. SATHIVEL S, BECHTEL P J, BABBITT J, PRINYAWIWATKUL W, NEGULESCU I and REPPOND K D (2004), `Properties of protein powders from arrowtooth flounder (Atheresthes stomias) and herring (Clupea harengus) by-products', J. Agric. Food Chem., 52, 5040±5046. SHAHIDI F (1994), `Protein concentrates from underutilized aquatic species', in Food flavor-generation, analysis, and process influence. Proceedings of the 8th International flavor conference, Developments In Food Science, 37, 1441±1451. SHAHIDI F, HAND X Q and SYNOWIECKI J (1995), `Production and characteristics of protein hydrolysates from capelin (Mallotus villosus)', Food Chem, 53, 285±293. SKREDE A and KJOS N P (1995), `Digestibility of amino acids in fish silage', European association for animal production, 81, 205±208. SMILEY S, BABBITT J, DIVAKARAN S, FORSTER I and OLIVEIRA A (2003), `Analysis of groundfish meals made in Alaska', in Bechtel P J, Advances in seafood byproducts: 2002 conference proceedings, Alaska Sea Grant College Program, University of Alaska Fairbanks, 431±454. SOARES J JR, MILLER D, CUPPETT S and BAUERSFELD P JR (1973), `A review of the chemical and nutritive properties of condensed fish solubles', Fishery Bull, 71, 255±265. STANSBY M E (1976), `Chemical characteristics of fish caught in the northeast Pacific ocean', Marine Fisheries Rev., 38(9), 1±11, MFR paper No. 1198. STONE F E and HARDY R W (1986), `Nutritional value of acid stabilized silage and liquefied fish protein', J. Sci. Food Agric., 37, 797±803. STONE F E and HARDY R W (1989), `Plasma amino acid changes in rainbow trout (Salmo gairdneri) fed freeze-dried fish silage, liquefied fish, and fish meal', in Proceedings of the aquaculture international congress, Vancouver, BC, Canada, Sept. 1988, 419±426. SUGIURA S H, DONG F M, RATHBONE C K and HARDY R W (1998), `Apparent protein digestibility and mineral availability in various feed ingredients for salmon feeds', Aquaculture, 159, 177±202. TARR H L A (1982), `Effects of processing on the nutritive value of fish products used for animal feeding', in Handbook of nutritive value of processed food. Vol. II. Animal feedstuffs, CRC Press, Boca Raton, FL, 283±303. UNDERLAND I, KELLEHER S and HULTIN H O (2002), `Recovery of functional proteins from herring (Clupea harengus) light muscle by an acid or alkali solubilization process', J. Agric. Food Chem., 50, 7371±7379. VALLE J M (1990), `Recovery of liquid by-products from fish meal factories: a review', Process Biochem. Int., 25(4), 122±131. WINDSOR M and BARLOW S (1981), Introduction to fishery by-products, Fishing News Books, Ltd. Farnham, Surrey, UK. ZARKADAS C G, HULAN H W and PROUDFOOT F G (1986), `The amino acid and mineral composition of white fish meal containing enzyme-digested or untreated stickwater solids', Anim. Feed Sci. Technol., 14, 291±305. SARGENT J R

21 Using marine by-products in pharmaceutical, medical, and cosmetic products J. Losso, Louisiana State University, USA

21.1

Introduction

The concept of utilizing bioactive compounds of marine origin in cosmetic, medical, and pharmaceutical applications is an ancient tradition. Historical records indicate that marine organisms were once items of commerce for biomedical uses to cure exotic diseases.1 For some time, bioactive compounds from the sea were used as drug carriers, demulcents, and nutritional supplements. Advances in marine biotechnology have increased the efficiency and profitability of the seafood industry. The USDA has estimated that by the year 2025 global aquaculture will provide 50 to 60% of the world food supply.2 Increasing food and human health products will originate from the sea for several reasons including the outbreak of bovine spongiform encephalopathy (BSE) also known as mad cow disease and the need for more efficient healthcare products from the sea.3 The search for novel antibiotics that can overcome the problem of multi-drug resistant bacterial strains is a well enunciated reason for investigation of the bioactivity of marine bioactive compounds. Currently, harvested seafood products generate significant amounts of by-products from which the seafood industry, scientists, government agencies, conservationists, consumer advocates and seafood processors would like to obtain high value fine biochemicals.3±6 The disposal of seafood waste is a problem at every one of the seafood processing plants around the world. All seafood processors always stress that this is one of the larger problems they face. For instance, in 2002, disposal rates in the New Orleans, Louisiana area were $30/ton, and it was not uncommon for a large

Marine by-products in pharmaceutical, medical, and cosmetic products

451

plant to spend $2,000 to $3,000 per month in disposal fees.7 Very often, local landfills refuse seafood as well as fishery wastes because of the nitrogenous runoff that they can produce, and the fouling of truck scales. As a result, alternative uses of the by-products that can provide economic benefits to processors and reduce the waste disposal issue have been investigated. The following paragraphs describe some of the most studied bioactive compounds from edible marine food products.

21.2

Squalamine

Squalamine (7-24-dihydroxylated-24 cholestane sulfate; Fig. 21.1) is an aminosterol present in the liver, gallbladder, intestines, testes, and stomach of dogfish shark (Squalus acanthias).8±11 Squalamine is a cationic water-soluble steroid and is effective against both gram-negative and gram-positive bacteria, fungi, angiogenesis, and tumor activities.8,12±15 Squalamine has shown promise for the treatment of lung and ovarian cancer.8 Phase I clinical trials using squalamine in patients with a variety of solid malignancies who had failed conventional therapies showed that squalamine was well tolerated by humans.16 Bayes et al.17 reported that squalamine is still among the bioactive compounds undergoing clinical trials for various types of cancer. Clinical trials are also underway for the regression of retinopathy.17 When used as inhibitor of agerelated macular degeneration, squalamine at a concentration as low as 1 ppm adminsitered once intravenously promoted shrinkage of the choroidal neovascularization lesion associated with macular degeneration and an improvement of 3 to 8 lines of vision with most patients having stabilization of vision.18±21 Another aminosterol with antimicrobial and anticancer activities similar to squalamine is MSI-1436 which differs from squalamine by the presence of a spermine side chain at C-3 on the cholesterol A ring8,14 (Fig. 21.2). Appetite suppression is another health enhancing property ascribed to squalamine and MSI-1436.8,22 MSI-1436 decreases body fat content by decreasing mRNA levels of agouti-related peptide and neuropeptide Y in the hypothalamus of mice; human trials are awaiting. Squalamine and MSI-1436 selectively inhibit bacteria with low minimum inhibition concentration of about 5 g/ml.8

Fig. 21.1 Structure of squalamine.

452

Maximising the value of marine by-products

Fig. 21.2 Structure of MSI-1436.

Squalamine is a very attractive bioactive compound for several reasons. First, as an inhibitor of cancer progression, the opportunities are enormous. Second, as an antimicrobial compound of marine origin, it offers a marketing advantage because there is no current evidence to suggest that viral or sub-viral particles, that are adapted to cold-blooded (poikilothermic) organisms, can be transmitted to humans. Third, as an appetite suppressing molecule, the possibilities are enormous because obesity and diabetes are on the rise worldwide. Obesity can lead to diabetes and possibly to cancer.23 Whereas squalamine has interesting biomedical properties, only microgram quantities of the amino steroid can be isolated from one shark. Tissue concentration of squalamine is very low (liver and gallbladder: 10±20 g/g; spleen and testes: 2 g/g; stomach: 1 g/g; gill: 0.5 g/g; and bowels: 0.02 g/g). Chemical synthesis is being used to produce analogs of squalamine; however, yields have been very low so far.

21.3

Collagen

Collagen is unique among body proteins because it is the single most important protein of connective tissues. Collagen molecules are classified into 21 different types and differ in their sequences, molecular weights, structures, and functions, but they can be broadly subdivided into families. Type I and II collagens are mostly found in the skins and bones whereas type IV, VI, VIII, X, and dogfish egg case collagens belong to the network-forming family. Collagen molecules are covalently cross-linked into fibrils that may swell, but do not dissolve. Commercially available collagen has several applications in the cosmetic, medical, and pharmaceutical industries. Successful medical and pharmaceutical applications of collagen include the treatment of urinary incontinence and pain associated with osteoarthritis, scaffolding biomaterials for ligaments replacement, cartilage engineering to repair join cartilage defects, other implants in humans, and inhibition of angiogenesis as means of preventing cancer metastasis.24±28 A matrix of collagen containing calcium phosphate (CaP-Gelfix(R)) was produced to create a cartilage via tissue engineering.24 Fibrocartilage

Marine by-products in pharmaceutical, medical, and cosmetic products

453

formation and bone invasion was observed in 20 weeks. Cells maintained their phenotype in the matrix; the matrix had a good healing response, was effective in cartilage regeneration, and showed potential for use to repair defective joint cartilage. Collagen-based tissue-engineered blood vessels (TEBVs) containing elastin have been suggested as a better mimic of arterial physiology with improved mechanical properties for use as bypass grafts in in vivo investigations.29 The cosmetic industry uses collagen extensively in personal care products. Antiangiogenic activity of type I collagen has been reported.30 Oral administration of type II collagen to adjuvant arthritis (AA) rats alleviated both distinct articular and general symptoms of arthritis.26 The authors suggested that the effectiveness of collagen was associated with downregulation of both IFN- and TNF-, and suppression of cell immunity. Traditionally, collagen has been obtained from the skins of land-based animals such as bovine and porcine. In light of recent reports on mad cow disease around the world, the use of collagen and collagen-derived products from land-based animal skins has been called into question.31,32 As a result, marine collagen and its by-products are in very high demand. Ogawa et al.33,34 isolated and purified type I and II collagens from warm water black drum (Pogonias cromis) and sheepshead (Archosargus probatocephalus) skins and bones and identified biochemical and thermal properties that were similar to bovine collagen. In an effort to find additional non-land-based sources of collagens, cartilages from farm-raised alligators have been used as sources of collagen (Losso, unpublished). However, the availability of collagens from the skins of warm water fish will not offset the huge demands for collagen in biochemical, biomedical, functional food, and pharmaceutical industries. Shark skin collagen is one of the most abundant and important sources of marine collagen because large numbers of sharks are caught as by-product of tuna fishing. The collagen from shark skin is of type I with similar properties as land-based animal type I collagen. However, shark skin collagen contains fewer imino acid residues and has a lower denaturation temperature than collagen from land vertebrates.35 Shark skin collagen hydrolyzates are sold as cosmetics and dietary supplements for the treatment of osteoporosis in Japan.35 Shark cartilage is known for its anti-angiogenic properties. Trade names such as Neovastat and AE 941 have been used by manufacturers to suggest probably a similar product obtained from shark cartilage. AE 941 [Arthrovas, Neoretna, Psovascar] inhibits angiogenesis by at least four molecular mechanisms: blockade of endothelial cell signaling via inhibition of vascular endothelial growth factor binding to its receptor vascular endothelial growth factor (VEGFR); modulation of matrix metalloproteinase MMP-2 and MMP-9 pathways; induction of endothelial cell apoptosis; and stimulation of angiostatin production.36,37 AEterna, the company which appears to own the exclusivity for the commercialization of AE 941 is right now focusing on non-small cell lung cancer as target for AE 941.37

454

21.4

Maximising the value of marine by-products

Elastin

Elastin is a cross-linked protein in the extracellular matrix that provides elasticity for many tissues. Elastin is a component of fish skin along with collagen and dermatan sulfate. Elastin fibers consist of an amorphous material as well as of 10±12 nm microfilaments which provide unique mechanical properties to those body tissues where they are present: the lung, skin, cartilage and vessels walls.38 The unique physicochemical property of elastin can be attributed to its characteristic amino acid composition, rich in glycine, proline and other hydrophobic amino acids. The high hydrophobicity and cross-linked character confers to the protein a high resistance to protease degradation.39 Elastin-based therapeutic approaches showed that soluble elastin and its peptide VAPG at 1 mg/ml inhibited lung colony formation of human melanoma HT168-M1 and the whole elastin protein, -elastin was a strong inhibitor of murine lung carcinoma.40 Elastin peptides have the unique property of being thermosensitive at high temperature and become insoluble by coarcervation.39 This unique property of elastin is an attractive new field of clinical research that is being exploited in cancer treatment to create hyperthermia to improve the permeability of the tumor cell and enhance the delivery of circulating anticancer agents.41 Commercial applications of elastin include skin care products, treatment of stretch marks (striae gravidarum) in pregnant women, and protein hydrolysates added to hair-care products to repair broken hair.

21.5

Proteoglycans

Proteoglycans contain more than 90% polysaccharides with about 5% polypeptide bound along the linear carbohydrate chain. Other members of the family include heparin, heparin sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, and hyaluronan. Proteoglycans are found extracellularly. Proteoglycans are major molecules involved in angiogenesis. Dermatan sulfate (DS) is one of the most important antithrombotic agents that has been approved for the prophylaxis and treatment of deep vein thrombosis (DVT) in patients undergoing elective hip replacement surgery, and for the treatment of disseminated intravascular coagulation (DIC).42 DS has important anticoagulant and antithrombotic activities.43,44 DS accelerates heparin cofactor II mediated inhibition of thrombin.45,46 Based on these findings, there are a number of future applications for DS including tissue transplantations, preparation of medical devices and artificial tissues.46±48 DS is commonly obtained from porcine skin as well as porcine and bovine intestinal mucosa. However, the ongoing episodes of BSE in cattle around the world is forcing researchers, manufacturers, and government officials to find new and safe sources of DS and other bioactive compounds that were traditionally obtained from land-based mammals. Because of the phylogeny distance between fish and mammals, it is so far assumed that bioactive compounds from

Marine by-products in pharmaceutical, medical, and cosmetic products

455

marine-based resources may be less prone to viral infections that could be pathogenic to humans. Proteoglycans possess a growth factor-dependent activity and in the presence of growth factors such as fibroblast growth factor -1, they modulate endothelial cell proliferation and migration. Fucosylated chondroitin sulfate (FucCS), a glycosaminoglycan obtained from sea cucumber (Ludwigothurea grisea) has the same structure as mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches.49 FucCS inhibits smooth muscle cell proliferation as heparin and has a potent enhancing effect on endothelial cell proliferation and migration in the presence of heparin-binding growth factors. The sulfated fucose branches are the structural motif for the proangiogenic activity of this chondroitin sulfate. FucCS also prevents venous and arterial thrombosis, in animal models. FucCS may be a promising glycosaminoglycan and as a promising molecule with possible beneficial effects in pathological conditions affecting blood vessels such as the neovascularization of ischemic areas.

21.6

Protamine

Protamine, a naturally occurring cationic polypeptide of about 30±65 amino acid residues is found in mammals, fish, birds, and reptiles. Protamines package DNA of most vertebrates sperm in a highly condensed and genetically inactive state.50 The protein has a very high content of arginine residues which inhibits both Gram positive and Gram negative bacteria.51 The protein is also highly basic protein, lysine deficient, high in cysteine, pI 12±13, and structurally diverse from species to species. The amino acid sequence from different species is given in Table 21.1. Human and experimental gliomas spread and grow in response to both paracrine and autocrine release of endothelial, fibroblast and platelet growth factors. Protamine dose-dependently reduced tumor volume, mitotic index, vascular density, and cell viability of highly malignant C6 glioblastoma in Wistar rats at a dose lower than toxic dose of suramin.52 Many types of carcinoma accumulate large numbers of degranulating mast cells which will release heparin. Protamine binds to heparin and neutralizes heparin anticoagulant effects and may therefore induce selective tumor cell thrombosis. Intravenously injected protamine induced selective thrombosis in tumors, and the effect lasted for several hours.53 Antithrombic compounds can also prevent lipid rich plaque rupture in diabetic and dyslipidaemic patients. Ornithine decarboxylase (ODC) which is associated with the onset and progression of a variety of cancers including colon, prostate, and breast was inhibited by protamine.54 Inhibition of ODC leads to polyamine depletion in cells, a cytostatic effect on proliferating endothelial cells, and the inhibition of angiogenesis.55 Nitric oxide (NO) is a pivotal factor for gastric ulcer healing. Protamine sulfate has been reported to stimulate NO production and potentiate the effect of heparin.

456

Maximising the value of marine by-products

Table 21.1 Amino acid sequence of protamine from different species 5 Stallion

10 15 20 25 30 ARYRC CRSQS QSRCR RRRRR RCRRR RRRSV RQRR--

Bull

ARYRC CLTHS GSRCR RRRRR RCRRR RRR- F GRRR

Salmon

PRRRR RASRP VRRRR RARRS TAVRR RRRVV RRRR

Perch

PRRRR HAARP VRRRR RTRRS SRVHR RRRAV RRRR

21.7

Future trends

Searching for bioactive compounds from the sea is an ancient tradition that will not end anytime soon. Bioactive compounds from marine origin appear to possess, in some cases, stronger biological activities than their land-based counterparts. Because of phylogeny differences, disease resistance appears to be a remote concern. However, supply of these marine bioactive compounds may become a problem in the future. Genetic engineering is already looking into designing and biosynthesizing analogs of bioactive compounds in short supply. Elastin and squalamine are two prototype products for which laboratory synthesis and biological engineering of analogs are already underway.8,56 Protamine bioactivity is mostly associated with the repeated chains of arginine. Arginine is a nitric oxide precursor. Biosynthesis of polyarginine molecules that mimic protamine bioactivity may provide a way to replace bioseparation of the polypeptide from aquatic sources.

21.8 1. 2. 3. 4.

5. 6. 7.

References

Drugs from the Sea. Transactions of the drugs from the sea symposium. University of Rhode Island. August 27±29, 1967. Marine Technology Society 1968. iii. AVAULT J JR. Perspective ± Aquaculture Development: Potential for Growth in the New Millennium. Louisiana Agriculture 1999, 42: 7. BRUDNAK MA. Weight-loss drugs and supplements: are there safer alternatives? Med Hypotheses. 2002, 58: 28±33. BORRESEN T. More efficient utilization of fish and fisheries products toward zero emission. International Symposium on `More Efficient Utilization of Fish and Fisheries Products'. October 7±10, 2001, Kyoto, Japan. Japanese Society of Fisheries Science. WIJIKSTROM UN. Future demand for, and supply of fish and shellfish as food. International Symposium on `More Efficient Utilization of Fish and Fisheries Products'. October 7±10, 2001, Kyoto, Japan. Japanese Society of Fisheries Science. MORRISEY MT. Full Utilization. J. Aquatic Food Product Technology. 1999, 8: 1±2. SCHEXNAYDER M. LSU AgCenter. Personal Communication 2002. FREUNDENTHAL HD.

Marine by-products in pharmaceutical, medical, and cosmetic products 8.

457

Squalamine: a polyvalent drug of the future? Curr Cancer Drug Targets. 2005, 5: 267±272. 9. SHEPHERD FA, SRIDHAR SS. Angiogenesis inhibitors under study for the treatment of lung cancer. Lung Cancer 2003, 41: S63±S72. 10. SAVAGE PB, LI C, TAOTAFA U, DING B, GUAN Q. Antibacterial properties of cationic steroid antibiotics. FEMS Microbiol Lett. 2002, 217: 1±7. 11. MOORE KS, WEHRLI S, RODER H, ROGERS M, FORREST JN JR, MCCRIMMON D, ZASLOFF M. Squalamine: an aminosterol antibiotic from the shark. Proc. Natl. Acad. Sci. USA. 1993, 90: 1354±1348. 12. PIETRAS RJ, WEINBERG OK. Antiangiogenic Steroids in Human Cancer Therapy. Evid Based Complement Alternat Med. 2005, 2: 49±57. 13. MARWICK C. Natural compounds show antiangiogenic activity. J Natl Cancer Inst. 2001, 93: 1685. 14. RAO MN, SHINNAR AE, NOECKER LA, CHAO TL, FEIBUSH B, SNYDER B, SHARKANSKY I, SARKAHIAN A, ZHANG X, JONES SR, KINNEY WA, ZASLOFF M. Aminosterols from the dogfish shark Squalus acanthias. J Nat Prod. 2000, 63: 631±635. 15. KIKUCHI K, BERNARD EM, SADOWNIK A, REGEN SL, ARMSTRONG D. Antimicrobial activities of squalamine mimics. Antimicrob Agents Chemother. 1997, 41: 1433±1438. 16. HAO D, HAMMOND LA, ECKHARDT SG, PATNAIK A, TAKIMOTO CH, SCHWARTZ GH, GOETZ AD, TOLCHER AW, MCCREERY HA, MAMUN K, WILLIAMS JI, HOLROYD KJ, ROWINSKY EK. A Phase I and pharmacokinetic study of squalamine, an aminosterol angiogenesis inhibitor. Clin Cancer Res. 2003, 9: 2465±2471. 17. BAYES M, RABASSEDA X, PROUS JR. Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 2005, 27: 193±219. 18. HIGGINS RD, YAN Y, GENG Y, ZASLOFF M, WILLIAMS JI. Regression of retinopathy by squalamine in a mouse model. Pediatr Res. 2004, 56: 144±149. 19. GENAIDY M, KAZI AA, PEYMAN GA, PASSOS-MACHADO E, FARAHAT HG, WILLIAMS JI, HOLROYD KJ, BLAKE DA. Effect of squalamine on iris neovascularization in monkeys. Retina. 2002, 22: 772±778. 20. CIULLA TA, CRISWELL MH, DANIS RP, WILLIAMS JI, MCLANE MP, HOLROYD KJ. Squalamine lactate reduces choroidal neovascularization in a laser-injury model in the rat. Retina. 2003, 23: 808±814. 21. HIGGINS RD, SANDERS RJ, YAN Y, ZASLOFF M, WILLIAMS JI. Squalamine improves retinal neovascularization. Invest Ophthalmol Vis Sci. 2000, 41: 1507±1512. 22. AHIMA RS, PATEL HR, TAKAHASHI N, QI Y, HILEMAN SM, ZASLOFF MA. Appetite suppression and weight reduction by a centrally active aminosterol. Diabetes. 2002, 51: 2099±2104. 23. RUPNICK MA, PANIGRAHY D, ZHANG CY, DALLABRIDA SM, LOWELL BB, LANGER R, FOLKMAN MJ. Adipose tissue mass can be regulated through the vasculature. Proc Natl Acad Sci USA. 2002, 99: 10730±10735. 24. KOSE GT, KORKUSUZ F, OZKUL A, SOYSAL Y, OZDEMIR T, YILDIZ C, HASIRCI V. Tissue engineered cartilage on collagen and PHBV matrices. Biomaterials. 2005, 26: 5187± 5197. 25. GENTLEMAN E, LAY AN, DICKERSON DA, NAUMAN EA, LIVESAY GA, DEE KC. Mechanical characterization of collagen fibers and scaffolds for tissue engineering. Biomaterials. 2003, 24: 3805±3813. 26. HU Y, ZHAO W, QIAN X, ZHANG L. Effects of oral administration of type II collagen on adjuvant arthritis in rats and its mechanisms. Chin Med J (Engl). 2003, 116: 284± 287. BRUNEL JM, SALMI C, LONCLE C, VIDAL N, LETOURNEUX Y.

458 27.

Maximising the value of marine by-products RHEN M, VEIKKOLA T, KUKK-VALDRE E, NAKAMURA H, IIMONEN M, LOMBARDO C,

Interaction of endostatin with integrins implicated in angiogenesis. Proc Natl Acad Sci USA. 2001, 98: 1024±1029. 28. TONNESEN MG, FENG X, CLARK RA. Angiogenesis in wound healing. J Invest Dermatol Symp Proc 2000, 5: 40±46. 29. BERGLUND JD, NEREM RM, SAMBANIS A. Incorporation of intact elastin scaffolds in tissue-engineered collagen-based vascular grafts. Tissue Eng. 2004, 10: 1526±1535. 30. KROON ME, VAN SCHIE ML, VAN DER VECHT B, VAN HINSBERGH VW, KOOLWIJK P. Collagen type 1 retards tube formation by human microvascular endothelial cells in a fibrin matrix. Angiogenesis. 2002, 5: 257±265. 31. HOAG H. BSE case rattles Canadian officials. Nature. 2003, 423: 469. 32. HELCKE, T. Gelatine, The food technologist's friend or foe? International Food Ingredients 2000, 1: 6±8. 33. OGAWA M, MOODY MW, PORTIER RJ, BELL J, SCHEXNAYDER MA, LOSSO JN. Biochemical properties of black drum and sheepshead seabream skin collagen. J. Agric. Food Chem. 2003, 51: 8088±8092. 34. OGAWA M, PORTIER RJ, MOODY MW, BELL J, SCHEXNAYDER M, LOSSO JN. Biochemical properties of bone and scale collagens isolated from subtropical fish black drum (Pogonias cromis) and sheepshead seabream (Archosargus probatocephalus). Food Chem. 2004. 88: 495±501. 35. NOMURA Y, OOHASHI K, WATANABE M, KASUGAI S. Increase in bone mineral density through oral administration of shark gelatin to ovariectomized rats. Nutrition. 2005, 21: 1120±1126. 36. BUKOWSKI RM. AE-941, a multifunctional antiangiogenic compound: trials in renal cell carcinoma. Expert Opin Investig Drugs. 2003, 12: 1403±1411. 37. DREDGE K. AE-941 (AEterna). Curr Opin Investig Drugs. 2004, 5: 668±677. 38. ROBERT L, JACOB MP, FUÈLOÈP T, TIÂMAÂR J, HORNEBECK W. Elastonectin and the elastin receptor. Pathol. Biol. (Paris) 1989, 37: 736±741. 39. HORNEBECK W, ROBINET A, DUCA L, ANTONICELLI F, WALLACH J, BELLON G. The elastin connection and melanoma progression. Anticancer Res. 2005, 25: 2617±2625. 40. TIÂMAÂR J, DICZHAÂZI C, LADAÂNYI A, RAÂSOÂ E, HORNEBECK W, ROBERT L, LAPIS K. Interaction of tumour cells with elastin and metastatic phenotype. Ciba Found. Symp. 1995, 192: 321±335. 41. MEYER DE, SHIN BC, KONG GA, DEWHIRST MW, CHILKOTI A. Drug targeting using thermally responsive polymers and local hyperthermia. J. Control Release. 2001, 74: 213±224. 42. IBBOTSON T, PERRY CM. Danaparoid: a review of its use in thromboembolic and coagulation disorders. Drugs. 2002, 62: 2283±2314. 43. WHINNA HC, CHOI HU, ROSENBERG LC, CHURCH FC. Interaction of heparin cofactor II with biglycan and decorin. J. Biol. Chem. 1993, 268: 3920±3924. 44. PAVAO MS, AIELLO KR, WERNECK CC, SILVA LC, VALENTE AP, MULLOY B, COLWELL NS, TOLLEFSEN DM, MOURAO PA. Highly sulfated dermatan sulfates from Ascidians. Structure versus anticoagulant activity of these glycosaminoglycans. J. Biol. Chem. 1998, 273: 27848±27857. 45. MASCELLANI G, LIVERANI L, PARMA B, BERGONZINI G, BIANCHINI P. Active site for heparin cofactor II in low molecular mass dermatan sulfate. Contribution to the antithrombotic activity of fractions with high affinity for heparin cofactor II. Thromb Res. 1996, 84: 21±32. 46. LINHARDT RJ, HILEMAN RE. Dermatan sulfate as a potential therapeutic agent. Gen PIHLAJANIEM T, ALITALO K, VUORI K.

Marine by-products in pharmaceutical, medical, and cosmetic products Pharmacol. 1995, 26: 443±451.

459

Which glycosaminoglycans are suitable for antithrombogenic or athrombogenic coatings of biomaterials? Part I: Basic concepts of immobilized GAGs on partially cationized cellulose membrane. Semin. Thromb. Hemost. 1997, 23: 203±213. 48. MIWA H, MATSUDA T. An integrated approach to the design and engineering of hybrid arterial prostheses. J. Vasc. Surg. 1994, 19: 658±667. 49. TAPON-BRETAUDIEÁRE J, CHABUT D, ZIERER M, MATOU S, HELLEY D, BROS D, MOURAÄO PAS, FISCHER AM. Molecular Cancer Research. 2002, 1: 96±102. 50. BALHORN R, CORZET M, MAZRIMAS JA. Formation of intraprotamine disulfides in vitro. Arch. Biochem. Biophys. 1992, 296: 384±393. 51. JOHANSEN C, VERHEUL A, GRAM L, GILL T, ABEE T. Protamine-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria. Appl Environ Microbiol. 1997, 63: 1155±1159. 52. ARRIETA O, GUEVARA P, REYES S, ORTIZ A, REMBAO D, SOTELO J. Protamine inhibits angiogenesis and growth of C6 rat glioma; a synergistic effect when combined with carmustine. Eur. J. Cancer. 1998, 34: 2101±2106. 53. SAMOSZUK MK, SU MY, NAJAFI A, NALCIOGLU O. Selective thrombosis of tumor blood vessels in mammary adenocarcinoma implants in rats. Am. J. Pathol. 2001, 159: 245±251. 54. FLAMIGNI F, GUARNIERI C, MARMIROLI S, CALDARERA CM. Inhibition of rat heart ornithine decarboxylase by basic polypeptides. Biochem. J. 1985, 229: 807±810. 55. TAKAHASHI Y, MAI M, NISHIOKA K. Alpha-difluoromethylornithine induces apoptosis as well as anti-angiogenesis in the inhibition of tumor growth and metastasis in a human gastric cancer model. Int. J. Cancer. 2000, 85: 243±247. 56. GIROTTI A, REGUERA J, RODRIGUEZ-CABELLO JC, ARIAS FJ, ALONSO M, MATESTERA A. Design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes. J. Mater. Sci. Mater. Med. 2004, 15: 479±484. 47.

BAUMANN H, MUELLER U, KELLER R.

22 Bio-diesel and bio-gas production from seafood processing by-products R. Zhang and H. M. El-Mashad, University of California Davis, USA

22.1

Introduction

Food processing by-products are largely organic in nature and can be potentially utilized for production of valuable products. For example, many by-products produced by the seafood industry may be used for production of food or feed products and energy ± a very desirable outcome from both environmental and sustainability points of view. The seafood industry produces many kinds of by-products. In addition to liquid effluent, solid materials are also produced, which include crab, prawns, lobster body parts, damaged shellfish, heads, tails, frames, offal (guts, kidney, liver, etc.) of fish as well as fish that do not satisfy the quality standards (Mhara, 2005). Lindsay (1975) stated that in the processing of most fish species for food purposes, the by-products represent 30 to 80% of the raw material. The seafood by-products typically have high contents of organic matter and salts and are highly putrifiable. Some of these materials are treated prior to discharge, used for fish meal production or directly disposed of by landfilling, land application, or marine dumping. According to Champ et al. (1981), the impacts of ocean disposal of fish by-product can include: 1. 2. 3. 4. 5.

high oxygen demand on receiving water visible surface slicks turbidity plumes organic enrichment and the attraction of undesirable predator species such as sharks.

Regulations in many countries have become more restrictive, with the aim of preventing or reducing the contamination caused by discharging seafood

Bio-diesel and bio-gas production from seafood processing by-products 461 processing by-products into the sea. In 2001, the Northwest regional office of the United States Environmental Protection Agency issued a new, stricter permit system for Alaska's seafood processing facilities, which requires that seafood processing by-products be discharged at least one nautical mile (1852 m) from shore in waters at least 36.6 m deep. However, some environmental impact is still expected from such ocean disposal. Fish oil is one of the major by-products produced by the seafood industry. It is generally produced in conjunction with the production of fish meal; it is the lipid fraction that can be extracted from fish or fish by-products (Aidos, 2002). Fish oil can be produced both from fish species specifically harvested for this purpose and from the offal and waste resulting from processing higher value species such as salmon, halibut and hake (Boyd et al., 2004). For example, Alaskan seafood processing operations produce approximately 30 million liters of fish oil annually. Fish oil has many food and non-food uses. Fish oil and other seafood processing by-products can be used as a source of renewable energy. The main objective of this chapter is to identify the theoretical and practical issues concerning utilization of seafood processing byproducts as valuable resources for energy production, and review the conversion technologies that could be used to convert such materials into bio-fuels in the forms of bio-diesel and bio-gas. Both bio-diesel and bio-gas are renewable biofuels. Many studies have been carried out to evaluate the potential of using seafood processing by-products for bio-diesel and bio-gas production. The biodiesel can be used as a fuel directly or blended with petroleum diesel and used in conventional diesel engines with few or no modifications. Bio-gas can be used as a fuel for internal combustion engines, turbines, or fuel cells for heat and electrical power generation. In the latter application, part of the heat produced by the electric generator is recovered and used to heat digesters. Removal of moisture and other constituents, such as hydrogen sulfide and ammonia, may be required prior to the end use. Many articles have been published on the importance and methods of bio-gas purification. The bio-gas can also be purified to produce methane gas that can be used as a fuel for transportation vehicles. The detailed descriptions of bio-gas cleaning, purification, and utilization methods are provided by Ravishanker and Hills (1984) and Jensen and Jensen (2000).

22.2 Quantity and quality of various seafood processing by-products The amount and characteristics of effluent streams produced at seafood processing plants largely depend on the type of animals being processed, the parts of animals that are left over, and amount of water added into the byproducts as a result of animal handling and plant facility wash-down. In addition to the quantity, the following characteristics of by-products are important for initial evaluation of their suitability as substrates for production of bio-diesel and

462

Maximising the value of marine by-products

bio-gas: moisture content, oil content, volatile solids or ash content, and protein content. Due to the large variability among different seafood by-products, it is recommended that laboratory or pilot trials be performed to determine the biodiesel or bio-gas yields from actual materials to obtain accurate information for performing the energy production calculations and designing the conversion processes. 22.2.1 Characteristics of fish oil Many factors affect the characteristics of fish oil, including species of origin, the season and the unit operations used by the processors. According to Hardy and Masumoto (1990), fish oils do not have significant differences among species in caloric content but they do differ in the content of essential fatty acids. Steigers (2003) cited the characteristics of fish oil, produced from many species, such as pollock, produced in Alaska: color is amber to light orange; density is 923 kg/ m3; sulfur content is 0.004±0.0084% by weight and the gross heat of combustion is 36.4±36.8 MJ/l. Garcia-Sanda et al. (2003) found that the oil separated from the waste streams of the seafood canning industries had very good characteristics as a fuel. The characteristics of fish oil and fuel-oil No. 1 in Spain, as an example, are compared in Table 22.1. The seafood oil has lower contents of sulfur and ash. It also has a relatively high caloric value, though lower than the value of fuel-oil No. 1. It should be mentioned that while the gross caloric value represents the total heat available in combustion of material, net caloric value represents the energy value after subtracting the energy required for water evaporation in the reaction from the total caloric value. 22.2.2 Characteristics of fish wastes The average composition of various parts of fish carcasses for Pacific cod, pollock and salmon, as reviewed by Babbitt (1990), is shown in Table 22.2. As can be seen, all materials have relatively high moisture contents (>70%) and very low ash contents (<4.1%), indicating that these materials could be highly Table 22.1 Comparison of oil separated from seafood processing by-products and fueloil No. 1 in Spain (Garcia-Sanda et al., 2003) Parameter Gross caloric value, kJ/g Net caloric value, kJ/g Sulfur, % Carbon and hydrogen, % Ash, % Freezing point, ëC Density at 15ëC, kg/m3 Viscosity at 23ëC, N.sec/m2

Seafood oil

No. 1 fuel oil

38.82 36.49 0.09 89 0.28 6.7 957 0.08

42.29 40.19 2.7 ± 10 ± ± ±

Bio-diesel and bio-gas production from seafood processing by-products 463 Table 22.2 Average composition (% wet base) of some fish by-products (averages from data reviewed by Babbitt, 1990) Fish species

Protein

Oil

Ash

Moisture

Pacific cod

Hand filleting Machine filleting

15.0 14.1

4.1 3.8

4.1 3.8

79.2 79.4

Pollock

Hand filleting Machine filleting

11.3 12.5

3.6 3.7

3.6 3.7

81.3 82.0

Salmon

Head Viscera

14.2 17.1

3.9 1.8

3.9 1.8

71.4 78.3

desirable for use as substrates for biological conversion processes. Salmon heads have the highest oil content among the three species examined. There is no significant composition difference between hand and machine filleting. 22.2.3 Characteristics of wastewater from seafood processing plants Carawan (1991) mentioned that the major types of by-products found in seafood processing effluent are blood, offal products, viscera, fins, fish heads, shells, skins and meat `fines'. These constituents contribute significantly to the suspended solids concentration of the effluent stream. The characteristics of liquid effluents (wastewater) from several seafood products are shown in Table 22.3, indicating high organic and oil contents in most streams. The high-strength wastewater shows high turbidity, strong greenish yellow color and strong odors. Moreover, seafood processing wastewater may contain high concentrations of organic nitrogen (up to 0.300 g/L), sulfate (0.6±2.7 g/L), chlorides (8±19 g/L) and brine solution from processing water (Carawan et al., 1979; Omil et al., 1995). Fortunately, these effluents, unlike many industrial effluents, normally do not contain toxic or carcinogenic substances (Afonso and BoÂrquez, 2002).

22.3 Theories and technologies for production of bio-diesel and bio-gas fuels 22.3.1 Bio-diesel production Bio-diesel (fatty acid methyl esters) is attractive because it is made from renewable resources, giving it many environmental and sustainability benefits. Chemically, bio-diesel is made up of alkyl esters of long-chain fatty acids (Meher et al., 2004). The required properties for bio-diesel are specified in the ASTM D6751-03a Standard (ASTM, 2003). According to this standard, some of these properties are required to obtain the best engine performance (e.g., flash point, viscosity, cetane number, carbon residue and free glycerin). Others are needed to assure environmentally friendly fuel usage (e.g., phosphorus content). According to the ASTM D6751-03a standard, a maximum value of 10 ppm is not problematic.

464

Maximising the value of marine by-products

Table 22.3 Characteristics of wastewater from fish processing plants (Carawan et al., 1979) Subcategory Farm-raised catfish Conventional blue crab Mechanized blue crab Alaskan crab meat Alaskan whole crab and crab section Dungeness and Tanner crab Alaskan shrimp West coast shrimp Southern non-breaded shrimp Breaded shrimp Tuna processing Fish meal All salmon Bottom and fin fish All sardines All herring Hand shucked clam Mechanized clam All oysters All scallops Abalone

BOD5 (mg/L)

COD (mg/L)

TSS (mg/L)

Oil and grease (mg/L)

340 4400 600 270

700 6300 1000 430

400 620 330 170

200 220 150 22

330 280±1200 1000±2000 2000 1000 720 700 100±24000 253±2600 200±1000 1300 1200±6000 800±2500 500±1200 250±800 200±10000 430±580

710 550±2000 2000±3700 3300 2300 1200 1600 150±42000 300±5500 400±2000 2500 3000±10000 1000±4000 700±1500 500±2000 300±11000 800±1000

210 60±130 1300±3000 900 800 800 500 70±20000 120±1400 100±800 921 600±5000 600±6000 200±400 200±2000 27±4000 200±300

30 28±600 100±270 700 250 ± 250 20±5000 20±550 40±300 250 600±800 16±50 20±25 10±30 15±25 22±30

Many types of oils and fats have been used for bio-diesel production, including vegetable oils, food grade cooking oils, off-quality and rancid vegetable oils, used cooking oil and animal fats such as lard, tallow, chicken fat and fish oils (Coltrain, 2001). Basically, there are at least three methods for converting oils and fats into bio-diesel, namely, microemulsions, pyrolysis (i.e., thermal cracking) and, the most common, transesterification (Ma and Hanna, 1999; Ghadge and Raheman, 2005). The main purpose of these methods is to lower the viscosity of oils and fats so that they can be combusted in engines without the problems that have been experienced with direct combustion of vegetable oils and animal fat, such as choking of fuel injectors and build-up of carbon deposit in the combustion chambers of engines. Microemulsions and pyrolysis processes Microemulsions made with solvents such as methanol, ethanol and ionic or nonionic amphiphiles have been studied as methods to overcome the high viscosity of vegetable oils (DemirbasÎ, 2003). Amphiphiles are defined as molecules that have an affinity for both aqueous and non-aqueous media. Pyrolysis is the thermal degradation, at high temperature, of vegetable oils by heat in the absence of oxygen, which results in the production of alkenes,

Bio-diesel and bio-gas production from seafood processing by-products 465 alkadienes, carboxylic acids, aromatics and small amounts of gaseous products. Depending on the operating conditions, the pyrolysis process can be divided into three subclasses: conventional pyrolysis, fast pyrolysis and flash pyrolysis (DemirbasÎ, 2003). Ma and Hanna (1999) have mentioned that the equipment for pyrolysis can be costly. In addition, pyrolysis has produced more bio-gasoline than diesel fuel. Thus the product should be examined for its suitability as a fuel for either diesel or gasoline engines. Transesterification As stated above, transesterification is the common method for bio-diesel production; it is also called alcoholysis, and when methanol is used it is called methanolysis (Meher et al., 2004). Although blending of oils with other solvents, and microemulsions lower the viscosity, engine performance problems (e.g., carbon deposit and lubricating oil contamination) still occur (Ma and Hanna, 1999). In transesterification, bio-diesel is produced by chemically reacting vegetable oil or animal fat with an alcohol in the presence of a catalyst (Gerpen, 2005). Several parameters influence the reaction. Among them are the mole ratio of oil to alcohol, reaction temperature, type and concentration of catalyst, mixing intensity, reaction time and free fatty acid and moisture content of the reactants (Madras et al., 2004; Meher et al., 2004). The stoichiometry of the reaction requires 3 mol of methanol and 1 mol of triglyceride to give 3 mol of fatty acid methyl ester and 1 mol of glycerol (Vicente et al., 2004). In practice, the amount of methanol needs to be higher in order to drive the equilibrium to a maximum ester yield (Ma and Hanna, 1999). The general equation of transesterification can be formulated as follows: RCOOR1 + R2OH Ester

Alcohol

Catalyst

ÿÿ! RCOOR2 + R1OH Ester

Alcohol

A transesterification process for biodiesel production using methanol as the reacting alcohol is shown in Fig. 22.1 (Gerpen, 2005). Typically for oils having low levels (less than 1%) of free fatty acids, the one-step esterification reaction is used, in which alcohol, oil and catalyst are combined in a reactor and agitated for some time (e.g., 0.5±1 hour) at an elevated temperature (e.g., 50±60ëC). While for oils having high concentrations of free fatty acids, a two- or three-step process is used. In the two-step process, oil and alcohol react first in the presence of acid catalyst, and then more alcohol is added and allowed to react in the presence of a caustic catalyst. During the three-step process, oil reacts with alcohol first, and then an acid catalyst is added to allow for more reaction. Afterwards, more alcohol is added to react with the product of the first two steps in the presence of a caustic catalyst (Canakci and Van Gerpen, 2001). Following the esterification reaction, glycerol is removed from the alkyl esters using either a settling tank or a centrifuge. After separation, alkyl esters pass through a methanol stripper (either vacuum flash process or falling film

466

Maximising the value of marine by-products

Fig. 22.1

Typical procedure for bio-diesel production (adapted from Gerpen, 2005).

evaporator) for alcohol removal. Then alkyl esters enter a neutralization step, where acid is added to the bio-diesel to neutralize any residual catalyst and to split any soap that may have formed during the reaction. The salts are removed during the water washing step and the free fatty acids stay in the bio-diesel. The water washing step is intended to remove any remaining catalyst, soap, salts, methanol, or free glycerol from the bio-diesel. Following the wash process, any remaining water is removed from the bio-diesel using a vacuum flash process. In some systems the bio-diesel is distilled in an additional step to produce a colorless bio-diesel. Methanol and ethanol are commonly used alcohols, with methanol being the most often used because of its low cost, and its physical and chemical advantages. The reaction can be catalyzed by an alkali (e.g., sodium methoxide, sodium ethoxide, sodium propoxide and sodium butoxide), acids (sulfuric acid, sulfonic acids and hydrochloric acid), or enzymes such as lipases (Ma and Hanna, 1999). The mechanisms of the different catalyzed transesterification processes are described by Meher et al. (2004). Alkali-catalyzed transesterification is much faster than acid-catalyzed transesterification and is most often used commercially. One limitation to the alkali-catalyzed process is its sensitivity to the purity of reactants. It is very sensitive to both water and free fatty acids (Zhang et al., 2003), and has some other drawbacks, including the difficulty of recycling glycerol, as it has some impurities, and the need for removal of the catalyst and treatment of wastewater produced during the production and washing of biodiesel. In particular, several steps such as the evaporation of methanol, removal

Bio-diesel and bio-gas production from seafood processing by-products 467 of saponified products, neutralization, and concentration are needed to recover glycerol as a by-product. 22.3.2 Bio-gas production Bio-gas is produced via anaerobic digestion processes and mainly consists of methane and carbon dioxide. Depending on the chemical composition of substrate and the operating conditions of the digestion process, methane content of bio-gas can range from 50 to 80%. The anaerobic digestion process involves transformation of organic material by a mixed culture of bacteria in the absence of oxygen. It consists of a number of sub-processes that occur in series and parallel manner (Pavlostathis and Giraldo-Gomez, 1991; Batstone et al., 2002) as shown in Fig. 22.2. The particulate organic matter is hydrolyzed by extracellular enzymes of microorganisms to soluble compounds such as amino acids, sugars and long-chain fatty acids. Then the products of the hydrolysis step are fermented into short-chain volatile fatty acids (VFAs), alcohols, ammonia and hydrogen sulfide. The VFAs (other than acetate) and alcohols are further converted by acetogenesis bacteria to acetic acid, hydrogen and carbon dioxide, which are then converted by methanogenic bacteria to methane and carbon dioxide. The anaerobic digestion process is normally carried out in an anaerobic digester (bioreactor) that is a closed vessel, which is gas-tight, thermally insulated in most cases, and equipped with a heating and a mixing device (Demuynck et al., 1984). The digesters are often cylindrical for achieving good mixing and some of them use cone-shaped bottoms to facilitate sludge removal (Metcalf and Eddy, 2003). The digester top can be fixed or floating. A floating

Fig. 22.2 Main sub-processes involved in anaerobic digestion process (adapted from Pavlostathis and Giraldo-Gomez, 1991; Elmitwalli, 2000; Batstone et al., 2002).

468

Maximising the value of marine by-products

top provides expandable gas storage with pressure control but is more expensive and difficult to manage (Erickson et al., 2004). The major factors that need to be considered when designing and operating anaerobic digesters include temperature, pH, organic loading rate, biodegradability and nutrient availability of substrate, and retention time. Anaerobic digestion occurs in a wide range of temperatures. It can occur at a temperature as low as 4ëC, but the digestion rate increases with the increase of temperature. Three temperature ranges have been explored for anaerobic digestion, including the psychrophilic range (10±20ëC), the mesophilic range (20±45ëC, typically 35ëC), and thermophilic range (45±60ëC, typically 55ëC). Psychrophilic digestion has been primarily associated with covered lagoon digesters operating at ambient temperature. Anaerobic digesters, however, are commonly designed to operate in either mesophilic or thermophilic range. Digestion at these higher temperatures handles higher organic loading rates and requires shorter solids retention time and is therefore more space-efficient. Also, a higher temperature increases the destruction of pathogens that may be present in wastewater. To reduce the heat loss and increase net energy production, digesters should be insulated. Many types of insulation materials, including both natural and synthetic materials, are used (Demuynck et al., 1984). The schematics of some commercially available anaerobic digesters used for wastewater treatment are shown in Fig. 22.3. Conventional anaerobic digesters used for wastewater treatment include batch or fed-batch, completely mixed, and plug-flow digesters. These digesters are suitable for treating concentrated waste. To improve the economics of treating dilute wastewater, a number of new biomassretaining digesters, often called high-rate digesters, are being developed. These biomass-retaining digesters are designed to provide special mechanisms to keep bacteria and solids in the digesters longer than the treated liquid fraction. The biomass-retaining mechanisms include internal solids settling, external solids separation and recycling, and biomass immobilization with a fixed or suspended medium. Major types of biomass-retaining digesters include anaerobic contact reactor (Defour et al., 1994; Masse and Masse, 2000), anaerobic sequencing batch reactor (ASBR) (Zhang et al., 1997, 2001; Dugba et al., 1999), upflow sludge blanket reactor (UASB) (Lettinga et al., 1980; Boardman et al., 1995; PunÄal and Lema, 1999; Field, 2004), anaerobic filter (Masse and Masse, 2000; Frankin, 2001), fluidized bed reactor, and anaerobic mixed biofilm reactor (Romano and Zhang, 2005). For space limited areas, these digesters are more suitable for treating dilute wastewater, for example, such as the wastewater from fish canning plants (Palenzuela-RolloÂn, 1999). According to the survey conducted by Demuynck et al. (1984) in Europe, 41% of the wastewater is treated using UASBs and 19% of industrial bio-gas plants use ACRs. Anaerobic biofilter and completely mixed digesters represent 15 and 14%, respectively. These percentages are changing, as the application of different digestion technologies evolves. Compared to anaerobic digesters that are designed for wastewater treatment, there are fewer digester designs available for treatment of solid wastes, which typically have less than 90% moisture. Different digester designs for treating

Bio-diesel and bio-gas production from seafood processing by-products 469

Fig. 22.3

Schematics of different designs of anaerobic digesters for wastewater treatment.

solid wastes are shown in Fig. 22.4. The simplest design is the batch digester with leachate recirculation. In such a system, the feedstock is loaded with a certain amount of inoculum at the beginning of the digestion time. Then the digester is closed and maintained at the desired temperature. During the digestion course, leachate is recalculated to provide the mixing. At the end of the digestion period, the digester is emptied, keeping a certain amount of the digestate as inoculum for the next batch. An improvement of batch digester is the sequential batch anaerobic composting (SEBAC) (Chynoweth et al., 1991) which consists of three batch digesters working in sequential order. The new batch digester is inoculated with the liquid from the old batch digester. Lissens et al. (2001) described the

470

Maximising the value of marine by-products

Fig. 22.4 Schematics of different designs of anaerobic digesters for solid waste treatment.

designs of Kompogas, Dranco, and Valorga digesters. In the Kompogas process, the wastes move via horizontal plug-flow in cylindrical digesters. The flow is aided by slowly rotating impellers. In the Dranco process, the plug-flow occurred vertically. Mixing is provided by recirculation of digesting waste between the reactor bottom and top. The flow of digesting waste in the Valorga process is circular plug-flow in cylindrical reactors. The mixing in this system is provided by bio-gas injection from the reactor bottom. The anaerobic phased solids digester (APS-Digester) as shown in Fig. 22.5 is a new digester design which combines the features of batch and continuous digesters (Zhang and Zhang, 1999; Hartman, 2004). The system typically consists of five reactors, including four hydrolysis reactors and one bio-gasification reactor. Feedstock is loaded into each of the hydrolysis reactors, acted on by extra-cellular

Bio-diesel and bio-gas production from seafood processing by-products 471

Fig. 22.5

Schematic of anaerobic phased solids digester system.

enzymes and acidogenic bacteria, thereby liquefied and converted to simple organic acids. These acids are collected and transferred to the bio-gasification reactor, where they are reduced further into bio-gas by methanogenic bacteria. Multiple hydrolysis reactors allow for a time separation between the beginnings of different batch hydrolysis reactions. This time separation contributes to a relatively constant level bio-gas production rate despite the batch loading and operational schedule. After digestion is complete for each batch feedstock, the digested solids and liquid are removed from the respective hydrolysis reactor. The APS-Digester has been tested in the laboratory with a variety of organic solid wastes and is now being scaled up for commercial applications.

22.4 Potential yields and quality of bio-diesel and bio-gas fuels 22.4.1 Yields, and chemical and physical properties of bio-diesel The literature on production of bio-diesel from fish oil is scarce. Recently research was conducted at the University of California, Davis, to produce bio-

472

Maximising the value of marine by-products

Table 22.4 Bio-diesel yield from salmon oil separated from fish hydrolysate

First step Oil, (g) Methanol, g (% w of oil) H2SO4, g (% w of oil) Second step Pretreated oil, g Methanol, g (% w of oil) KOH, g (% w of oil) H2SO4, g (% w of oil) Third step Pretreated oil, (g) Methanol, g (% w of oil) KOH, g (% w of oil) Biodiesel yield, g (%) Glycerol yield, g (%)

Two steps

Three steps

30.1 9.58 (31.8) 0.27 (0.9)

30.1 6.25 (20.8)

31.5 5.25 (16.7) 0.24 (0.7)

32.8 3.3 (10.1) ± 0.27 (0.8)

± ± ± 29.4 (97.5) 5.33 (17.7)

31.0 5.32 (17.2) 0.24 (0.8) 29.1 (96.6) 4.85 (16.1)

diesel from the oil separated from salmon hydrolysate. The bio-diesel yields from the two- and three-step procedures were compared as shown in Table 22.4. In the two-step procedure, sulfuric acid was added with methanol in the first step. Then potassium hydroxide was added later. In the three-step procedure, no acid was added during the first step because the pH of the salmon hydrolysate was about 3.7 due to formic acid addition during its production. In the second step, sulfuric acid was added to reduce the level of free fatty acids, via converting the free fatty acids to esters, to a level at which soap is not formed during the alkaline treatment (Canakci and Van Gerpen, 2001). In the third step, potassium hydroxide was added as a caustic catalyst. All experiments were performed at a temperature of 52±55ëC with a mixing intensity of 600 rpm. The oil and chemical reagents used, and the bio-diesel and glycerol yields are shown in Table 22.4. There was no significant difference in the bio-diesel yield between the two procedures. The bio-diesel produced was orange-red in color. It should be mentioned that the yield presented here is the yield without washing of the bio-diesel; a slightly lower yield is expected after a washing step, as some of the soap and other impurities will be removed. A more detailed study is under way to optimize the yield using the minimum amount of catalysts and to determine the characteristics of bio-diesel produced from salmon oil. Meher et al. (2004) showed the general characteristics of bio-diesel in different countries. Some selected properties of No. 2 diesel and biodiesel are compared in Table 22.5. Tyson (2004) stated that the biodiesel characteristics (Table 22.5) are not based on the specific raw materials or the manufacturing process used to produce the bio-diesel. However, the ASTM D6751 standard is based on the physical and chemical properties needed for safe and satisfactory

Bio-diesel and bio-gas production from seafood processing by-products 473 Table 22.5 Selected properties of typical No. 2 diesel and bio-diesel fuels (Tyson, 2004) Fuel property Fuel standard Net heating value, MJ/L Kinematic viscosity, mm2/sec at 40ëC Specific gravity, at 15ëC Density, kg/m3 at 15ëC Water and sediment, vol % Carbon, wt % Hydrogen, wt % Boiling point, ëC Flash point, ëC Cloud point, ëC Pour point, ëC Cetane number

Diesel

Bio-diesel

ASTM D975 ~36.0 1.3±4.1 0.85 848 0.05 max 87 13 180±340 60±80 ÿ15 to 5 ÿ35 to ÿ15 40±55

ASTM D6751 ~33.0 4.0±6.0 0.88 878 0.05 max 77 12 315±350 100±170 ÿ3 to 12 ÿ15 to 10 48±65

diesel engine operation. No data are available in the literature about the characteristics of bio-diesel produced from fish oil. 22.4.2 Yields, and chemical and physical properties of bio-gas Many studies have been made of methane production from fish by-products. Folkecenter (2005) identified some values for the bio-gas yield from fish oil and different fish by-products (Table 22.6). Some of the published data on bio-gas production from anaerobic digestion and codigestion of fish by-products under different experimental conditions are given in Table 22.7. The bio-gas yield and composition varied with the actual material digested. Lanari and Franci (1998) studied the anaerobic digestion of solid materials removed from fish-farm effluents, under psychrophilic conditions (24±25ëC). Their results showed that methane yield ranged from 0.4 to 0.46 L/gVS. Table 22.6 Some values of bio-gas and methane yields from fish oil and fish processing by-products (Folkecenter, 2005) By-product type

Fish oil/meal industry Fish filleting industry Herring cannery Mackerel cannery Shellfish industry Smoke fish industry

Volatile solids content %

Methane yield L/gVS

Bio-gas production m3/ton (wet substrate)

8±24 7±20 8±11 17±23 20±26 8±44

0.36 0.45 0.55 0.55 0.75 0.59

43±136 47±135 66±91 140±190 225±281 71±389

Table 22.7 Bio-gas production from fish by-products under different experimental conditions Feedstock

Reactor type

Tempera- HRT, SRT, OLR, VS ture, ëC day day g[VS]/L/day destruction, %

Fish by-product Fish by-product Fish by-product Fish by-product + wood Saline fish by-products Saline fish by-products Saline fish by-products Saline fish by-products Saline fish by-products Sisal pulp (SP) and fish by-products (FW) Sisal pulp (SP) and fish by-products (FW) Sisal pulp (SP) and fish by-products (FW) Sisal pulp (SP) and fish by-products (FW) Solid by-product removed from fish farm Solid by-product removed from fish farm Solid by-product removed from fish farm

ASBR ASBR ASBR ASBR Semi-continuous Semi-continuous Semi-continuous Semi-continuous Semi-continuous Batch

35 35 35 35 35 35 35 35 35 27

12 5 7 7 24 27.5 27.9 41.2 60.0 24

Batch

27

Batch Batch

0.7 1.6 0.83 1.5 2.2 1.8 1.8 1.1 0.9

75.9 87.9 89.2 89.7 48.2 48.2 47.4 59.0 61.9

1.1 2.3 1.0 1.1 0.81 0.79 0.58 0.51 0.42

Methane content, %

1.57 1.44 1.21 0.73 0.39 0.43 0.33 0.43 0.45 0.31

78.6 76.5 81.3 79.3 50.9 51.7 48.9 50.0 54.1 61

24

0.62

64

27

24

0.48

65

27

24

0.44

58

Upflow 24±25 anaerobic reactor Upflow 24±25 anaerobic reactor Upflow 24±25 anaerobic reactor

68 70 66 52

Bio-gas Bio-gas production yield rate (L/g[VS (L/L. day) added]

38

0.227

about 98

0.13

0.46

80

31

0.345

about 96

0.21

0.45

80

22

0.751

about 93

0.38

0.4

80

Reference

Hartman et al. (2001) Hartman et al. (2001) Hartman et al. (2001) Hartman et al. (2001) Gebauer (2004) Gebauer (2004) Gebauer (2004) Gebauer (2004) Gebauer (2004) Mdhandete et al. (2004) Mdhandete et al. (2004) Mdhandete et al. (2004) Mdhandete et al. (2004) Lanari and Franci (1998) Lanari and Franci (1998) Lanari and Franci (1998)

Bio-diesel and bio-gas production from seafood processing by-products 475 During anaerobic digestion of blue crab cooking wastewater in laboratoryscale, upflow anaerobic reactors, Rodenhizer and Boardman (1999) found that bio-gas production ranged from 6.6 L [gas]/L [feed] to 10.0 L [gas]/L [feed]. In the bio-gas, methane, carbon dioxide and hydrogen sulfide comprised 68, 28 and 1.5% of the gas, respectively. O'Keefe et al. (1996) studied the feasibility of using a hybrid sludge-bed filter (HSBF) reactor for anaerobic composting of crab residuals at 35ëC. It should be mentioned that the HSBF is a tank containing granular sludge in the bottom and biofilters are installed on the top. Thus it has the advantages that come with both the UASB and anaerobic filter. The reactor system consisted of a leach bed reactor and an HSBF reactor. A leachate volume of about 4 liters was maintained in the HSBF reactor. Two volumes (4 L and 10.5 L) of leachate were maintained in the leach bed reactor. With the smaller leachate volume, the operation was characterized as a percolating operation, whereas with the larger leachate volume, the operation was characterized as a flooded operation. During the percolating operation, the methane yield of 0.25 L/gVS and VS destruction of 78% were obtained after 47 days. During the flooded operation, an average methane yield of 0.29 L/gVS and a VS destruction of 50% were obtained after 28 days. The reactors were stable, producing a biogas with high methane content (>70%). Hartman et al. (2001) studied the codigestion of fish (salmon) and wood wastes in an anaerobic sequencing batch reactor (ASBR) at 35ëC. They found that stable bio-gas production could be obtained at loading rates of less than 2 g VS/L/day. At loading rates of 2 g VS/L/day, reactor failure occurred. The ammonia concentration was 700 mg/L. The authors attributed this failure to the high level of long-chain fatty acids in the fish oils. Adding the steam exploded wood waste resulted in a 10% increase in bio-gas production from 1.0 to 1.1 L/L/ day (Table 22.7). The methane content of the biogas was 81.3% for fish waste, and averaged 78.5% for the fish and wood waste. The addition of wood waste may contribute to reactor stability without necessitating an increase in reactor volume, which could allow a higher loading rate, thereby decreasing the reactor volume required. During batch codigestion at 35C, Callaghan et al. (1999) found that codigestion of fish offal with cattle slurry resulted in an increase in the methane yield compared with the digestion of cattle slurry alone. VS reductions of 47.3 and 31.1% were achieved, respectively. Palenzuela-Rollon et al. (2002) treated a synthetic wastewater process, simulating that from canning sardines and tuna, using an upflow anaerobic sludge blanket (UASB) reactor operated at 30ëC. Total ammonia concentration ranged from 243±299 mg/L. A methane yield of 0.23 L/g was calculated. Achour et al. (2000) studied the anaerobic digestion of tuna processing liquid effluent in an upflow anaerobic cylindrical fixed bed reactor operated at 30ëC. The results showed that a methane yield of 0.25 m3/kg COD degraded (0.18 m3 CH4/kg VS degraded) was achieved. Fish-oil by-products were used as an additional feedstock during the anaerobic codigestion. According to Francese et al. (2000), fish oil by-product is a residue of the manufacturing of fish oil having 32.8% TS and 91.2% VS.

476

Maximising the value of marine by-products

During the anaerobic codigestion of pig manure (97% v/v), fish oil by-product (2% v/v) and bentonite-bound oil (1%v/v), a bio-gas yield of 0.184 L/g VS with 65% CH4 in the bio-gas was obtained at 30ëC temperature and a 15-day hydraulic retention time (HRT).The HRT is defined as the average time that a liquid substrate is retained in the reactor and calculated by dividing the reactor volume by daily feeding rate. The codigestion of fish oil by-product and bentonite-bound oil with pig manure increased the net daily bio-gas production four-fold compared to digestion of pig manure alone.

22.5 Problems encountered and possible approaches for overcoming them 22.5.1 Problems concerning bio-diesel production Generally, free fatty acids in the oil are known to react with the alkaline catalyst and form saponified products during transesterification reactions. They also lead to longer production processes and an increase in the production cost (Meher et al., 2004). Therefore, purification of the bio-diesel produced is essential. To overcome the drawbacks of using alkali-catalysis, enzymatic processes using both extracellular and intracellular lipases have recently been developed (Fukuda et al., 2001). A comparison between the alkali-catalysis and lipasecatalysis methods for bio-diesel fuel production is shown in Table 22.8. A lipase-catalysis process seems to be superior in terms of the quality of the final product and recovery of glycerol. However, the application of enzymatic catalysis on an industrial scale may not be feasible because of the high cost associated with enzymes (Fukuda et al., 2001; Jaeger and Eggert, 2002). Use of the supercritical methanol method, without any catalyst, is proposed as a way to solve the problems encountered in alkali-catalysis method for biodiesel production. The study performed by Meher et al. (2004) on the transesterification of rapeseed oil showed that the supercritical methanol method had a higher bio-diesel yield than conventional methods, due to the higher conversion (95%) of free fatty acids to methyl esters. The saturated fatty acids Table 22.8 A comparison between alkali-catalysis and lipase-catalysis transesterification (Fukuda et al., 2001)

Reaction temperature Free fatty acids in raw materials Water in raw materials Yield of methyl esters Recovery of glycerol Purification of methyl esters Production cost of catalyst

Alkali-catalysis process

Lipase-catalysis process

60±70ëC saponified products interference with the reaction normal difficult repeated washing cheap

30±40ëC methyl esters no influence higher easy none relatively expensive

Bio-diesel and bio-gas production from seafood processing by-products 477 were completely converted to methyl esters at temperatures above 400ëC, while the unsaturated fatty acids required a lower temperature of 350ëC. Thus it is expected that use of the supercritical methanol method at a temperature below 350ëC will be suitable for bio-diesel production from fish oil due to the relatively high content of unsaturated fatty acids in fish oil. According to Aidos (2002), the saturated fraction of fish oil ranged from 22.5 to 35.8%. The amount and variety of fatty acids present in fish oil depend on fish species and the biological stage, as well as fish diet, fishing location, ocean temperature, and nutritional and spawning state. Cao et al. (2005) studied the production of bio-diesel via non-catalytic transesterification using supercritical methanol with propane as a co-solvent. The reaction could be completed in a very short time. They found that at 280ëC more than 98% of triglyceride was converted to methyl esters after 10 minutes, while at 300ëC, the total conversion was achieved within 5 minutes. Compared with the catalytic processes, purification of products is much simpler and more environmentally friendly. However, the process requires high temperatures and pressures, which means higher production costs and energy consumption. Research is still being pursued by various researchers to determine the most effective method for producing a high quality and low cost bio-diesel from fish oil. 22.5.2 Problems concerning biogas production Anaerobic digestion has been proven to be an excellent method for treating highstrength wastewater from the economic and sustainability points of view (Lettinga, 2001). However, anaerobic treatment of marine wastewater can be inhibited by high concentrations of sodium and sulfide (Soto et al., 1991; Vidal et al., 1997). Feijoo et al. (1995) mentioned that seafood-processing wastewater contains high concentrations of different ions, mainly Na+, Clÿ and SO42ÿ. The sodium concentration can be as high as 12 g Na+/L in seawater. Thus careful operation of the digester and selection of the appropriate anaerobic inoculum are required (Aspe et al., 1997). Boardman et al. (1995) studied the effect of different sodium concentrations on the specific methanogenic activity (SMA) during digestion of clamprocessing wastewater at 32ëC. Their data showed that methanogenesis was inhibited by NaCl added to the wastewater. A ten-fold decrease in methanogenic activity was observed for a three-fold increase in Na+ concentration: the SMAs were 0.4, 0.24, 0.18, 0.06 and 0.04 g CH4 COD/g VSS/day at salt concentrations of 4.2, 5.3, 6.3, 8.4 and 12.6 g Na+/L, respectively. Also, total cumulative methane production was 78, 55, 47, 10 and 5% of the theoretically possible production at the various Na+ concentrations above. Sodium levels at about 5250 mg/L significantly impacted the performance of a UASB reactor operated at 32ëC. Boardman et al. (1995) also found that the methane production efficiency of the UASB reactor was somewhat better at an organic loading rate (OLR) of 13.8 g COD/L/day than at an OLR of 16.3 g COD/L/day. The methane content in biogas ranged between 70 and 80%.

478

Maximising the value of marine by-products

Gebauer (2004) studied the mesophilic (35ëC) anaerobic treatment of sludge from saline fish farms under different HRTs (24, 27.5, 27.9, 41.2 and 60 days) in a semi-continuous digester. Using undiluted influent having a salinity of 35% and Na concentration of 10.2 g/L, an average methane content of 51.1% could be achieved. The process was stable with a VFA concentration of 4.2±7.2 g/L as acetic acid, indicating that the digestion was inhibited. Methane yield ranged from 0.16 to 0.24 L/gVS. While using water-diluted substrate that had 17.5% salinity and 5.3 g [Na]/L, a stable operation was obtained at 30-day HRT with total the VFA concentration being 0.6 g/L as acetic acid and a methane yield of 0.22 L/gVS. The methane content of the biogas was 57.6%. With this diluted substrate the hydrogen sulfide content of the biogas was almost half (1±1.6%) of that obtained with the undiluted substrate, which is an advantage for the final use of the bio-gas. Vidal et al. (1997) studied the anaerobic digestion of wastewater from a fish meal processing factory. An anaerobic filter was used, which was started with marine sediments as an inoculum. The reactor was operated at 37ëC under OLRs. Concentrations of 30 g [NaCl]/L in this wastewater had no toxic effect as indicated by the methane production. The authors attributed this either to the use of marine sediment as the inoculum or to the adaptation of bacteria to concentrations over 10 g Na+/L. Methane production yield expressed as L/g COD are calculated from the data of Vidal et al. (1997) at different OLRs (Fig. 22.6). The maximum methane yield was obtained at an OLR of 5.7±7.1 g/L/day. Sulfate abatement also increased with greater OLRs. A reduction of methane

Fig. 22.6

Methane yield from anaerobic digestion of fish meal wastewater in an anaerobic filter (calculated from Vidal et al., 1997).

Bio-diesel and bio-gas production from seafood processing by-products 479 yield per unit of converted COD was observed due to the effect of sulfate on methanogenesis. Aspe et al. (1997) mentioned that marine sediment adapted better and faster to the saline substrate at 37ëC. They observed a 50% inhibition of methanogenic activity at 0.22 g [H2S]/L, 53 g [Na+]/L and 10 g [SO4ÿ2]/L. According to Ward and Slater (2002), high levels of ammonia are released during anaerobic digestion of fish by-products, which then inhibits the digestion process. High ammonia concentrations coincide with high pH values and cause inhibition or total cessation of anaerobic digestion, especially at high operating temperatures (Van Velsen, 1981). This is due to the fact that at high digestion temperatures and pH values, the free ammonia concentration increases. McCarty (1964) reported that free ammonia concentrations exceeding 150 mg/L were toxic to methanogenesis. However, the adaptation of the inoculum and many other factors could affect the response of bacteria to ammonia. It is important to have adapted inoculum, which can handle high concentrations of ammonia, sulfate and salts, for starting an anaerobic digester to treat seafood processing by-products that normally have high concentrations of these constituents. According to Omil et al. (1995), the adaptation to high salinity, with the antagonistic effects on sodium caused by the presence of other ions, makes it possible to operate reactors at high sodium concentrations (5±12 g/L). Moreover, marine sediment inoculum appears to be a good option as well. There are other techniques to overcome the problems of inhibition in anaerobic digesters. One of them is dilution of the feedstock with fresh water. However, this option needs to be carefully assessed, because increased wastewater volume may require greater reactor volume to treat, which in turn increases the total cost of the reactor and perhaps affects the total economics of the digester systems. Besides the aforementioned challenges, there could be other constraints on producing bio-gas and bio-diesel from seafood by-products. In fact, these constraints apply to other biomass technologies as well. Among them are the high capital cost of the processing plant and lack of financial incentives.

22.6

Future research needs

More research is needed in the future to study and optimize various parameters involved in the bio-diesel production from fish oil and develop cost-effective technologies. Optimum quantities of alcohol and the best particular catalyst for producing bio-diesel from fish oil via transesterification still need to be determined. A continuous production process needs to be developed. Yields and quality of bio-diesel produced using different catalysts should be evaluated. An economic analysis of production in both batch and continuous reactors is required. Compared to fish oil, other seafood processing by-products are much more variable in their moisture content and chemical composition. Application of anaerobic digestion technologies is often site-specific. More research is needed to demonstrate the application of various anaerobic digesters for treatment of

480

Maximising the value of marine by-products

both liquid and solid streams. Process parameters, such as organic loading rate (OLR), and hydraulic and solid retention times, should be investigated for specific materials. Scientific documentation and report of by-product characteristics and the performance of anaerobic digesters in terms of bio-gas and methane yields, total solid and volatile solid destruction, and stability of the process, will benefit the further development of science and engineering involved in anaerobic digestion technologies.

22.7

Summary

Utilization of various seafood processing by-products as valuable resources instead of throwing them away as wastes is important from the standpoint of both sustainability, and environmental and public health protection. Many seafood byproducts have a high organic content and are biological degradable, and therefore, are desirable substrates for the production of bio-fuels. Fish oil can be converted into bio-diesel and many liquid or solid materials can be converted into bio-gas, though some unique characteristics of such materials, such as high salt, sulfur and nitrogen contents, need to be considered. In the decision-making process for assessing the costs and benefits of bio-diesel or bio-gas production, performing energy and mass balance calculations is often necessary. As pointed out in this chapter, many factors influence the characteristics of fish oil and other by-products and should be considered during the selection of an existing conversion technology or development of new technologies. As the seafood industry puts more emphasis on the collection and utilization of their by-products, the demand for efficient and cost effective conversion technologies will grow. There are only limited research data available in the literature on biodiesel production from fish oil. Most of the past research on biogas production has been concentrated on the treatment of wastewater generated at seafood processing plants. Based on the energy density of the materials, the by-product steams that have lower moisture content are preferred substrates for bio-gas production. More research is needed to investigate the conversion of these materials for energy production. More scientific publications that document the characteristics of specific seafood by-products and performance of conversion technologies are needed to increase the knowledge and data bases, which will aid in the development and application of conversion technologies for bio-diesel or bio-gas production.

22.8

Sources of further information and advice

Among the useful books on the basics of anaerobic digestion and solving problems in the operation of bio-gas plants are Demuynck et al. (1984) and Metcalf and Eddy (2003). More information about anaerobic digestion of industrial wastewater using UASB reactors can be found on the website

Bio-diesel and bio-gas production from seafood processing by-products 481 developed by Field (2004). Advice on implementing sustainable and robust environmental protection technologies (e.g., anaerobic reactors) can be obtained from the Lettinga Associates Foundation for Environmental Protection & Resource Conservation, at Wageningen University, The Netherlands. Guidance and recommendations on the application of anaerobic reactors, especially the anaerobic phased solids digester system, can be obtained from the research group of Prof. Ruihong Zhang, Department of Biological and Agricultural Engineering, University of California, Davis. More information about the benefits of bio-diesel, methods for bio-diesel production, making bio-diesel at a lab-scale, applications of bio-diesel, environmental impacts of bio-diesel applications, and suppliers in different countries can also be found on: http://journeytoforever.org/biodiesel_meth.html. http://journeytoforever.org/biodiesel_link.html Many articles have been published in journals and magazines such as Bioresource Technology, Transactions of the ASAE, and Biocycle. Among them, two excellent review articles (Ma and Hanna, 1999; Meher et al., 2004) which contain much valuable theoretical and practical information about biodiesel production. More information about bio-diesel production and purification can be found in Biodiesel Industry Directory (2005), on the website of http:// bdid.texterity.com/bdid/2005/. Many useful links to this site can be found regarding many aspects of bio-diesel production, consulting and application.

22.9

List of abbreviations

ACR AMBR APS-Digester ASBR BOD COD CSTR HRT HSBF LCFA OLR SEBAC SMA TS TSS UASB VFA VS

anaerobic contact reactor anaerobic mixed biofilm reactor anaerobic phased solids digester anaerobic sequencing batch reactor biological oxygen demand chemical oxygen demand constantly stirred tank reactor hydraulic retention time hybrid sludge-bed filter long chain fatty acids organic loading rate sequential batch anaerobic composting specific methanogenic activity total solids total suspended solids upflow anaerobic sludge blanket reactor volatile fatty acids volatile solids

482

Maximising the value of marine by-products

22.10

References

and HAMDI, M. (2000). Design of an integrated bioprocess for the treatment of tuna processing liquid effluents. Process Biochemistry 35: 1013±1017. Â RQUEZ, R. (2002). Review of the treatment of seafood processing AFONSO, M.D. and BO wastewaters and recovery of proteins therein by membrane separation processes ± prospects of the ultrafiltration of wastewaters from the fish meal industry. Desalination 142: 29±45. AIDOS, I. (2002). Production of high-quality fish oil from herring byproducts. PhD Thesis, Wageningen University, Wageningen, The Netherlands. ASPEÂ, E., MARTI, M.C. and ROECKEL, M. (1997). Anaerobic treatment of fishery wastewater using a marine sediment inoculum. Water Research 31 (9): 2147±2160. ASTM, AMERICAN SOCIETY FOR TESTING AND MATERIALS. (2003). Standard specification for biodiesel fuel blend stock (B100) for middle distillate fuels. ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, USA. BABBITT, J.K. (1990). Intrinsic quality and species of North Pacific fish. In Proceedings of International By-products Conference, April 25±27, 1990, Alaska Sea Grant College Program, University of Alaska Fairbanks, Alaska, USA. ACHOUR, M., KHELIFI, O., BOUAZIZI, I.

BATSTONE, D.J., KELLER, J., ANGELIDAKI, I., KALYUZHNYI, S.V., PAVLOSTATHIS, S.G., ROZZI, A.,

and VAVILIN, V.A. (2002). Anaerobic Digestion Model No. 1. International Water Association (IWA) task group for mathematical modeling of anaerobic digestion processes. IWA Publishing, London, UK. BIODIESEL INDUSTRY DIRECTORY (2005). http://bdid.texterity.com/bdid/2005/ accessed on 18/10/2005. BOARDMAN, G.D., TISINGER, J.L. and GALLAGHER, D.L. (1995). Treatment of clam processing wastewaters by means of upflow anaerobic sludge blanket technology. Water Research 29(6): 1483±1490. BOYD, M., MURRAY-HILL, A. and SCHADDELEE, K. (2004). Biodiesel in British Columbia feasibility study report. WISE Energy Co-op/Eco-Literacy Canada. http:// www.citygreen.ca/pdfs/Biodiesel-in-BC-Appendices.pdf. CALLAGHAN, F.J., WASE, D.A.J., THAYANITHY, K. and FORSTER, C.F. (1999). Co-digestion of waste organic solids: batch studies. Bioresource Technology 67: 117±122. CANAKCI, M. and VAN GERPEN, J. (2001). Biodiesel production from oils and fats with high free fatty acids. Transactions of the ASAE 44(6): 1429±1436. CAO, W., HAN, H. and ZHANG, J. (2005). Preparation of biodiesel from soybean oil using supercritical methanol and co-solvent. Fuel 84: 347±351. CARAWAN, R.E. (1991). Plants wastes management guidelines. Aquatic fishery products. Department of Food Science, North Carolina State University. http:// www.p2pays.org/ref/02/01796.pdf. Accessed on 15/3/2005. CARAWAN, R.E., CHAMBERS, J.V., ZALL, R.R. and WILKOWSKE (1979). Water and wastewater management in food processing. Spin-off on seafood water and wastewater management. Extension special report No. AM-18F, January, North Carolina State University, Cornell University and Purdue University, Raleigh, North Carolina, USA. CHAMP, M.A., O'CONNOR, T.P. and KILHOPARK, P. (1981). Ocean dumping of seafood wastes as a waste management alternative. In: (Otwell, W.S., Ed.): The Proceedings of Seafood Waste Management in the 1980s Conference, Orlando, Florida, September SANDERS, W.T.M., SIEGRIST, H.

Bio-diesel and bio-gas production from seafood processing by-products 483 23±25, 1980.

and LIU, K. (1991). A novel process for anaerobic composting of municipal solid waste. Applied Biochemistry and Biotechnology 28/29: 421±432. COLTRAIN, D. (2001). Biodiesel: is it worth considering. Department of Agricultural Economics, Kansas Cooperative Development Center, Kansas State University, Manhattan, Kansas, USA. http://www.agecon.ksu.edu/renewableenergy/pdfs/ Biodiesel%20Is%20it%20Worth%20Considering.pdf. DEFOUR, D., DERYCKE, D., LIESSENS, J. and PIPYN, P. (1994). Field experience with different systems for biomass accumulation in anaerobic reactor technology. Water Science and Technology 30(12): 181±191. DEMIRBASÎ, A. (2003). Biodiesel fuels from vegetable oils via catalytic and non-catalytic supercritical alcohol transesterifications and other methods: a survey. Energy Conversion and Management 44: 2093±2109. DEMUYNCK, M., NYNS, E.J. and PALZ, W. (1984). Biogas Plants in Europe. A Practical Handbook. Solar energy R&D in the European community. Series E, Energy from biogas: v.6. D. Reidel Publishing Company, Dordrecht, Holland. DUGBA, P.N., ZHANG, R.H., RUMSEY, T.R. and ELLIS, T.G. (1999). Computer simulation of a two-stage anaerobic sequencing batch reactor system for animal wastewater treatment. Transactions of the ASAE 42(2): 471±477. ELMITWALLI, T.A. (2000). Anaerobic treatment of domestic sewage at low temperature. PhD Thesis, Wageningen University, Wageningen, The Netherlands. ERICKSON, L.E., FAYET, E., KAKUMANU, B.K. and LAWRENCE, C. (2004). In: Carcass Disposal: A Comprehensive Review National Agricultural Biosecurity Agricultural Biosecurity Center, Kansas State University, Manhattan, Kansas, USA. FEIJOO, G., SOTO, M., MENDEZ, R. and LEMA, J.M. (1995). Sodium inhibition in the anaerobic digestion process: Antagonism and adaptation phenomena. Enzyme and Microbial Technology 17: 180±188. FIELD, J. (2004). Anaerobic sludge bed technology pages. http://www.uasb.org/. Accessed 29/5/2005. FOLKECENTER (2005). Folkecenter biogas technology for licence production. http:// www.folkecenter.dk/en/biogas/Folkecenter_biogas_technology.pdf. 18/10/2005 Â RDOBA, P.R. and SIN Ä ERIZ, F. (2000). FRANCESE, A.P., ABOAGYE-MATHIESEN, G., OLESEN, T., CO Feeding approaches for biogas production from animal wastes and industrial effluents. World Journal of Microboiology & Biotechnology 16: 147±150. FRANKIN, R.J. (2001). Full-scale experiences with anaerobic treatment of industrial wastewater. Water Science and Technology 44 (8): 1±6. FUKUDA, H., KONDO, A. and NODA, H. (2001). Biodiesel fuel production by transesterification of oils. Journal of Biosience and Bioengineering 92 (5): 405±416. GARCIA-SANDA, E., OMIL, F. and LEMA, J.M. (2003). Clean production in fish canning industries: recovery and reuse of selected wastes. Clean Technologies and Environmental Policy 5 (3±4): 289±294. GEBAUER, R. (2004). Mesophilic anaerobic treatment of sludge from saline fish farm effluents with biogas production. Bioresource Technology 93: 155±167. GERPEN, J.V. (2005). Biodiesel processing and production. Fuel Processing Technology 86 (10): 1097±1107. GHADGE, S.V. and RAHEMAN, H. (2005). Biodiesel production from mahua (Madhuca indica) oil having high free fatty acids. Biomass and Bioenergy 28 (6): 601±605. HARDY, R.W. and MASUMOTO, T. (1990). Specifications for marine by-products for CHYNOWETH, D.P., BOSCH, G., EARLE, J.F.K., LEGRAND, R.

484

Maximising the value of marine by-products

aquaculture. In the proceedings of International by-products conference, April 1990, Anchorage, Alaska. HARTMAN, K., ZHANG, R., ANTUNES, M., CHORNET, E., TURNBULL, J. and SHOEMAKER, S. (2001). Anaerobic digestion of fish and wood wastes for biogas production. ASAE paper No 01-6014. HARTMAN, K.A. (2004). An APS-Digester for the treatment of organic solid waste and power generation: UCD digester design. MSc thesis, Department of Biological and Agricultural Engineering, University of California Davis, Davis, California, USA. JAEGER, K. and EGGERT, T. (2002). Lipases for biotechnology. Current Opinion in Biotechnology 13(4): 390±397. JENSEN, J.K. and JENSEN, A.B. (2000). Biogas and natural gas fuel mixture for the future. In the Proceedings of 1st World Conference and Exhinition on Biomass for Energy and Industry, Sevilla, Spain, 5±9 June 2000. LANARI, D. and FRANCI, C. (1998). Biogas production from solid wastes removed from fish farm effluent. Aquatic Living Resources 11(4):289-295. LETTINGA, G. (2001). Digestion and degradation, air for life. Water Science and Technology 44 (8): 157±176. LETTINGA, G., VAN VELSEN, A. F. M., HOBMA, S. W., DE ZEEUW, W. and KLAPWIJK, A. (1980). Use of upflow sludge blanket reactor concept for biological waste water treatment, especially for anaerobic treatment. Biotechnology Bioengineering 22: 699±734. LINDSAY, G.D. (1975). East coast fish processing ± Effluent characterization. In the Proceedings of fish processing plant effluent treatment and guidelines seminar, held in Vancouver, British Colombia, January 15±16, 1974. LISSENS, G., VANDEVIVERE, P., DEBAERE, L., BIEY, E.M. and VERSTRATE, W. (2001). Solid waste digestors: process performance and practice for municipal solid waste digestion. Water Science and Technology 44(8): 91±102. MA, F. and HANNA, M.A. (1999). Biodiesel production: a review. Bioresource Technology 70: 1±15. MADRAS, G., KOLLURU, C. and KUMAR, R. (2004). Synthesis of biodiesel in supercritical fluids. Fuel 83: 2029±2033. MASSEÂ , D.I. and MASSE, L. (2000). Characterization of wastewater from hog slaughterhouses in Eastern Canada and evaluation of their in-plant wastewater treatment systems. Canadian Agricultural Engineering 42 (3): 139±146. MCCARTY, P.L. (1964). Anaerobic waste treatment fundamentals. Part 3: Toxic materials and their controls. Pub. Works, Nov. 9. MDHANDETE, A., KIVAISI, A., RUBINDAMAYUGI, M. and MATTIASSON, B. (2004). Anaerobic batch co-digestion of sisal pulp and fish wastes. Bioresource Technology 95: 19±24. MEHER, L.C., VIDYA SAGAR, D. and NAIK, S.N. (2004). Technical aspects of biodiesel production by transesterification: a review. Renewable and Sustainable Energy Reviews xx: 1±21. METCALF AND EDDY, INC. (2003). Wastewater Engineering, Treatment and Reuse, 4th edn. McGraw-Hill, New York, USA. MHARA, B.I. (2005). Information note on the composting of organic waste from seafood processing. http://www.bim.ie/uploads/text_content/docs/InfoNote.pdf. Accessed on 16/10/2005. O'KEEFE, D.M., OWENS, J.M. and CHYNOWETH, D.P. (1996). Anaerobic composting of crabpicking wastes for byproduct recovery. Bioresource Technology 58: 265±272. OMIL, F., MEÂNDEZ, R. and LEMA, J.M. (1995). Anaerobic treatment of saline wastewaters under high sulphide and ammonia content. Bioresource Technology 54: 269±278.

Bio-diesel and bio-gas production from seafood processing by-products 485 (1999). Anaerobic Treatment of Fish Processing Wastewater with Special Emphasis on Hydrolysis of Suspended Solids. PhD Thesis, Wageningen University, Wageningen, The Netherlands. PALENZUELA-ROLLOÂN, A., ZEEMAN, G., LUBBERDING, H.J., LETTINGA, G. and ALAERTS, G.J. (2002). Treatment of fish processing wastewater in a one or two-step upflow anaerobic sludge blanket (UASB) reactor. Water Science and Technology 45 (10): 207±212. PAVLOSTATHIS, S.G. and GIRALDO-GOMEZ, E. (1991). Kinetics of anaerobic treatment: A critical review. Critical Reviews in Environmental Control 21(5/6): 411±490. Ä AL, A. and LEMA, J.M. (1999). Anaerobic treatment of wastewater from a fish canning PUN factory in a full scale upflow anaerobic sludge blanket (UASB) reactor. Water Science and Technology 40 (8): 57±62. RAVISHANKER, P. and HILLS, D. (1984). Hydrogen sulfide removal from anaerobic digester gas. Agricultural Wastes 11(3): 167±179. RODENHIZER, J.S. and BOARDMAN, G.D. (1999). Collection, analysis, and utilization of biogas from anaerobic treatment of crab processing waters. Journal of Aquatic Food Product Technology 8 (2): 59±67. ROMANO, R.T. and ZHANG, R.H. (2005). Anaerobic digestion of juice from pressed onion waste using a Mixed Biofilm Reactor. Paper Presented at ASAE Annual International Meeting. July 17±20, Tampa, FL. ASAE Paper Number: 057038. SOTO, M., MENDEZ, R. and LEMA, J.M. (1991). Biodegradability and toxicity in the anaerobic treatment of fish canning wastewater. Environmental Technology 12: 669±677. STEIGERS, J.A. (2003). Demonstrating the use of fish oil as fuel in a large stationary diesel engine. In Advances in Seafood Byproducts (Ed. P.J. Bechtel). Proceedings of the 2nd International seafood byproduct conference November 10±13, 2002, Anchorage, Alaska, USA. TYSON, K.S. (2004). Biodiesel handling and use guidelines. US Department of Energy Office of Scientific and Technical Information. http://www.nrel.gov/ vehiclesandfuels/npbf/pdfs/tp36182.pdf .Accessed on 13/9/2005. VAN VELSEN, A.F.M. (1981). Anaerobic digestion of piggery waste. PhD Thesis, Wageningen University, Wageningen, The Netherlands. VICENTE, G., MARTIÂNEZ, M. and ARACIL, J. (2004). Integrated biodiesel production: a comparison of different homogeneous catalysts systems. Bioresource Technology 92: 297±305. VIDAL, G., ASPEÂ, E., CRISTINA MARTIÂ, M. and ROECKEL, M. (1997). Treatment of recycled wastewaters from fishmeal factory by an anaerobic filter. Biotechnology Letters 19 (2): 117±121. WARD, C. and SLATER, B. (2002). Anaerobic digestion of fish processing by-products. Nutrition and Food Science 32(2): 51±53. ZHANG, R. and ZHANG, Z. (1999). Biogasification of rice straw with an anaerobic-phased solids digester system. Bioresource Technology 68: 235±245. ZHANG, R.H., YIN, Y., SUNG, S. and DAGUE, R.R. (1997). Anaerobic treatment of swine waste by the anaerobic sequencing batch reactor. Transactions of the ASAE 40(3): 761±767. ZHANG, R.H., TAO, J. and DUGBA, P.N. (2001). Evaluation of two-stage anaerobic sequencing batch reactor systems for animal wastewater treatment. Transactions of the ASAE 43(6): 1795±1801. ZHANG, Y., DUBEÂ, M.A., MCLEAN, D.D. and KATES, M. (2003). Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresource Technology 89: 1±16. PALENZUELA-ROLLOÂN, A.

23 Composting of seafood wastes A. M. Martin, Memorial University of Newfoundland, Canada

23.1

Introduction

In many areas around the world where commercial fish processing is conducted, large amounts of seafood solid wastes are created. Some reports indicate that as much as 60% of the finfish catch is not used, and a potentially higher amount of waste can be found in shellfish processing (Green and Mattick, 1979). Although a fraction of seafood waste and by-products is recovered and processed to make products such as fishmeal, this is not always possible, owing to economic and geographical considerations. Because of the biological characteristics of the waste, it rapidly decomposes; therefore, much of this waste is disposed of by returning it to the sea which, in addition to serving as a nutrient for marine life, in some circumstances results in potential pollution problems. With its high nitrogen composition, seafood waste has the potential to be used as a plant fertilizer. However, its sensory characteristics, i.e., odoriferous nitrogen compounds work against this use, and this type of application is only done on a small scale. Some processing of solid seafood waste, either by adding acids or lactic acid bacteria, results in the production of liquefied fish silage, which can be used as either feed or fertilizer. Although fish fertilizer products have been developed and are commercially available, the economics of their production and marketing do not meet the needs of all seafood processors. Moreover, because many fisheries activities are located in remote areas, on many occasions there are few alternatives to dumping the wastes at sea or to disposing of them in landfill sites. However, environmental concerns and increasing regulations regarding the disposal of organic wastes have resulted in the need to find better alternatives to these practices, based on ecologically responsible methods.

Composting of seafood wastes 487 This chapter will present and discuss the composting of seafood wastes, including vermicomposting operations, as a potential economic and environmentally-friendly way of solving the problem created by the accumulation of fisheries biomass from offal, by-catch, and from undesirable by-products of further processing. The characteristics and potential uses of the compost produced will also be presented. 23.1.1 General characteristics of seafood wastes Seafood processing wastes can account for a large proportion of the seafood biomass processed. In some cases, well-ground and dispersed waste added to some environments might be beneficial by enhancing the nutrient content of the waters. However, as indicated above, the traditional way of disposing of this waste in the sea, harbours, landfills or rivers is becoming unsustainable because of their biological characteristics, which results in a high organic content at the disposal sites. Subsequently, high values of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) at those sites will result from the dumping of non-treated solid seafood wastes. Treatment of these wastes could be considered a more acceptable option from an environmental point of view, if a simple and inexpensive treatment is employed (Veiga et al., 1994). The fisheries industry has been recognized as a potential large source of waste materials (Martin, 1998). Fish frames, guts, heads and fins are commonly not used, and generally constitute the largest quantity of seafood wastes. In the filleting industry, the frame, which is the portion left after the fillets have been removed, could amount to over half of the initial biomass. Martin and Patel (1991) reported that in 1988, in the Canadian Province of Newfoundland alone, from approximately 351 000 tonnes of landed ground fish, approximately 140 000 tonnes of fish frames and 32 000 tonnes of fish guts became wastes. In general, large amounts of seafood wastes can be found in any fishing area around the world. However, given the nutrient value of fisheries by-products and their potential use in feed formulations or compost (Shahidi, 1992), there are opportunities for finding uses for those wastes.

23.2

Biodegradation of seafood wastes by composting

23.2.1 Composting Among the many definitions of composting available in the literature, Mathur (1991) stated: `Composting is the biological conversion of waste materials, under controlled conditions, into a hygienic, humus rich, relatively biostable product that conditions soils and nourishes plants'. Also, in general terms, composting has been defined as a `low cost, environmentally beneficial and potentially profitable use of fisheries products' (Anonymous, 1992). Several comprehensive works about the different aspects of composting operations, including those for seafood wastes, have been published (Minnich and Hunt, 1979; Mathur 1991, 1998; Miller 1992; Gershuny and Martin, 1992), including

488

Maximising the value of marine by-products

Table 23.1 Main characteristics of the composting process, raw materials and final product Process description

Raw material features

Impact on final compost

Carbon dioxide production Heat generation

Appropriate carbon to nitrogen ratio Appropriate moisture content

Microbial degradation Mineralization

Appropriate pH Organic substrate

Able to enhance soil conditions Appropriate nutrient content for plants Free of pathogens Mature and stable organic matter

Oxidative reactions Thermophilic reactions

Solid state Wastes and/or by-products

practical handbooks such as that published by Kreith (1994). Table 23.1 presents a synthesis of the main characteristics of the composting process, raw materials and final product. Owing to their nature as a high-concentration animal-protein product, seafood wastes have specific characteristics that could affect the success of a composting operation. Among these, the presence of products from fish protein decomposition, such as ammonia, have important potential effects on the biodegradation reactions. A typical composting process is the microbial degradation of biological organic material under aerobic conditions. Thermophilic microorganisms will do much of the biodegradation. Heat will be generated during the process, which should end when the materials added to the composting process have been transformed into a useful product. It is expected that this product can be used as a soil conditioner, given its physicochemical characteristics such as porosity and water retention, as a soil enhancer by adding organic matter to the soil, or as a fertilizer if it contributes substantial nutrients to the soil (generally, by supplementing the compost with other nutrient sources). Also, it has been pointed out that the final product from a composting process should be free of viable plant seeds and human, animal, and plant pathogens to avoid lessening its beneficial impact when applied to land (Haug, 1993). In addition to the benefits from avoiding pollution caused by the alternative disposal of waste materials, the production of compost from seafood wastes could be of interest to regions with both fisheries industries and agriculture activities. This is specifically true, for example, in areas with limited amounts of soil, or with poor soil characteristics, which could benefit from the application of products such as compost. 23.2.2 Other alternatives for the use of seafood wastes A number of studies have been published regarding alternatives for seafood waste and by-product utilization, with an emphasis on solid wastes, such as

Composting of seafood wastes 489 those by Green and Mattick (1979), Martin (1994), and Shahidi (1995). Martin (1998) presented the options available for the bioconversion of seafood wastes to industrial products. The following paragraphs will briefly introduce processes that result in the degradation (by chemical, physical and biological methods) of seafood waste biomass, as some of them are treated extensively elsewhere in this book. Discarding wastes on land In many areas of the world, seafood wastes have been used directly, without further processing, to add nutrients to cultivated land. For example, in the island of Newfoundland, Canada, the direct use on gardens and fields of capelin (Mallotus villosus), harvested close to shore during their yearly migration to spawn, has been a practice for many years. This kind of spreading of undigested biological material rich in protein and lipid components has, as expected, many drawbacks, including negative odour effects produced by their sensory characteristics. Also, much of the nutrient value of the fish waste is lost using this method. Some components of the waste fish biomass, such as viscera and even muscles, are rich in proteolytic and other hydrolytic enzymes, some of which are psychrophilic (Martin and Patel, 1991). As a result, even under low-temperature conditions, enzymatic activity in the waste fish biomass will rapidly result in the release of volatile ammonia from protein-rich wastes, and high ammonia concentration could overcome the capacity of microorganisms present to produce nitrates. Moreover, calcium from the fish bones will result in an alkaline medium, facilitating the loss of ammonia gas created (Hayes et al., 1993). Nevertheless, this alkaline characteristic could be welcome in acidic soils. Landfilling Landfilling implies the deposition of waste on specially prepared sites where the wastes are accumulated on top of previous loads, sometimes with the addition of layers of soil between loads. Therefore, landfilling means burying the seafood wastes. Under these conditions, anaerobic degradation will prevail, with subsequent production of bad-smelling gases rich in reduced nitrogen and sulphur compounds. Hayes et al. (1993) reported the generation of compounds with very descriptive names such as `cadaverine' (also, `putrescine'), and the well known `rotten egg gas', hydrogen sulphide. Although well-designed landfill operations can minimize this negative effect, this operation interferes with the recovery of valuable organic materials. Many of these materials could be transformed by aerobic composting into a valuable product, rich in humic compounds. Production of fishmeal Using seafood wastes as a raw material for the production of fishmeal is a common practice in many countries. Fishmeal production involves heat treatment of the fish biomass, among other process operations. Although fishmeal is more valuable than compost in terms of nutrient content, its production could be limited by economic factors such as energy costs and, in some cases,

490

Maximising the value of marine by-products

geographical ones, which limit its potential as a solution to the disposal of seafood waste biomass. Other technologies Martin (1998) refers to the considerable work that has been conducted on the production of fish protein concentrates, fish protein hydrolysates, and fish silage, from seafood wastes, by-products, and species that are caught but have no market. Also, significant work has been done with minced fish (Regenstein, 1986). These products, some of them based on the liquefaction of seafood wastes by chemical or biological hydrolytic processes, have been developed with the aim of recovering valuable protein present in seafood biomass. Their intended use has been as ingredients in food or feeds, with the only probable exception being that of liquid fertilizer products. Given these objectives, it is obvious that the materials involved in the formulation of food products for humans and feed products such as pet food or feed for aquaculture operations should be of acceptable quality and sanitary standards (in the case of fishmeal, although it is mostly marketed as a feed component, the heat treatment involved in its production allows, in some cases, for the use of materials of lesser quality). Therefore, the previously mentioned technologies generally exclude the use of rotten, semi-decomposed, smelly fish offal and the like, as raw materials. These are the kind of materials more properly destined to composting, spreading on land, and landfilling operations. An overview of the main alternative uses of seafood wastes is presented in Table 23.2.

23.3

Composting operational parameters

A composting operation can be interpreted as the combination of a series of physical, chemical and biological reactions occurring in the materials being composted. These reactions depend on a number of variables or parameters, the most important of which will be presented in the following subsections. In addition to these parameters, others such as the particle size of the materials being composted, are important if a comprehensive optimization of the process is required (Mathur, 1998). It is important to point out that this section refers to composting due to the action of microorganisms, which can be defined as `microbial composting.' The characteristics of what is known as `vermicomposting' will be presented in another section of this chapter. 23.3.1 Aeration Composting is an aerobic process (although an `anaerobic compost' process could be considered as equivalent to what happens in operations such as landfilling, some of them are more complex than that and are inoculated with specific microbial cultures to optimize the operation; many aerobic composting

Composting of seafood wastes 491 Table 23.2

Alternative uses of seafood wastes

Technology

Advantages

Disadvantages

References

Discarding of wastes on land (spreading)

Simplicity

Foul odours from rotten wastes Ammonia loss to air Invasion of pests such as rodents Habitat for insects

Hayes et al. (1993)

Landfilling or burial

Simplicity

Fetid odours from anaerobic decomposition

Production of fishmeal

Product can be commercialized

Requires relatively high capital investment Not economical for small seafood operators

Martin and Patel (1991)

Production of fish protein concentrates and hydrolysates

Potential for high quality product

Cost, requires precise control of the process

Martin (1998)

Production of fish silage

Low capital investment

Needs to be dried unless used near production site

Martin (1996)

Potential loss of much of the nitrogen Lower value than alternatives Requires careful management of production

Mathur (1991, 1998), Martin (1998)

Can be used as feed or fertilizer Composting

Simplicity Generally, a low capital investment Product · Adds nutrients to the soil · Enhances soil properties · Can be commercialized · With hygienic characteristics

systems probably have a fair bit of anaerobic activity, also). Consequently, the presence of air is required as a provider of oxygen, which is needed for the growth, and other metabolic reactions of the aerobic microorganisms responsible for the biodegradation of the waste material being composted. An insufficient

492

Maximising the value of marine by-products

supply of air will result in the composting mixture adopting some of the characteristics of an anaerobic degradation process, including the release of malodorous gases. Aeration contributes to maintaining an adequate moisture level (by water being evaporated into the air inside the mixture), to water vapour at equilibrium conditions. Excessive aeration could dry out the composting mixture. Aeration also acts as a temperature control mechanism, by dissipating and transporting the heat generated in the degradation reactions (excessive aeration can also result in temperatures too low for the composting process). 23.3.2 Carbon to nitrogen ratio The carbon to nitrogen (C/N) ratio is an important parameter, which will relate the composting reactions to the relative concentrations of essential chemical constituents required for the growth and metabolic reactions of the microbial population. Compounds such as carbohydrates, in addition to being sources of carbon for the microbial biomass, will generate energy required for the microbial metabolic activity. Nitrogen is an essential component of proteins and amino acids required for the growth of the microbial biomass. Generally, it is recommended that, to maintain an active microbial population in a composting operation, the available carbon to nitrogen ratio should be kept at appropriate levels. Lower ratios will result in losses of nitrogenous compounds, while higher ratios will retard the composting reactions (Inbar et al., 1991). 23.3.3 Level of moisture Water is essential to the viability of microbial populations and is a medium for the biodegradative reactions in the compost mixture. While low moisture levels will affect the speed of decomposition in the compost, flooding of a compost site will interfere with the gas exchange required in aerobic processes. For example, if the air-filled empty or free spaces inside the compost mixture are filled with water, the amount of oxygen available to be transferred to the compost biological phase could be affected. Hobson and Wheatley (1993) indicated a range of 40± 60% moisture content as the appropriate one for composting operations. 23.3.4 Temperature Temperature is a significant parameter due to its role in the regulation of the composting reactions. These reactions are multiple, some occurring in parallel and others in a sequential way. Therefore, the range of adequate temperatures for the composting process is wide. Some biodegradation can occur at low ambient temperatures, such as 20ëC, however, most of the composting reactions will be conducted at higher temperatures, including thermophilic conditions of as high as 65ëC. Temperature has an important effect in controlling the kind of microbial population present and the reaction rates of the degradation taking place during the composting process (Nakasaki et al., 1985a; Golueke and Diaz, 1996).

Composting of seafood wastes 493 23.3.5 pH levels Biodegradation in composting depends on microbial activity, and similar to the effect of temperature, pH contributes to regulating microbial reactions. Microbial resistance to acidic and alkaline conditions is a function of the type of microbial population present, with individual species having optimum values of pH for growth and for their metabolic reactions. In general, the optimum pH for many microbial species, including bacteria and fungi, are, approximately, in the range of pH values from 5 to 8. Extremes values of pH affect the composting process. However, the organic nature of the wastes composted, specifically if they contain protein compounds (such as is the case of seafood wastes), will provide buffering qualities to the composting mixture, i.e. the materials themselves will establish the pH. The release of carbon dioxide and ammonia during the degradation of the wastes will impart, respectively, acidic and alkaline characteristics, which tend to neutralize the pH value of the mixture without the need of external adjustment (Haug, 1993). Table 23.3 presents a summary of the most important operational parameters for composting. Table 23.3

Composting operational parameters

Parameter

Effect on process

References

Aeration

Provide oxygen to aerobic microorganisms Control water content Control temperature by removing heat

Lau et al. (1992), Haug (1993)

Carbon content

Source of carbon and energy for microorganisms

Haug (1993)

Nitrogen content

Necessary for microbial biomass growth

Decker (2000)

C/N ratio

Active microbial aerobic metabolism aided by an appropriate ratio Higher ratio slows process Too low ratio leads to loss of nitrogen

Inbar et al. (1991), Haug (1993)

Moisture content

Necessary to microbial activity If too high, can block airspace, reducing oxygen transfer Required by aerobic microorganisms Prevent anaerobic conditions

Haug (1993), Hobson and Wheatley (1993)

PH

pH control not required due to compost buffer properties

Haug (1993)

Temperature

Influences type of microbial population and biodegradation rates Sanitizes compost

Golueke and Diaz (1996), Stentiford (1996)

Oxygen

Haug (1993)

494

23.4

Maximising the value of marine by-products

Characteristics of the composting of seafood wastes

Fisheries waste biomass contains valuable macronutrients including protein and elements such as phosphorus, among others. The degradation of materials rich in protein results in the production of gases rich in ammonia and other compounds, some of them producing putrid smells. Moreover, the release of ammonia and other nitrogen-containing gases will make the process pointless from the point of view of nutrient recovery, as the main element contributed by the protein, nitrogen, will be lost in the gas. Another characteristic of proteinrich composting mixtures is that, owing to its chemical composition, it tends to have a low C/N ratio. To avoid these problems, another main ingredient is required for successful composting of seafood wastes: what is known as a `bulking agent'. Bulking agents have a major role in composting operations, as their presence will facilitate the aeration of the mixture, avoid the release of offensive gases, and contribute the required carbon for an adequate carbon to nitrogen ratio (Frederick et al., 1989; Imbeah, 1998). Examples of suggested bulking agents that could accomplish these objectives are forestry and wood processing wastes (such as size-reduced or shredded brush, bark, wood chips, sawdust), agricultural wastes, and peat. Some materials, if used as bulking agents, will fulfil only some of the above-mentioned criteria for a good bulking agent. For example, some non-biodegradable (non-digestive) material can be used to facilitate the aeration of the mixture, although it will not contribute nutrients to it. The release of ammonia from protein compounds in seafood wastes and of calcium from fish bones, tends to shift the pH of the mixture towards the alkaline range. Therefore, it would be convenient if the bulking agent employed, in addition to increasing the carbon to nitrogen ratio of the mixture, could contribute to decreasing its pH by having acidic characteristics. Another requisite for the bulk agent is to be able to absorb water, as seafood wastes generally tend to have high moisture contents (Mathur et al., 1986). Therefore, the requirements for an effective bulking agent are complex, and not all potentially available materials will be successful in this role. For example, Hayes et al. (1993) reported that, in many cases, the addition to seafood waste composts of weak acidic materials might fail to avoid the loss of much of the ammonia. The authors presented, as examples of materials with low buffering capacity, river mud, citrus wastes, and banana wastes. A study of several bulking agents for the composting of fish offal was conducted by Liao et al. (1995), who reported that peat moss, compared to sawdust and wood shavings, gave the best results from the point of view of nitrogen conservation. 23.4.1 Other substrates of marine origin for composting operations In this chapter, the term seafood wastes includes wastes from the processing of shellfish, as the main features for the composting of finfish and shellfish wastes are the same (Mathur et al., 1986), the only exception being the presence of chitin in the latter. The processing of shellfish could result in chitin-rich wastes,

Composting of seafood wastes 495 which could be composted (Kuo, 1995). Slow chitin degradation rates would delay the compost process; thus consideration could be given to the separation of the shells (for which perhaps other uses can be found) from the wastes, before composting. In addition to seafood wastes, marine algae biomass can be added to composting operations to enhance the nutrient value of the final product. Some seaweed species could contribute elements such as calcium, copper, iodine, magnesium, phosphorus and potassium (Gershuny and Martin, 1992).

23.5

Technological aspects

23.5.1 Limitations of traditional methods for the composting of seafood wastes Through the years, many composting systems have been developed, from the most unsophisticated ones (such as those in household backyards), to recent systems that incorporate control equipment and closed environments. In general, most of the composting technologies have been based on the treatment of organic materials of plant origin, and in many cases (mostly at the household composting level) it has been advised not to include wastes of animal origin in the materials to be composted. The rationale for this is related to the requirements for aeration of the composting mixture. Traditional composting operations are aerated by what can be called `active aeration' methods, i.e. by periodically turning of the compost or by forcing air through the mixture. This will result in increased discharge of gases from the compost, contributing to the loss of ammonia and if improperly managed, as indicated above, the release of smelly compounds in the air. Therefore, not only will valuable nitrogen be lost from the compost, but also the environment surrounding the compost operation will be affected. Many of the problems arising when new composting technologies are introduced are caused by ineffective operational designs. To overcome some of these problems, quality control methods need to be applied (SereÂs-Aspax and AlcanÄiz-Baldellou, 1985). Moreover, because of its biological characteristics, on many occasions it is difficult to predict the behaviour of a composting operation, as it is dependent on numerous variables, many of them not easy to control. For example, combinations of organic wastes with sufficient nitrogen content, and with similar conditions of bulk density, carbon and water content, could result in different rates of composting (Mote and Griffis, 1980). Weather conditions are also an important factor for outdoor composting operations. 23.5.2 Passive aeration composting A compost process can be conducted in enclosed conditions (in barrels, wood enclosures, or the like), or on the soil in piles (heaps or mounds). In the latter type, in many cases the system employed is identified as a `windrow' (Kuhlman,

496

Maximising the value of marine by-products

1990). A windrow system, on occasions, could benefit by an appropriate containment of the system, or enveloping. Because the aim of the passively aerated system is to increase the retention of the gases produced inside the composting mixture, it is important to find a good cover for the windrow. However, by interfering with the mass and heat transfer mechanisms between the composting material and the environment, covering could result in overheating of the compost pile (it could limit the flow of fresh air to the inside of the pile, and of hot gases, produced in the compost operation, to the outside). The use of covering materials will also add costs to the operation. A solution to these limitations could be found if the same bulk agent employed in the composting process is also used to envelope the composting mixture, by placing it as the most exterior layer of the composting pile. Another possibility is the use of finished compost as the cover. When the bulk agent or finished compost are used with this aim, they and the microbial population attached to them act as a biofilter (Martin, 1991) for the gases produced in the composting operation. Generally, the aeration of the composting pile will be achieved by the penetration of air inside it. To facilitate this process, the bulk material should be of loose characteristics, to allow for the formation of void spaces where the air can penetrate the pile. After the decomposing reactions become established in the composting mixture, it will increase its temperature due to the metabolic energy liberated by the biophase. If an appropriate flow of air is present, the temperature rise will be highest roughly in the centre of the pile. This is because the dissipation of heat from the innermost sections will be more difficult than the dissipation of heat from the more external layers of the compost mixture. Therefore, the flow of air, initially mostly caused by gas diffusion, will also be aided by convective currents, as the hot gases from inside the pile will tend to move up to colder layers of the pile, being replaced by cooler air from the external layers of the pile. At this stage, particularly in larger piles, the flow of air could become the limiting factor in the supply of oxygen to the biodegradation process, which will result in the development of anaerobic reactions. Because, in the case of animal wastes, including seafood wastes, no turning of the pile in aid of the aeration is advised to avoid the release of odorous gases, new solutions need to be found. One solution, as indicated above, is the use of cover material with biofilter properties. Another possibility is to enhance the flow of air by mechanical aeration devices, which will result in forcedconvection conditions. However, this method implies additional capital expenditure (fans, air compressors, pumps, or combinations of them), and operating expenses (energy). Economic considerations in a composting operation need to consider the expenses involved in setting up the pile and the cost of maintaining the operation for a given period of time. Therefore, the latter solution could be unaffordable. To avoid this problem, the fundamentals of passive aeration composting have been applied to the design of what is known as Passively Aerated Windrow System (PAWS), as reported by Mathur (1991, 1998).

Composting of seafood wastes 497 23.5.3 Passively aerated windrow system (PAWS) The PAWS composting system, as its name indicates, avoids any mechanical action to promote aeration, such as turning, or pumping or blowing air. Instead, the design of the PAWS incorporates elements that will allow the flow of air inside the composting pile without forced convention. Therefore, to convey air deep inside the composting process, pipes with their ends opening outside the pile are placed at the base. These ends should allow the air to enter the pile, and may be covered with a mesh or lattice screen to avoid the intrusion of pests to the pile. In addition, the pipes should have perforations directed towards the top of the pile, through which the air will diffuse to the composting mixture. To avoid letting liquefied fish waste go through these pipes, they should be inserted in a section of the pile covered with the bulking agent. Another possibility is to build the compost pile on top of a perforated plate or base, which will allow the introduction of air to the system. As indicated above, the aeration will be further aided by convective currents created by the differences in temperature between the core of the composting mixture and the exterior layers, and finally, the surroundings. These added features characterizing the PAWS should result in avoiding two of the main problems in composting: overheating, and the development of anaerobic zones. It has been reported that the PAWS has been successfully applied for the composting of a number of farming and industrial wastes, including those from the pulp and paper industry, and from seafood processing wastes (Mathur, 1991). As well as the material to be composted, the selection of the bulking agent is of paramount importance for the success of the PAWS. The bulking agent, in addition to its operational role in enveloping the composting pile, can be used to increase the carbon to nitrogen ratio of the blend (although not all the carbon in the bulking agent will be available for the composting reactions, so the full carbon content should not be counted when calculating the ratio). In Table 23.4, some potential bulking agents, and their characteristics, are presented. Hayes et al. (1993) pointed out the effect of materials such as peat, mature compost, and by-products and wastes from the forestry and wood industry when used as PAWS envelope or covering. The authors claim that these materials are hygienic and free of seeds and pathogens, act as walls for the pile, and function as a biofilter by retaining vapours liberated from the composting reactions. Being closer to the outside temperature, the envelope can sustain mesophilic microbial populations, including those involved in the transformation of ammonia to nitrates. The proportional amount of bulk material required for a composting operation needs to be calculated by means of materials balances, based on its moisture content and the carbon to nitrogen ratio required. Application of PAWS to the composting of seafood wastes A composting process has been developed in which seafood wastes are mixed with peat, which acts as a bulking agent. It has been reported that the compost produced this way is of good quality and has appropriate concentrations of nutrients. Moreover, no foul odours were produced in the composting operation (Mathur et al., 1986). As already mentioned, one of the advantages of this

498

Maximising the value of marine by-products

Table 23.4 Overview of some potential degrading bulking agents to be used in the composting of seafood wastes Bulking agent

Characteristics

References

Peat

Several types can be employed Established market as a soil conditioner Not available in all areas Does not significantly degrade during composting Low fertilizer value Generally needs to be purchased

Dayegamiye and Isfan (1991), Mathur (1991), Martin et al. (1993a), Liao et al. (1995)

Wastes and byproducts from the forestry and wood industry (sawdust, shavings, tree bark)

Less acidity and buffering capacity than peat Higher bulk density than peat Low fertilizer value (lower nitrogen content than peat) Lignins contribute to humus formation Tannins, abundant in tree barks, could slow biodegradation

Janssen (1984), Frederick et al. (1989), Dayegamiye and Isfan (1991), Mathur (1991), Martin et al. (1993a), Hayes et al. (1993), Liao et al. (1995)

process is the retention of part of the nitrogen present in the seafood biomass. This technology is based on some specific properties of the bulking agent incorporated into the degradation process. When peat is used, its fibres will interfere, due to sorption reactions, with the loss of the ammonia produced in the composting reactions. It is possible that both adsorption and absorption mechanisms take part in this process, resulting in the low pH peat fibres being able to interact with the alkaline ammonia gas. It appears that peat is, among the potential bulk materials to be employed in composting, probably the one which possesses the best characteristics for an effective operation of PAWS, and specifically for the composting of seafood wastes. It has been reported that both horticultural sphagnum peat, and light brown peat that is not used for fuel or horticulture (and therefore, should be less expensive), do meet the requirements for an appropriate bulk material (Mathur et al., 1988). The same authors indicated that peat has a higher buffering action and higher nitrogen concentration than wood waste products, and that peat moss is a product traditionally employed to ameliorate soil conditions. The use of peat is fitting in places where it is available (as a local resource or as a commercial product), such as in some areas of Canada; however, it should be mentioned that other materials (mentioned elsewhere in this chapter) could be used successfully as bulking agents. Use of peat as a bulking agent Several studies have been conducted on the potential of peat to act as a filter material by adsorbing organic and inorganic compounds (Mueller, 1972;

Composting of seafood wastes 499 Viraraghavan and Ayyaswami, 1987). A comprehensive review on the role of peat in waste biodegradation was presented by Martin (1991), who reported that peat is characterized by being acidic, and by possessing high adsorptive and absorptive capabilities. Basically, the solid phase of peat is composed of organic material, with some ash content. When compared with standard soils, peat ash contents are low. The chemical composition of peat is the result of the decomposition of organic materials in the absence of oxygen, which results in a material with a particular chemical profile, characterized by the presence of humic materials and bitumens. The degree of decomposition of the peat is defined by its degree of humification, and this factor is important in the evaluation of the type, quality and specific applications of the peat. The determination of the degree of humification of a peat sample is generally conducted following the von Post system of classification, consisting of ten levels, from low decomposed, low humification, H-1, to high decomposition and humification, H-10. Each level represents approximately 10% decomposition. The extent of decomposition is found by squeezing recently harvested peat and analysing the water squeezed and the compressed peat (Anonymous, 2005). Equally, the concentration of ash in peat varies with the degree of humification (Fuchsman, 1980). As expected, the most decomposed peat is that found at the lower layers in a peat bog. A peat good for horticultural applications (H-1 to H2) has limited humification, with a pale brown colour. Lower layers of peat will contain darker deposits, and the deepest levels will correspond to values of H-5 and higher, a peat appropriate for fuel applications. The type of peat known as sphagnum will be found in the higher layers. The characteristics of peat are related to the biosystem from which it is derived. In general, peat can be considered a renewable resource, as it accumulates if there is adequate plant biomass and growing conditions present on site as well as the availability of water (Fuchsman, 1980). For a composting process using the technology of PAWS, there is flexibility in the use of peat. Although horticultural-type peat is generally preferred, peat from lower layers could be employed in the base of the pile and for enveloping. However, the peat fibres should have appropriate particle sizes and moisture contents to avoid clumping, and to facilitate the gas exchange in the pile. Raw peat is low in nutrients, and its main established commercial use is as a soil conditioner, which includes its valued capacity for the retention of moisture in the soil. However, the composting process with seafood wastes should increase its nutrient content, namely its nitrogen concentration. Mathur et al. (1986) indicated that the moist, acidic peat fibres adsorb the ammonia from protein degradation as ammonium ion. Therefore, although not necessarily possessing the chemical composition of a standard commercial fertilizer product, the resulting compost will be a nutrient source for plants, which require nitrogen in nitrate or ammonium forms. In general, composting with peat is a good technology for nitrogen conservation (Liao et al., 1995). Table 23.5 presents information on the chemical composition of some seafood wastes

500

Maximising the value of marine by-products

Table 23.5 Chemical composition of seafood waste composts with peat and with a combination of peat and sawdust as bulk materials Components

Concentration (% dry weight)a Bulking agent b

Ash Carbohydratesd Lipids Nitrogen

Peat

Peat and sawdustc

19.5  2.8 63.9 0.9  0.0 2.5  0.1

23.9  3.7 62.3 3.2  0.0 1.7  0.0

a

Mean values of three determinations  standard deviations. From Martin and Chintalapati (1989) c Equal proportions of peat and sawdust, from Martin et al. (1993a) d Calculated by difference, assuming a protein content of 6.25  % nitrogen b

composts prepared with peat, and with a combination of peat and sawdust, as bulking agents. Phenolic compounds in lignocellulosic materials could act as inhibitors of biological reactions. Although there are phenolic compounds in peat, Mathur (1991) indicated that calcium and proteins could deactivate them. Seafood wastes, in addition to their high protein content, also contain calcium compounds from the fish bones. Martin and Patel (1991) reported that the final product obtained from peat composted with seafood fisheries could potentially be a good commercial soil product due to its physical, nutritional, and functional characteristics. A comparative study on the use of sphagnum peat and sawdust in the composting of fisheries and other food processing wastes was presented by Martin et al. (1993a). Environmental impact The composting process is a natural way of disposing of solid organic waste and of producing materials rich in humus with soil-amelioration properties and thus is an environmentally-friendly operation. As indicated above, potential problems related to the production of odorous gases, common in ill-designed composting operations, can be avoided by correctly applying the PAW technology. Frederick et al. (1989) indicated that a suitable composting pile design with appropriate bulking agent would result in an efficient, odour-free biodegradation process. Another potential problem, the production of leachates, can be dealt with by building a good base to the composting pile (as indicated elsewhere in this chapter), by installing impermeable films at the base, or by adding an appropriate leachate recovery system connected to, for example, an oxidation lagoon. However, besides the environmental advantages brought by composting and related biodegradation operations, consideration should be given to the environ-

Composting of seafood wastes 501 mental impact associated with the use of bulking agents, specifically, what kind of material is to be used, and its source. The quantities of bulking agent required by a sizeable composting operation have both economic and environmental consequences, which could limit the viability of the operation. The ideal bulking agent will be one which is also an industrial waste or by-product available in the proximity of the composting operation. In this category, the best candidates are, possibly, lignocellulosic materials including straw and other agriculture residues, as well as forestry waste organic materials such as sawdust, bark, and wood chips. Peat, which possesses very good characteristics as a bulk material for the composting of seafood wastes, is a material not present in all environments. However, it is generally abundant in some northern regions, close to locations with important fish harvesting and processing operations (Canada, northern regions of Europe, among others). These regions are, generally, characterized by a restricted amount of cultivable land and a subsequent shortage of agricultural and in many cases, forestry waste material, factors that hamper the availability of other bulking agents. Although, as indicated above, peat can be considered as a renewable material due to its natural regeneration, there have been concerns about how bogs and wetlands, where peat is deposited, can be affected by its extraction. However, Hayes et al. (1993) report that peatland ecosystems where peat has been extracted can be restored, with the new wetland presenting better ecological characteristics than virgin peatlands due to a diminished production of methane. The same authors point out the benefits of peat in the restoration of organic matter in soils, benefits that will add value to a peat-containing compost product. As an answer to the above-mentioned concerns related to peat extraction, materials with peat-like characteristics can be used as alternatives. For example, Briddlestone and Gray (1991) suggested the use of a highly stabilized product, produced by aerobic processing of organic wastes, as a substitute for peat.

23.6

Biological aspects

Composting is a very complex biological process, difficult to control even when the chemical composition of the mixture, including the carbon to nitrogen ratio, are the result of careful calculations to determine, from the amounts of nutrients present, how much of them are available (i.e., how much can be metabolized during the composting process), and the process conditions are monitored. The biological phase (consisting of multiple biological reactions) of the operation is ultimately the active agent determining the outcome of the whole operation. 23.6.1 The role of microorganisms in composting Composting is a biodegradation process, its active element being the microbial population that does the digestion of the organic materials present, which

502

Maximising the value of marine by-products

generally are composed of wastes of biological origin. A secondary active element is the presence of hydrolytic enzymes in the wastes, such as those in plant material and animal tissues. In seafood wastes, this element is of particular importance, due to the presence of enzymes in fish viscera, which accounts for a good fraction of the fish-offal mass. Biological degradation is also contributed by other non-microbial entities such as insects and worms. As already indicated, composting by worms (vermicomposting) is discussed elsewhere in this chapter. Enzymatic contributions from the decaying waste biomass are only relevant at the beginning of the composting operation, as enzymes will be inhibited and destroyed as a result of the biological degradation process. Similar fates await protozoa and insects, mostly due to the heat liberated by bacterial and fungal degradation, which will be, by far, the main contributor to the composting process (because these latter organisms can withstand higher temperatures). Given the appropriate operating parameters, some kinds of microorganisms (such as bacteria and fungi) have the ability to adapt to the environmental conditions of the process, and to interact among them in symbiotic and other types of relationships, which will result in an efficient and complete degradation or the waste biomass. Microorganisms, being such a relevant factor in the composting process, their role should be understood from a basic science point of view (Miller, 1992). However, in terms of practical applications, the use of microorganisms in composting is studied from what can be called a macro or overall approach (Davis et al., 1992a,b). Composting, as happens in other biological waste treatment operations such as sewage treatment, anaerobic digestion, and the like, is done by a large number of species, working in mixed culture conditions, and in parallel and consecutive reactions. Therefore, the experimental methodology employed implies working with mixed populations of microorganisms, most of them unknown at a given time, as they develop naturally in the open environment of the composting operation. This does not exclude the possibility of seeding with some microbial inoculums specially formulated for the compost operation. Some authors indicate that inoculation or seeding has been effective, however some negative results have been also reported (Nakasaki et al., 1985b). These authors indicate that, in their research, no clear difference in the overall rate of composting or in the quality of the compost resulted by seeding. If it is decided to accelerate the composting reaction by introducing microorganisms specialized in the degradation of a given waste material at the beginning of the process, often the easiest way to accomplish this is to add some amount of not totally matured (or uncured) compost from another composting operation. In vermicomposting, which will be discussed later in this chapter, the actions of both worms and microorganims are complementary. Bacteria, fungi and protozoa populate the digestive system of earthworms, and they contribute to their diet.

Composting of seafood wastes 503 23.6.2 Biodegradation of bulk materials The use of lignocellulosic wastes or by-products as bulking agent will in general prolong the complete decomposition of all of the materials in the composting blend, as these bulking agents will degrade more slowly than other biological wastes, such as those of animal origin. Also, phenolic compounds present in lignocellulosic materials could inhibit some of the microorganisms present in the compost. However, Mathur (1998) pointed out that the mixing of this kind of materials with ammonia-generating wastes would result in the neutralization and auto-oxidation of the phenolics, due to the ammonia released. In the case of peat, several studies have been conducted on the use of some peat hydrolysation products, such as peat extracts, in fermentation processes (Manu-Tawiah and Martin, 1987; Martin and Chintalapati, 1989; Martin and Bemister, 1994, Martin et al., 1993b; VaÂzquez and Martin, 1998, among others). These studies could be a base for future work in understanding the potential microbial degradation of peat in a composting operation.

23.7

Vermicomposting

Vermicomposting is the use of earthworms in the degradation and stabilization of organic wastes. This use is based on the properties of some earthworm species to quickly ingest organic wastes and break them into small particles. In general, the same principles involved with traditional composting apply to vermicomposting, although there are some specific characteristics that need to be taken into consideration due to the presence of worms in the composting pile. A historical overview of vermicomposting has been presented by Edwards (1995). Vermicomposting happens in combination with the traditional composting process based on the degradation of wastes by microorganisms; both processes complement each other. The microbial population pre-digest the organic materials upon which the worms feed, and the worms enhance the aerobic conditions in the composting medium by tunnelling through it. Waste materials at various degrees of decomposition from microbial action are ingested by the worms, metabolized and expelled at a higher degree of degradation in the form of particles identified as castings (Loehr et al., 1985). The presence of degrading microorganisms in the earthworm digestive system should also be taken into consideration (Satchell, 1983). Albanell et al. (1988) studied the chemical changes happening during some vermicomposting processes, and Edwards and Fletcher (1988) reported on the relationships between earthworms and microorganisms in this process. The parallel actions of microbial degradation and worm digestion act in a mutually beneficial way. However, in spite of the potential advantages of this symbiotic relationship, the presence of two mechanisms for biodegradation in a close environment adds complexity to the overall design of the composting process. For example, the optimum parameters for one mechanism may not correspond to the optimum for the other, and the metabolites produced by one

504

Maximising the value of marine by-products

type of degradation could be inhibitory to the other. Loehr et al. (1985) discussed the factors affecting the vermicomposting operation, such as the worms' requirement for aeration and the need to prevent toxic conditions. The role of biological mechanisms in the degradation of the wastes in the compost is also enhanced by the worm digestive enzymes that are present in the worm excretions or casts (Edwards et al., 1995). Those enzymes, many of them hydrolytic, continue to act after being expelled from the worm's body (Edwards and Lofty, 1977). Many earthworms are found in soils, and indeed some may be present at times in any compost operation. In general, they thrive and reproduce abundantly in the presence of high concentrations of organic matter. An assessment of earthworms and their relationships with soils was presented by Lee (1985). Among the earthworm species suitable for vermicomposting, Eisenia fetida, also known as the tiger or brandling worm (Edwards, 1995) has been found to possess good attributes such as adaptation to a range of temperatures and water content in the composting medium. Also, it becomes predominant in mixed populations, and its metabolic rates are high (Edwards, 1988). Other important species include Lumbricus rubellus (the red worm), Eudrilus eugeniae (the African night crawler), Perionyx excavatus (Edwards, 1995), and Eisenia andrei (Frederickson et al., 1997), among others. The positive effect of earthworms on soil characteristics has been presented in several studies (Lee, 1985; Blair et al., 1995; Edwards et al., 1995; Syers and Springett, 1984; Tomlin et al., 1995). Similar effects also occur with the compost produced with the aid of earthworms, resulting in the potential for worms to produce compost with better growth improvement characteristics for plants (Harris et al., 1991). The main characteristics of a vermicomposting process and its final product are presented in Table 23.6. 23.7.1 Vermicomposting process parameters Aeration Worms require air in the same way as the aerobic microoganisms involved in composting do, therefore any improvement in the aerobic conditions of the composting medium will be beneficial for both composting mechanisms, i.e. microbial composting and vermicomposting. Indeed, the presence of worms improves the aeration of the composting operation. Through their digestive action, worms transform the waste material not only chemically but physically as well, resulting in reduced sized porous particles with good water-holding capacity. The presence of these kinds of smaller excreted particles, together with the tunnelling and turning produced by the earthworms' presence and movement, could eliminate the need for aeration technologies, either based on forced or passive principles. This is valid unless the temperature increases to levels that could negatively affect the worm population. In this case, it would be necessary to introduce aeration mechanisms to help with the dissipation of the heat generated in the operation.

Composting of seafood wastes 505 Table 23.6

Main characteristics of the vermicomposting process

Factors

Features

Benefits

Aeration

Increased due to worm action Affected by secretions from worms'calciferous glands Should be kept below 35ëC

Enhanced waste degradation Moderate soil acidity

Increased due to worm action

Enhanced waste degradation

Neuhauser et al. (1988)

Supply hydrolytic enzymes

Enzymatic action contributes to waste degradation

Loehr et al. (1985), Edwards and Fletcher (1988), Mishra and Tiwari (1993), Edward et al. (1995) Mishra and Tiwari (1993), Tan (1996) Edwards and Lofty (1977), Lee (1985) Edwards and Lofty (1977) Blair et al. (1995) Syers and Springett (1984), Berry (1994), Edwards (1995)

pH

Temperature Digestion of organic materials Casts excreted by worms

Final product

Finely divided, humus material Improved soil structure, porosity High water holding capacity Increased soil respiration Nutrients converted into forms more available to plants

Drawbacks References Edwards (1995)

Pathogens are not killed

Blair et al. (1995), Mishra and Tiwari (1993) Edwards (1995)

Chemical composition of the composting medium Because of the high water content in the worm biomass, the water content required by a vermicomposting operation is somewhat higher than in traditional compost, in the range of approximately 70 to 90% moisture content. Dehydration of earthworms is a possibility if high temperatures and drying of the medium happens during the composting operation. Also, earthworms exhibit lesser ability than microorganisms to tolerate and adapt to adverse chemicals in the medium (Edwards, 1995). In particular, earthworms are sensitive to the

506

Maximising the value of marine by-products

presence of ammonia (Edwards, 1998). These characteristics could limit the amount of organic matter that can be processed by vermicomposting, and could restrict the use of this method. Edwards and Bohlen (1995) reported on the relationships between earthworms and soil nutrients. Indeed, the spectrum of organic materials ingested by earthworms is quite comprehensive, including decomposing animal and plant residues. This makes vermicomposting a suitable step to be added to and integrated with a process for the degradation of wastes of animal origin. In addition to organic solid waste, earthworms have been used in the stabilization of sewage sludge (Neuhauser et al., 1988) pH The pH of the composting medium controls the kind of earthworm species inhabiting a particular environment, and the size of its population. It has been reported that the optimum pH for E. fetida lies at a pH around neutrality (Edwards, 1988). Moreover, the same author indicated that this earthworm tends to migrate to acidic environments of pH 5.0. This could validate the use of some acidic bulking agents such as peat in composting operations. Temperature An important difference between a traditional composting process and vermicomposting is the range of temperatures at which the reactions take place. While the former reaches temperatures in the thermophilic range, due to the heat released by the microbial degradation reactions, the latter needs to adapt to the viable temperatures for earthworms. It is reported that the maximum temperature endured by earthworms is 35ëC, while their activity is optimum in the range of 15 to 25ëC (Edwards, 1995). The lower temperatures required for vermicomposting compared with those for thermophilic microbial degradation results in the need to have some phasing of the process, allowing an initial microbial composting or precomposting phase without worms, where high temperatures will be able to degrade certain thermosensitive compounds and inactivate pathogens (to simplify the operation, this phase is not generally found in home vermicomposting). Afterwards, the vermicomposting phase should be monitored to avoid increases of temperature above those tolerated by the earthworms, resulting from high microbial activity. The temperature can be kept at appropriate levels by ensuring good aeration conditions to dissipate the heat generated by the microbial activity. 23.7.2 Vermicomposting of seafood wastes Few studies have been conducted on the application of vermicomposting in the treatment of solid wastes from the seafood industry. Decker et al. (2000) pointed out the difficulties in using earthworms to decompose protein matter in animal biomass wastes because of the potential toxicity to the worms of the high concentration of ammonia liberated from protein degradation. A solution

Composting of seafood wastes 507 to this problem, as indicated above, is to run a precomposting step until the concentration of ammonia decreases to acceptable levels for the worms. However, in both the precomposting and vermicomposting stages the nitrogen to carbon ratio needs to be adjusted by the addition of materials high in carbon to compensate for the high nitrogen concentration in the seafood waste materials. This can be accomplished by the addition of, for example, lignocellulosic by-products or wastes, or peat as bulk material. Mathur et al. (1986) reported on the favourable characteristics of peat for this kind of operation, such as high air to water ratio in freshly harvested peat, broad carbon to nitrogen ratio, and suitable water, heat and odorous-gas retention properties of peat. Decker (2000) cited these same properties as convenient in the vermicomposting of seafood wastes. The main objectives of the research reported by Decker (2000) can be summarized as: (a) determination of the appropriate amount of peat to be added to a given amount of fish offal for a vermicomposting operation, (b) study of chemical changes in the vermicomposting medium, and (c) determination of maximum concentration of ammonia tolerated by E. fetida without compromising the survival of the earthworm. Decker et al. (2000) reported that vermicomposting was an effective method for stabilizing fresh fish offal, when composted with peat. The authors found that the maximum amount of fish offal that allowed a 100% survival of Eisenia fetida amounted to a 13% (dry weight) of the composting mixture, and that the level of ammonium should be kept no higher than 1.0 ppm.

23.8

Quality considerations

The success of a composting operation can be considered and analyzed from various points of view: economic, environmental, sociological, and technological. The main factors in these analyses are: (a) the elimination of waste materials and pollutant products, and (b) the creation of a valuable product, the compost. The first factor is dealt with in a straightforward way when the potentially pollutant materials are incorporated into the composting operation, and it is biodegraded as a result of the composting reactions. The second factor depends on the possibility of marketing and using the compost produced, and is linked to its quality. The definition of the quality of a compost product is complex. Not only is the quality of the compost a function of a number of biological, chemical and physical factors (Table 23.7), but it will be relative to the raw material used in the composting and the use intended for the final product. A classification of the various grades of compost in relation to their intended use has been presented by van der Werf (2004).

508

Maximising the value of marine by-products

Table 23.7 Main parameters determining quality in a compost product Biological

Chemical

Physical

Absence of pathogens and weed seeds Biological activity Maturity Stability

C/N ratio

Effect on soil structure

Gas generation (odour) Macroelements content Microelements content Nutrient value pH Water content

Particle size Presence of foreign objects Water retention

Some important compost characteristics, such as the stabilization of the biodegradation activities and its degree of humification or maturity, will act as a common denominator in defining its marketability (Golueke, 1977; IglesiasJimenez and Perez-Garcia, 1989, 1992). These characteristics will be reached during the last phase of the composting operation, known as curing or aging (Jenkins, 1999). During curing, the compost should become mature. A mature compost is the one that is ready to be applied to the soil. The final product should not develop microbial activity after being added to the soil (which could subtract nutrients such as nitrogen and oxygen, making them unavailable for plant growth), should be free of toxic compounds, and be free of ammonia, which should have been converted to nitrates (Haug, 1993). Additional considerations for compost quality are the absence of pathogenic organisms and weed seeds (Hayes et al., 1993). From the point of view of its use, if a compost is not completely mature, it should at least be stable enough to ensure that no nitrogen subtraction from the soil will result from its microbial activity. Standard analytical methods can be applied to evaluate many quality parameters of the compost. Vinceslas-Akpa and Loquet (1997) reported on the application of chemical and spectroscopic analyses to find the chemical composition of the composition mixtures, with the objective of determining the transformation of organic matter in composting and vermicomposting processes. Regarding toxicity, Mathur et al. (1986) suggested seed germination tests, which can be particularly important in the determination of the presence of aliphatic acids and phenolics (Devleeschauwer et al., 1981; Mathur 1991), which can inhibit plant growth. To confirm the absence of plant inhibitors, seeds are soaked with a water extract of the compost and their germination compared with seeds soaked in pure water; the level of germination should be the same in both groups if no inhibitors are present in the compost (Mathur et al., 1986). Also, Mathur and Johnson (1987) reported on the use of tissue-culture tests for detecting toxins in composts of peat, fisheries wastes and seaweeds. The level of success of a composting operation can also be measured by the degree to which the nutrient content of the degraded wastes has been preserved

Composting of seafood wastes 509 in the final product. However, unless the compost is supplemented with nutrients before being marketed, the nitrogen, phosphorus and potassium composition (NP-K, expressed in %), which is used to characterize fertilizer products, is generally low. Nevertheless, probably the main value of compost application to soils is reflected in the term `soil conditioner', which takes into consideration the extent to which compost products enhance the physical structure of the soil and characteristics such as water retention and slow release of nutrients (Gershuny and Martin, 1992). Reports about seafood waste compost indicate that the product is of high quality with an earthy odour and good concentrations of organic and inorganic nutrients (Mathur et al., 1986). As indicated before, shellfish wastes have also been composted, and Hountin et al. (1995) reported the use of compost prepared with shrimp wastes and peat on the growth of barley (Hordeum vulgare L.). Decker (2000) reported that vermicomposting increased the stabilization of organic matter when compared to experiments without earthworms.

23.9

Future trends

At present, in many areas of the world the use of appropriate technologies for seafood wastes treatment or recycling is lacking. However, an increasing awareness of the value of fisheries biomass and a willingness to take measures to reduce pollution of marine environments should encourage the recovery of seafood wastes. Physical and chemical processes for the recovery of seafood wastes and the production of acceptable products from them have been employed with limited success. Therefore, it is generally accepted that bioconversion operations should be applied to the processing of seafood wastes. For small-scale seafood processing plants, the best option for using wastes is the application of simple technologies resulting in products to be used as soil conditioner, fertilizer or feeds. In this context, the biodegradation of seafood wastes by composting has the advantages of being a low-cost operation. Andree (1992) discussed practical approaches for the development of composting applications to fisheries by-products. As presented in this chapter, appropriate technologies have been designed based on the study of the composting of seafood wastes with suitable bulk materials, and on vermicomposting, which have resulted in effective methods for stabilizing fresh fish offal (Martin and Decker, 2000). Therefore, it is expected that in the future biodegradation processes, such as the production of fish silage (Martin, 1996) and seafood waste composting, will be applied more broadly, particularly in small-scale seafood producing plants, and most probably in the developing countries.

510

Maximising the value of marine by-products

23.10

Sources of further information and advice

Composting is a technology that is used across the world. Also, in recent years interest has increased in many national and international institutions for environmentally friendly ways to deal with wastes. Therefore, it is possible to find in many countries both private and governmental agencies dealing with composting, and many sources of technical and scientific information and advice are available. In this section, some sources will be mentioned; however, they do not represent all the potential sources available. Further information about them can be obtained by contacting them directly. Composting Council of Canada 16 Northumberland Street Toronto, ON M6H 1P7, Canada Phones: (416) 535-0240; 1-877-571-GROW (4769) Fax: (416) 536-9892 E-mail: [email protected] Web page: http://wwwcompost.org The US Composting Council 4250 Veterans Memorial Highway, Suite 275 Holbrook, NY 11741, USA Phone: 631-737-4931 Fax: 631-737-4939 Web page: http://www.compostingcouncil.org/index.cfm European Compost Network Ecn/Orbit E.V. Postbox 22 29 D-99403 Weimar, Germany Phone: +49 (0) 25 22-96 03 41 Fax: +49 (0) 25 22-96 03 43 E-mail: [email protected] Web page: http://www.compostnetwork.info The Composting Association, UK Avon House Tithe Barn Road, Wellingborough, Northamptonshire, NN8 1DH UK Phone: +44 (0) 870160 3270 Fax: +44 (0) 870160 3280 E-mail: [email protected] Web page: http://www.compost.org.uk Also, institutions dealing with solid wastes will have interest in the development of composting. One example is the:

Composting of seafood wastes 511 Solid Waste Association of North America (SWANA) 1100 Wayne Ave, Suite 700 Silver Spring, MD 20910, USA Postal Address: P.O. Box 7219 Silver Spring, MD 20907-7219, USA Phone: 1-800-GO-SWANA (467-9262) Fax: (301) 589-7068 E-mail: [email protected] Web page: http://swana.org In the specific area of composting of seafood wastes, The National Sea Grant College Program of the United States, which includes a number of universities conducting research and other programmes on the use and conservation of aquatic resources, is a good source of expertise. National Sea Grant Office, NOAA/Sea Grant, R/SG 1315 East-West Highway, SSMC-3, Eleventh Floor Silver Spring, MD 20910, USA Phone: 301-713-2431 Fax: 301-713-0799 Web page: http://www.nsgo.seagrant.org

23.11

References

and CABRERO T (1988), `Chemical changes during vermicomposting (Eisenia fetida) of sheep manure mixed with cotton industrial wastes', Biology and Fertility of Soils, 6, 266±269. ANDREE S (1992), `Implementing fisheries by-products composting applications: the next step', Proceedings of the 1991 Fisheries By-Products Composting Conference, Madison, WI, Virginia, Sea Grant Institute, University of Wisconsin, 181±187. ANONYMOUS (1992), Proceedings of the 1991 Fisheries By-Products Composting Conference, Madison, Virginia, Sea Grant Institute, University of Wisconsin, vii. ANONYMOUS (2005), Canadian Sphagnum Peat Moss Association Website (http:// www.peatmoss.com) BERRY EC (1994), `Earthworms and other fauna in the soil', in Hatfield JL and Stewart BA Soil Biology: Effects on Soil Quality. Boca Raton, FL, CRC Press, Inc. 61±90. BLAIR JM, PARMELEE RW and LAVELLE P (1995), `Influences of earthworms on Biogeochemistry', in Hendrix PF Earthworm Ecology and Biogeography in North America, Boca Raton, FL, CRC Press Inc, 27±158. BRIDDLESTONE AJ and GRAY KR (1991), `Aerobic processing of solid organic wastes for the production of a peat alternative: a review', Process Biochemistry, 26, 275±279. DAVIS CL, HINCH SA, DONKIN CJ and GERMISHIZEN PJ (1992a), `Changes in microbial population numbers during the composting of pine bark', Bioresource Technology, 39, 85±92. ALBANELL E, PLAIXATS J

512

Maximising the value of marine by-products

and GERMISHIZEN PJ (1992b), `The microbiology of pine bark composting. An electron microscope and physiological study', Bioresource Technology, 40, 195±204. DAYEGAMIYE AN and ISFAN D (1991), `Chemical and biological changes in compost of wood shavings, sawdust and peat moss', Canadian Journal of Soil Science, 71, 475±484. DECKER S (2000), Vermicomposting of cod (Gadus morhua) offal mixed with sphagnum peat, M.Sc. (Environmental Sciences) Thesis, Memorial University, St. John's, NL. DECKER S, MARTIN AM and HELLEUR R (2000), `Influence of exchangeable ammonium on the survival of earthworms during the vermicomposting of fish offal mixed with peat', in Warman, PR and Taylor BR Proceedings of the International Composting Symposium, Truro, NS, CBA Press, Inc, 153±163. DEVLEESCHAUWER D, VERDONK O and VAN ASSCHE P (1981), `Phytotoxicity of refuse compost', Biocycle, 22, 44±46. EDWARDS CA (1988), `Breakdown of animal, vegetable and industrial organic wastes by Earthworms', in Edwards CA and Neuhauser EF Earthworms in Waste and Environmental Management, The Hague, SPB Academic Publishing bv, 21±31. EDWARDS CA (1995), `Historical overview of vermicomposting', Biocycle, 6, 56±58. EDWARDS CA and BOHLEN PJ (1995), `Earthworm effects on N dynamics and soil respiration in microcosms receiving organic and inorganic nutrients', Soil Biology and Biochemistry, 27, 1573±1580. EDWARDS CA and FLETCHER KE (1988), `Interactions between earthworms and microorganisms in organic-matter breakdown', Agriculture, Ecosystems and Environment, 24, 235±247. EDWARDS CA and LOFTY JR (1977), Biology of Earthworms, London, Chapman & Hall, Ltd. EDWARDS CA, BOHLEN PJ, LINDEN DR and SUBLER S (1995), `Earthworms in agroecosystems', in Hendrix PF Earthworm Ecology and Biogeography in North America. Boca Raton, FL, CRC Press Inc, 185±213. FREDERICK L, HARRIS R, PETERSON L and KEHRMEYER S (1989), `The compost solution to dockside fish wastes', University of Wisconsin Sea Grant Advisory Report No. WIS-SG-89-434. FREDERICKSON J, BUTT KR, MORRIS RM and DANIEL C (1997), `Combining vermiculture with traditional green waste composting systems', Soil Biology and Biochemistry, 29, 725±730. FUCHSMAN CH (1980), Peat: Industrial Chemistry and Technology. New York, Academic Press, Inc. GERSHUNY G and MARTIN DL (1992), The Rodale Book of Composting, Emmaus, PA, Rodale Press. GOLUEKE GC (1977), `Biological processing: composting and hydrolysis', in Wilson DG, Handbook of Solid Waste Management, New York, van Nostrand Reinhold, 197± 225. GOLUEKE CG and DIAZ LF (1996), `Historical review of composting', in de Bertoldi M, Sequi P, Lemmes B and Papi T The Science of Composting, Part 1, Glasgow, Blackie, 3±14. GREEN JH and MATTICK JF (1979), `Fishery waste management', in Green JH and Kramer, A Food Processing Waste Management, Westport, CT, AVI Publishing Co., 202± 227. DAVIS CL, HINCH SA, DONKIN CJ

Composting of seafood wastes 513 and PRICE BC (1991), `Vermicomposting in a rural community', in The Biocycle Guide to the Art and Science of Composting, 143±145. HAUG RT (1993), The Practical Handbook of Compost Engineering, Florida, USA, Lewis Publishers. HAYES LA, RICHARDS R and MATHUR SP (1993), `Economic viability of commercial composting of fisheries waste by passive aeration', Proceedings 3rd Annual Meeting Composting Council of Canada, 135±154. HOBSON PN and WHEATLEY AD (1993), Anaerobic Digestion: Modern Theory and Practice, London, Elsevier Science Publishers Ltd. HOUNTIN JA, KARAM A and PARENT LEÂ (1995), `Effect of peat moss-shrimp wastes compost on the growth of barley (Hordeum vulgare L.) on a loamy sand soil', Communications in Soil Science and Plant Analysis, 26, 3275±3289. IGLESIAS-JIMENEZ E and PEREZ-GARCIA V (1989), `Evaluation of city refuse compost maturity: a review', Biological Wastes, 27, 115±142. IGLESIAS-JIMENEZ E and PEREZ-GARCIA V (1992), `Determination of maturity indices for city refuse composts', Agriculture, Ecosystems and Environment, 38, 331±343. IMBEAH, M (1998), `Composting piggery waste: a review', Bioresource Technology, 63, 197±203. INBAR Y, CHEN Y, HADAR Y and HOITINK HAJ (1991), `Approaches to determining compost maturity', in The Biocycle Guide to the Art and Science of Composting, 183±187. JANSSEN BH (1984), `A simple method for calculating decomposition and accumulation of young soil organic matter', Plant & Soil, 76, 297±304. JENKINS J (1999), `The Humanure Handbook', Jenkins Publishing, Grove City, PA. KREITH F (1994), Handbook of Solid Waste Management, New York, McGraw-Hill. KUHLMAN LR (1990), `Windrow composting of agricultural and municipal wastes', Resources Conservation and Recycling, 4, 151±160. KUO S (1995), `Nitrogen and phosphorus availability in groundfish waste and chitinsludge cocompost', Compost Science and Utilization, 3, 19±29. LAU AK, LO KV, LIAU PH and YU CJ (1992), `Aeration experiments for swine waste composting', Bioresource Technology, 41, 142±152. LEE KE (1985), Earthworms: Their ecology and relationships with soils and land use, Sydney, Academic Press, Inc. LIAO PH, VIZCARRA AT, CHEN A and LO KV (1995), `A comparison of different bulking agents for the composting of fish offal', Compost Science & Utilization, 3, 80±86. LOEHR RC, NEUHAUSER EF and MALECKI MR (1985), `Factors affecting the vermistabilization process', Water Research, 19, 1311±1317. MANU-TAWIAH W and MARTIN AM (1987), `Study of operational variables in the submerged growth of Pleurotus ostreatus mushroom mycelium', Applied Biochemistry and Biotechnology, 14, 221±229. MARTIN AM (1991), `Peat as an agent in biological degradation: peat biofilters', in Martin AM Biological Degradation of Wastes, London, UK, Elsevier Applied Science, 341±362. MARTIN AM (1994), Fisheries Processing, Biotechnological Applications, London, Chapman & Hall Ltd. MARTIN AM (1996), `The role of lactic acid fermentation in bioconversion of wastes', in Bozoglu TF and Ray B. Lactic Acid Bacteria: Current Advances in Genetics, Metabolism and Applications, NATO ASI Series H, Cell Biology, Berlin, Springer-Verlag, 98, 219±252. MARTIN AM (1998) `Fisheries waste biomass: bioconversion alternatives', in Martin, AM HARRIS GD, PLATT WL

514

Maximising the value of marine by-products

Bioconversion of Waste Materials to Industrial Products, 2nd edn, London, Chapman & Hall, 449±479. MARTIN AM and BEMISTER PL (1994), `Use of peat extract in the ensiling of fisheries wastes', Waste Management & Research, 12, 467±479. MARTIN AM and CHINTALAPATI SP (1989), `Fish offal-peat compost extracts as fermentation substrate'. Biological Wastes, 27, 281±288. MARTIN AM and DECKER S (2000), Composting of fisheries wastes, in Shahidi F Seafood in Health and Nutrition, St. John's, Science and Technology Publishing Co, 387±394. MARTIN AM and PATEL TR (1991), `Bioconversion of wastes from marine organisms', in Martin AM Bioconversion of Waste Materials to Industrial Products, London, Elsevier Applied Science, 417±440. MARTIN AM, EVANS J, PORTER D and PATEL T R (1993a), `Comparative effects of peat and sawdust employed as bulking agents in composting', Bioresource Technology, 44, 65±69. MARTIN AM, ACHEAMPONG E and PATEL TR (1993b), `Production of astaxanthin by Phaffia rhodozyma using peat hydrolysates as substrate', Journal Chemical Technology Biotechnology, 58, 223±230. MATHUR SP (1991), `Composting processes', in: Martin AM Bioconversion of Waste Materials to Industrial Products. London, Elsevier Science Publishers Ltd, 147± 183. MATHUR SP (1998), `Composting processes', in Martin AM Bioconversion of Waste Materials to Industrial Products, 2nd edn, London, Blackie Academic & Professional, 154±193. MATHUR SP and JOHNSON WM (1987), `Tissue-culture and sucking mouse tests of toxigenicity in peat-based composts of fish and crab wastes', Biological Agriculture and Horticulture, 4, 235±242. MATHUR SP, DAIGLE JY, LEVESQUE M and DINEL H (1986), `The feasibility of preparing high quality composts from fish scrap and peat with seaweeds or crab scrap', Biological Agriculture and Horticulture, 4, 27±38. MATHUR SP, DAIGLE JY, BROOKS JL, LEVESQUE M and ARSENAULT J (1988), `Composting seafood wastes', Bio Cycle, 29, 44±49. MILLER FC (1992), `Biodegradation of solid wastes by composting', in Martin AM Biological Degradation of Wastes, London, Elsevier Applied Science, 1±30. MINNICH J and HUNT M (1979), The Rodale Guide to Composting, Emmaus, PA, Rodale Press. MISHRA SL and TIWARI TN (1993), `Ecological role and digestive enzymes of earthworms ± a review', in Tripathi AK, Srivastara AK and Pandey SN Advances in Environmental Sciences, New Delhi, Ashish Publishing House, 263±277. MOTE CR and GRIFFIS CL (1980), `Variations in the composting process for different organic carbon sources', Agricultural Wastes, 2, 215±223. MUELLER JC (1972), `Peat in pollution abatement', in Proceedings Symposium Peat Moss in Canada, University of Sherbrooke, QueÂbec, 274±289. NAKASAKI K, SHODA M and KUBOTA H (1985a), `Effect of temperature on composting of sewage sludge', Applied Environmental Microbiology, 50, 1526±1530. NAKASAKI K, SASAKI M, SHODA M and KUBOTA H (1985b), `Effect of seeding during thermophilic composting of sewage sludge', Applied Environmental Microbiology, 49, 724±726. NEUHAUSER EF, LOEHR RC and MALECKI MR (1988), `The potential of earthworms for managing sewage sludge', in Edwards CA and Neuhauser EF, Earthworms in

Composting of seafood wastes 515 Waste and Environmental Management, The Hague, SPB Academic Publishing bv, 9±20. REGENSTEIN JM (1986). `The potential of minced fish', Food Technology, 40, 101±106. SATCHELL JE (1983), `Earthworm microbiology', in Satchell JE Earthworm Ecology from Darwin to Vermiculture, New York, Chapman & Hall, 351±363. Ä IZ-BALDELLOU JM (1985), `Application of pyrolysis-gas SEREÂS-ASPAX A and ALCAN chromatography to the study of composting process of barley straw and peartree wood', Journal of Analytical and Applied Pyrolysis, 8, 415±426. SHAHIDI F (1992), `Nutrients of fisheries by-products and their potential use in feed and compost formulations', Proceedings of the 1991 Fisheries By-Products Composting Conference, Madison, Virginia, Sea Grant Institute, University of Wisconsin, 73±79. SHAHIDI F (1995), `Role of chemistry and biotechnology in value-added utilization of shellfish processing discards', Canadian Chemical News, 9, 25±29. STENTIFORD EI (1996), `Composting control: principles and practice', in de Bertoldi M, Sequi P, Lemmes B and Papi T, The Science of Composting, Part 1, Glasgow, Blackie, 29±59. SYERS JK and SPRINGETT JA (1984), `Earthworms and soil fertility', Plant and Soil, 76, 93± 104. TAN KH (1996), Soil Sampling. Preparation and Analysis, New York, Marcel Dekker Inc. TOMLIN AD, SHIPITALO MJ, EDWARDS WM and PROTZ R (1995), `Earthworms and their influence on soil structure and infiltration', in Hendrix P.F Earthworm Ecology and Biogeography in North America. Boca Raton, FL, CRC Press Inc, 159±183. VAN DER WERF P (2004), `Quality in the bag', Solid Waste Recycling, Oct/Nov, 46±52. Â ZQUEZ M and MARTIN AM (1998), `Mathematical model for Phaffia rhodozyma growth VA using peat hydrolysates as substrate', Journal Science of Food and Agriculture, 76, 481±487. VEIGA MC, MEÂNDEZ R and LEMA JM (1994), `Waste water treatment for fisheries operations', in Martin AM Fisheries Processing. Biotechnological Applications, London, Chapman & Hall Ltd, 344±369. VINCESLAS-AKPA M and LOQUET M (1997), `Organic matter transformations in lignocellulosic waste products composted or vermicomposted (Eisenia fetida andrei): Chemical analysis and 13C CPMAS NMR spectroscopy, Soil Biology and Biochemistry, 29, 751±758. VIRARAGHAVAN T and AYYASWAMI A (1987), `Use of peat in water pollution control: a review', Canadian Journal Civil Engineering, 14, 230±233.

Index

ACE activity inhibition 240±1 acetic acid 161±2, 332±3 acetolysis 351±2 acetyl CoA 414 acetylation 347±8 deacetylation 345±7 degree of 346±7 acid-aided method 75±84, 152±61, 199±200, 205±6, 215±16 acid ensilage method 419 acid hydrolysis 120, 148±9, 441±2 preparation of chitin and chitosan 345, 350±3 acid method for collagen 129±31, 283±4, 332±3 acid method for gelatin 285±7, 298, 332±3 acid-soluble collagen (ASC) 129±31, 132, 282, 283±4 acidic processes for protein recovery 161±2 see also acid-aided method acidification 56, 57 acidity adjustment 362±3 acidolysis 114 actin 70 degradation and temperature 213 actinidin 121 activated charcoal 299 active aeration 495 aeration 490±2, 493

active 495 passive 495±6 PAWS 497±501 vermicomposting 504, 505 aesthetic deterioration 96 affinity chromatography 384 agriculture 364 alanine 324 collagen and gelatin 288, 289, 290, 291, 292, 293, 294 Alaska 4, 435, 461 Alcalase 123, 124, 125±7, 232 alcoholysis (transesterification) 114, 465±7 aldehydes 31 algae antioxidants from marine algae 398±403 biomass added to composting operations 495 pigments from 424±5, 427, 428 algal oil 268±9, 270 alginate 86±7 alkali-aided method 75±84, 152±61, 199±200, 205±6, 215±16 alkali-catalysed transesterification 466±7, 476 alkaline hydrolysis 120, 149 alkaline pretreatment 282±3 alkaline process for gelatin 285, 286, 287, 298

Index alkaline processes for protein recovery 162 see also alkali-aided method alkylation 348±9 -carotene 415, 416 -linolenic acid (ALA) 23, 259 -tocopherol 29±30, 32±3 amino acids 12 antioxidant properties 404 collagen and gelatin composition 288, 289, 290, 291, 292, 293, 294 nutritional properties 292±3 recommended pattern for children and infants and 292, 295 content of fish mince/surimi 216±17 essential 80±2 hydrophilic 311±12, 320, 321, 323 hydrophobic 319, 320, 321, 323 seafood flavour amino acid compensation of hydrolysate 311±12 clam 318 lobster 309±10, 311±12, 319±20 red hake 320 taste active free amino acids 318 ammonia 479 vermicomposting and 505±7 ammonium sulphate 382±3 amphiphiles 464 anaerobic contact reactor (ACR) 468, 469 anaerobic digestion 467±71, 479±80 problems of bio-gas production 477±9 anaerobic filter 468, 469, 478±9 anaerobic phased solids digester (APS-Digester) 470±1 anaerobic sequencing batch reactor 468, 474, 475 anchovy 260 angiogenesis inhibition 453 animal feeds 66, 239±40, 435±49 antifungal activity 359±60, 362 antimicrobial activity 358±60 antioxidants 11, 32, 34, 35, 250, 397±412 chitosan 363, 406±7 FPH 237±9 from marine algae 398±403 from marine animals and their by-products 404±7 from other marine sources 407±8 appetite suppression 451, 452 aquaculture xxi, 17, 34±5, 61, 88, 145, 171 feeds 17, 435±49

517

FPH 239±40, 443 global production 66, 67, 374, 450 aqueous extraction 305 arachidonic acid 23 argentine 178 arginine 456 ash 462±3 aspartic proteinases 118 astaxanthin 134 antioxidant 400, 408 pigment 416, 418, 420, 421, 422, 427 ATPase activity 161 autolysis and FPH 14±15, 118, 120, 231 spoilage 48±9, 53, 56 see also fish sauce; fish silage avrainvilleol 402 Baader flesh-bone separator 94±5, 98 backbones 4, 5, 6, 7, 8 removal prior to flesh-bone separation 99 bacteria 408 see also microorganisms baffle flow reactor 469 bandages 355 batch digester with leachate recirculation 469, 470 batch reactor 469, 474 beef plasma protein (BPP) 82 belly flap/trimmings 186 belt and drum flesh-bone separators 93, 94±5, 98, 201 -carotene 415, 416 -N-acetylhexosaminidase 379 binary ice (slurry ice) 177 bioactive compounds 450±9 collagen 452±3 elastin 454, 456 FPH 128±9 future trends 456 protamine 455±6 protein powder as bioactive ingredient 250 proteoglycans 454±5 squalamine 451±2, 456 see also health; medical applications; pharmaceutical applications biodegradation of wastes 487±90, 491 see also composting bio-diesel 460±85 future research needs 479±80 problems concerning production of 476±7

518

Index

quality and quantity of seafood processing by-products 461±3 theories and technologies for producing 463±7 yields and properties 471±3 bio-gas 460±85 future research needs 479±80 problems concerning production of 477±9 quality and quantity of seafood processing by-products 461±3 theories and technologies for producing 467±71 yields and properties 473±6 biological oxygen demand (BOD) 464 biological quality of compost 507±8 biological separation techniques 92 biological value (BV) 69 biomass-retaining digesters 468 biotechnology 387±8 bitterness 236, 308, 315 black caviar 182 black scabbard fish 174±5 `blackspot' formation 425 bleaching 269±71 blood pressure 240±1 blood thinning 240 bloom strength 289±90 blubber oils 265, 267±8, 271±2 blue-green algae 424 body weight control 355 bone meal 440 bones 108, 109, 187±8 backbones see backbones calcium from 330±6 biochemical properties 330±1 calcium and phosphorus composition 330±1 calcium solubilisation using fish bone peptide 333±5 degradation of bone 331±3 in vivo availability of soluble calcium complex 335±6 utilisation of calcium and organic compound 331±5 enzymatic extraction of collagen 129±31, 132 flesh-bone separators 92, 93±5, 98, 99, 198, 201, 207 products and feeds 439 botargo 183±4 bovine spongiform encephalopathy (BSE) 280, 450, 453 boxes/boxing, fish 176

branched (cyclic) fatty acids 23 bromelain 121 bromophenols 401, 402 bulking agents 494, 503 PAWS 497, 498±501 vermicomposting 507 butylated hydroxyanisole (BHA) 34, 250, 397 butylated hydroxytoluene (BHT) 34, 250, 397 by-catch 107, 144±5, 171±9, 189±90 discards, by-products and 171±3 using 174±9 By-Catch Bank 173 by-products 304±5 by-catch, discards and 171±3 components and manufacture of feeds 437±8 composition of fish by-products 3, 4, 462±3 and their applications xxi±xxii definition 108 estimate of available quantities 108±9, 437 driving forces for utilisation of 436±7 general scheme for utilisation of by-products 180 quantity and quality 461±3 and their possible use 109 see also waste calcium xxii, xxiii±xxiv, 188, 328±39 biochemical properties of fish bone 330±1 in vivo availability of soluble calcium complex from fish bone 335±6 industrial uses 328 recommended intake 328±9 sources in foods 329 utilisation of fish bone calcium and organic compound 331±5 calcium-fortified foods 329, 335±6 calcium phosphate 329 cancer 451, 454 canthaxanthin 416, 418, 420, 421 capelin oil 261, 262 protein hydrolysates 404±5 roe 184 capsules 297 capture fisheries, global xxi, 3, 26, 47, 66, 67, 171 carbon dioxide 467 see also bio-gas

Index carbon to nitrogen ratio 492, 493 cardiovascular disease 272 Carophyll Pink 420, 421 Carophyll Red 420 carotenoids xxii, xxiii, 399±400, 414±22, 426±8 comparison with synthetic colorants 420±2 measurement of colour 422 preparation and stabilisation 419 properties and functions 417±18 uses in food and feed products 420 carotenoproteins xxiii, 133±4 carp cultured 5, 6, 7, 13 osteocalcin 330, 334±5 wild 5, 6, 7, 13 cartilage 131±3, 453 cartilaginous fish 292 casein phosphopeptides (CPP) 333±6 Casson equation 252 catalase 101 catching methods 61 catfish oil 263, 264 cattle 293 caviar 181±2 production using marine enzymes 386 see also roe cellulose 340, 341, 342 cellulose acetate 382 centrifugation 76±8, 155±6, 299 decanter centrifuges 84±5, 203, 206 `champagne' method 177 characterisation 16±17 cheeks 92±3 using 179±81 chemical hydrolysis 119, 120, 148±9 see also acid hydrolysis chemical oxygen demand (COD) 464 chemical preservation 53, 55±6 chemical processing methods 92, 144±67 chemical hydrolysis 119, 120, 148±9 see also acid hydrolysis fish protein concentrate 146±8 fish protein isolates 75±84, 152±62, 163, 199±200, 205±6, 215±16 future trends 162±3 surimi processing see surimi processing chemical quality of compost 507±8 chilling 52±5, 176±7 chitin xxii, xxiii±xxiv, 133±4, 340±73 applications of chitin and its oligomers 340±1, 344, 353±64

519

agricultural 344, 363 food 344, 357±63 industrial 344, 364 medical 344, 354±7 and composting 494±5 enzymatic hydrolysis and preparation of chitin and its oligomers 350±2 N-alkylation for improved solubility 348±9 production 345±6 safety and regulatory status 364±5 structure and properties 341±5 trapping-retention of heavy metals 349±50 chitinase 379 chitinolytic enzymes 379 chitosan xxii, xxiii±xxiv, 133±4, 340±73 antioxidant 363, 406±7 applications of chitosan and its oligomers 340±1, 344, 353±64 agricultural 344, 363 food 344, 357±63 industrial 344, 364 medical 344, 354±7 coatings see coatings comb-shaped 348 deacetylation and molecular weight and its activity 346±7 depolymerisation and N-acetylation 347±8 enzymatic hydrolysis and preparation of chitosan and its oligomers 352±3 production 345±6 safety and regulatory status 364±5 structure and properties 341±5 trapping-retention of heavy metals 349±50 chitosan-alginate treatment 86±7 chitosan-oxychitin 357 chlorella 423±4 chlorophylls 399, 414, 422±4 cholesterol 156, 241 chondroitin sulphate (CS) 131±3 chondroitins xxiv chromatin 444 chromatography 384 chymotrypsin 14, 387 citric acid 332±3 clam flavour production 316±19 clarification agents 295, 297 Clostridium botulinum 222 coastal fleet 47, 49±50 see also on-board handling

520

Index

coatings chitosan edible coatings 361, 406±7 chitosan-oxychitin for prosthetic articles 357 cod 5, 6, 7, 12±13, 290, 295 composition of by-products 463 enzymes from 377, 384, 386 oil from 260, 264 coffee 362±3 cold-adapted enzymes 375 cold-water fish 281, 288, 290 collagen 8±9, 188, 279±303, 330 enzymatic extraction of collagen and collagen-derived products 129±31, 132 food applications 295 gelatinisation into fish glue 15 key drivers of marine collagen 279±81 manufacture 281±5 stabilising 287 medical and pharmaceutical applications 452±3 properties 288±95 chemical 288 nutritional 292±5 physical 288±91 quality improvement 298±9 sources of marine collagen 281 collagenolytic enzymes 377±8 colorants see pigments colour fish mince 97, 98 masking colour 103 whitening methods 99±103 frame mince 209 measurement of 422 protein isolates and surimi 157±8 comb-shaped chitosan derivatives 348 composting 486±515 biodegradation of seafood wastes 487±8 biological aspects 501±3 characteristics of composting of seafood wastes 494±5 future trends 509 limitations of traditional methods 495 operational parameters 490±3 quality considerations 507±9 technological aspects 495±501 vermicomposting 502, 503±7 computer vision equipment 52, 175±6 concentration marine enzymes 381±3 and seafood flavour 306

quality 322±4 connective tissue (stroma proteins) 70 consistency index 252 consumers 196 safety concerns and collagen and gelatin 280 contamination 428 continuously stirred tank reactor 469 cooking juice 305, 321 cook loss 234±6 coral 407 Corolase 232 coronary heart disease 24 cosmetic products 450±9 covering compost 496 crab flavour 321 crab sticks, flakes and chunks 149±50, 220±2 crustaceans see shellfish cryoprotectants 33, 97 cryoprotective properties of FPH 236 surimi 150, 151±2, 199, 218 cryptoxanthin 415±16, 417 CSW (chilled sea water) 54, 55, 176±7 culture media 242 curcudiol 408 curcuphenol 408 cut-offs (trimmings) 4, 5, 6, 7, 8, 186 cuttlefish 294 cymopol 402 cysteine 71 cysteine proteinases 118 cystoketal 402 Cystoseira 398, 399, 401, 402 dairy products 296, 329 dark muscle 70, 73, 211±12 surimi from 152 deacetylation 345±7 decanter centrifuges 84±5, 203, 206 degree of acetylation (DA) 346±7 degree of hydrolysis (DH) 127, 232±3, 321±2 antioxidative properties of FPH 238 degumming 269 dehydration see drying demersal species 98 demineralisation 283 denaturation enzymes in enzymatic hydrolysis 233 proteins 161 protein powders 254±5 denaturation temperature 131, 132 deodorising 271

Index depolymerisation 347±8 dermatan sulphate (DS) 454 deskinning 386 deterioration lipids 30±2 see also oxidation of lipids marine biomass 47±9, 53 dewatering 151, 199 diesel 472, 473 see also bio-diesel diet (of fish) and fatty acid composition 28±30 modification and fish quality 32±3 see also feeds Digestase 386 digestive system 240 3,4-dimethoxyphenol 408 dimethylsulphoniopropionate (DMSP) 403 1,1-diphenyl-2-picrylhydrazyl (DPPH) 408 discards 171±3 see also by±catch; underutilised species disulphide bond 71 docosahexaenoic acid (DHA) 22, 23, 24, 25, 110, 259±61, 272 docosapentaenoic acid (DPA) 24, 259±61, 271±2 dogfish 292 Dranco digester 470 dried fish maws 185±6 dried heads 58 drying 53, 55 drying methods and seafood flavour quality 322±4 freeze-drying 322±4, 383 gelatin 287 low temperature (LT) drying 439 DSC thermograms 254±5 earthworms see vermicomposting economic factors 17 collagen and gelatin 280±1 pigments 426±7 eicosanoids 23 eicosapentaenoic acid (EPA) 22, 23, 24, 25, 110, 259±61, 271, 272 Eisenia fetida 504 elastin 454, 456 emulsification properties 237 emulsions containing protein powders 251±4 flow properties analysis 251±2 viscoelastic properties 253±4

521

endogenous enzymes see autolysis endopeptidase activity 231±2 endoproteases 118, 231±2, 306±8 environment disposal of waste at sea 174 impact of PAWS composting 500±1 pigments and 426±7, 428 enzymatic hydrolysis 10±11 chitin and chitosan 345, 350±3 for FPH 118±29, 135, 230±3, 442 lipids 31, 111±18, 135 seafood flavour manufacture 306±8 cooking juice vs hydrolysate in lobster and crab flavour production 321 hydrolysis conditions 321±2, 323 seafood flavours from processing by-products 308±19 source of enzymes 307±8 source of raw material 308 enzymatic methods 107±43 bio-diesel production 476±7 by-products extracted by 108±9 collagen manufacturing 129±31, 132, 282, 285 extraction of antioxidants 403 future trends 135±6 shellfish industry by-products 133±4 skin, bones, fin, scales, cartilage 129±33 TGase modification and gelatin 298±9 traceability of by-products 134±5 see also enzymatic hydrolysis enzymes and composting 502 enzymatic activities in protein-rich by-products 13±16 enzymatic spoilage 48±9, 53, 56 marine enzymes see marine enzymes Escherichia coli (E. coli) 359 essential amino acids 80±2 esterification 111, 113±18 ethanol 146, 147, 148 ethylenediaminetetraacetic acid (EDTA) 283 European Union (EU) regulations 174 exopeptidases 118, 231±2, 306±8 fatty acid methyl esters see bio-diesel fatty acids 22±4, 49 composition in selected marine organisms 260, 261 factors affecting fatty acid composition of fish and by-products 28±30 in fish by-products 26±7

522

Index

in fish muscle 25±6 marine oils 266 algal oils 269, 270 marine mammal oils 266±8 monounsaturated 23, 266, 271 omega-3 xxii, xxiii, 22, 23, 75, 258±61, 444 omega-6 23, 258±9 PUFAs see polyunsaturated fatty acids unsaturated 23±4, 403 see also fish oils; lipids; marine oils feeds 17, 388, 435±49 benefits of feeds made from seafood processing wastes 443±4 by-product components 437±8 driving forces for utilisation of by-products 436±7 feed products from fish by-products 438±41 FPH 239±40 future trends 444±5 methods of producing hydrosylates and silage 441±2 pigments in 420, 426±7 fermentation 305±6 production of hydrolysates 442 using marine enzymes 385 fertilisers 66, 486 FPH 241±2 spreading waste 489, 491 fillet 108, 109 fillet blocks 96 filleting waste 91±8 by-products from 92, 145 recovery of flesh from 92±8 filtration 299 membrane filtration 206, 381±2 microfiltration 207, 208, 209±10 finfish 304, 308 fins 129±33, 188 fish bone peptides (FBP) calcium solubilisation using 333±5 in vivo effects on calcium bioavailability 335±6 fish frame protein see frame mince fish mince 8, 58, 92, 95±104, 150, 196±228 advantages and disadvantages 198 by-products 204±5 functional properties 210±16 future trends 103±4 machinery for preparation 200±4 manufacturing 197±8 nutritional characteristics 216±17

problems of 97±8 protein recovery methods 98, 205±7 quality and improvement 99±103 storage stability 217±19 comparison with surimi 218±19 factors affecting 217±18 mince from surimi by-products 219 use in surimi 210 utilisation 219±23 fish oil by-products 475±6 fish oils 26, 33, 57, 58, 73±5, 461, 462±3 characteristics 462 enzymatic extraction methods 109±18, 135 feeds 435, 436±7, 439±40, 443±4 health effects 24±5, 109±11 from isoelectric precipitation and solubilisation 76±8, 80, 155±6 manufacturing process 269±71 from processing by-products 261±5 see also lipids; marine oils fish protein concentrates (FPC) 11, 146±8, 490, 491 fish protein hydrolysates (FPH) xxii±xxiii, 10±11, 146, 229±48, 250, 256, 490, 491 antioxidants 404±6 feeds 239±40, 443 FPC as substrate for making 148 future trends 242±3 methods of production 441±2 autolysis 14±15, 118, 120, 231 enzymatic hydrolysis 118±29, 135, 230±3, 442 on-board processing 56±8 physiological role in humans and animals 239±41 properties 128±9, 234 biological activities 128±9 functional properties 128 role in food systems 234±9 role as growth media for microorganisms 242 role in plant growth and propagation 241±2 using marine enzymes 385±6 fish protein isolates 152±62, 163 other processes using low or high pH 161±2 pH-shift processing 75±84, 152±61, 199±200, 205±6, 215±16 fish sauce 10, 58, 118, 188±9, 231, 305, 385

Index fish silage 9±10, 56, 57, 118, 441±2, 486, 490, 491 fish solubles 438, 440 Fishbase 59 fishing regions 28±30 fishmeal 58, 91, 338, 489±90, 491 and feeds 435, 436±7, 438±9, 443, 444 flavour, seafood see seafood flavour flavour enhancers 324 Flavourzyme 122, 232, 307±8, 314 seafood flavour clam flavour production 317±19 hydrolysis conditions 321±2, 323 lobster flavour extracts 309±10, 311 flesh-bone separators 92, 93±5, 98, 198, 201, 207 removal of backbone prior to separation 99 flesh recovery 91±106 from demersal species 98 from filleting waste 92±8 future trends 103±4 quality and improvement of fish mince 99±103 flocculants 84, 85, 86 flow behaviour index 252 flow properties analysis 251±2 fluidised-bed reactor 468, 469 fluorohydrolysis 352 Food and Environment Protection Act 1985 (UK) 174 food preservation methods see preservation methods food supplements 250 chitosan 355±6 collagen and gelatin 293±5 FPH 241, 243 food systems, role of FPH in 234±9 fractionation 383±4 frame mince 187, 206±7 characteristics 209 utilisation 210 frames 4, 5, 6, 7, 8, 487 fish flavour from white fish frames 313±15 using 187±8 frankfurter analogue 223 freeze-drying flavour products 322±4 marine enzymes 383 freezing/freeze-storage 13, 33, 53, 178 frozen mince blocks 96 stability of fish mince/surimi 217±19 surimi 203±4

523

freshness 212±13 freshwater fish 23±4 fruit, preservation of 362 fruit juices 362 fucolls 400±1 fucosylated chondroitin sulphate (FucCS) 455 fucoxanthin 414, 418 fuel oil 462 see also bio-diesel fuhalols 400±1 functional foods 249, 256 functional properties alkali-aided and acid-aided processes 158±61 enzymatically hydrolysed proteins 128 fish mince/surimi 210±16 protein powders 249±57 protein-rich by-products 11±16 fungal protease 122 Gadidae species 5, 6, 7 gadiform species by-products generated during filleting 50, 51 utilisation of by-products from 59±61

-carotene 415, 416 gear selectivity 175, 189 gel formation fish mince/surimi 211 gelatin 288±91 marine enzymes 386±7 pH-shift processes 158±61 gel strength 289±90 gelatin 8±9, 15, 129, 188, 279±303, 332±3 food applications 295±7 key drivers of marine gelatin 279±81 manufacture 281±2, 285±7 stabilising 287 non-food applications 297 properties 288±95 chemical 288 nutritional 292±5 physical 288±91 quality improvement 298±9 sources of marine gelatin 281 gelatin desserts 295±6 genetic engineering 456 genetic identification method 134±5, 136 global catch xxi, 3, 26, 47, 66, 67, 171 global fisheries production 66±8, 171, 172, 374 glucosamine xxiv glutamic acid 318±19, 324

524

Index

glycine 319, 324 collagen and gelatin 288, 289, 290, 291, 292, 293, 294 grading 175±6 Grateloupia filicina 398 greater sand eel 178 gummy-type confectionery products 296±7 gutting 16, 50±1 habitat 11±16 haddock 5, 6, 7 haem proteins 157, 211±12 haemoglobin 239 hagfish 292 hake 290 red hake flavour 313±15, 320 halal food certification 280 harp seal 293 header/gutter machine 200 heads 4, 6, 7, 109 dried heads 58 fatty acids 27 using 179±81 health FPH and 240±1, 250 lipids and 24±5, 109±11 pigments and 417±18, 423, 424 proteins and 250 PUFAs and 24±5, 271±2 health effects and PUFA sources 109±11 see also medical applications heat treatment 53, 55 heavy metals 428 chitin, chitosan and heavy-metal complexes 343 trapping-retention 349±50 water purification 364 heparin 354, 455 herring oil 263, 265 roe 184 high-pressure treatment 99 Hizikia fusiformis 398 homogenisation 76 hot-water fish 281, 288, 291 hurdle technology 55±6 hybrid sludge-bed filter (HSBF) reactor 475 hydraulic retention time (HRT) 475 hydrocarbons 266 hydrochloric acid 283 hydrocolloids 97

hydrogen bonds 69, 70±1 hydrogen peroxide 99±101, 102 hydrolysates see fish protein hydrolysates (FPH); protein hydrolysates hydrolysation plant 58±9 hydrolysis chemical 119, 120, 148±9 see also acid hydrolysis degree of 127, 232±3, 238, 321±2 enzymatic see enzymatic hydrolysis hydrolysis conditions 321±2, 323 hydrophilic amino acids 311±12, 320, 321, 323 hydrophilic colloids 101 hydrophobic amino acids 319, 320, 321, 323 hydroxyapatite (HA) crystals 330 7-hydroxycymopol 402 hydroxyproline 288, 289, 290, 291, 292, 293, 294 hypercholesterolemic activity 356 hyperoxidase (catalase) 101 ice/icing 176, 177 ice-water fish 281, 288, 289 Iceland 4, 174 By-Catch Bank 173 Individual Quota System 173 imaging technology 52, 175±6 immune system 240 impurity removal 299 individual variations 11±16 indoles 403, 408 industrial wastewater treatment 364 inoculation (seeding) 502 inoculum 477±9 interesterification 114 interfacial/surface properties 236±7 invertebrates antioxidants from 407±8 collagen and gelatin from 281, 288, 294 iodine 428 ion exchange chromatography 299, 383±4 ionic strength (IS) 70 isoelectric point 72, 73 isoelectric precipitation and solubilisation 75±84, 152±61 equipment considerations 82±4 isopentenyl pyrophosphate (IPP) 414, 415 isopropanol 146, 147 jawless fish 292 jellyfish 294

Index kamaboko 220 kazunoko 184 kazunoko kombu 184 kelp 184 ketones 31 4-ketozeaxanthin 408 Kjeldahl method 127 Kojizyme 122 Kompogas digester 470 kosher food certification 280 krill 67, 68, 260 processing 65 proteases 377, 388 proteins recovered from 80±2 land, discarding of wastes on 489, 491 landfilling 489, 491 leachates 500 lenses 425±6 ling 5, 6, 7 linolenic acid 23, 24 lipases 48±9, 378±9 lipase-catalysed hydrolysis and esterification 111, 113±18 lipase-catalysed transesterification 476 lipids xxii, xxiii, 4, 22±46, 211 composition of fish wastes 462, 463, 464 deterioration 30±2 enzymatic extraction 31, 109±18, 135 concentration of n±3 PUFAs 111 lipase-catalysed hydrolysis and esterification 111, 113±18 protease-catalysed hydrolysis 111±13, 115 factors affecting fatty acid composition of fish and by-products 28±30 fatty acids found in fish by-products 26±7 fatty acids found in fish muscle 25±6 future trends 34±5 health benefits 24±5, 109±11, 271±2 implications for fish fat by-product valorisation 32±4 mincing and degradation of 95±6, 97 oxidation see oxidation of lipids properties of lipids in seafood 73±5 recovery in acid- and alkali-aided processes 76±8, 80, 155±6 see also fish oils; marine oils; polyunsaturated fatty acids (PUFAs) lipolysis 31, 48±9 liquefaction 306

525

liver oils 186±7, 266 livers 27, 186±7 livestock feeds 66, 239±40, 435±49 lobster flavour extracts 309±13 cooking juice vs hydrolysate 321 flavour-imparting compounds and chemistry 319±20 loss modulus 253±4 low temperature (LT) drying 439 lumpfish 290 caviar 183 lungfish 291 lutein 416, 417 lycopene 415 lyophilisation (freeze±drying) 322±4, 383 lysine 82 maatjes 385 mackerel 291 crude proteinases from mackerel intestine 122 macular degeneration 451 mammals, marine see marine mammals marine-derived tocopherol (MDT) 29±30 marine enzymes 374±96 chitinolytic 379 collagenolytic 378 future trends 388±9 lipases 378±9 polyphenoloxidases 380±1 producing from seafood processing by-products 381±4 concentration 381±3 extraction/solubilisation 381 fractionation/purification 383±4 proteolytic xxiii, 11, 14, 376±7 transglutaminases 379±80, 386±7 utilisation of 375, 384±8 marine mammals collagen and gelatin 281, 288, 293 oils from 265±8 health effects 271±2 marine oils 258±78 algal oil 268±9, 270 fish oils see fish oils health effects 24±5, 109±11, 271±2 manufacturing process 269±71 marine mammal oils 265±8, 271±2 from processing by-products 261±5 see also fish oils; lipids; polyunsaturated fatty acids (PUFAs) marshmallows 296±7 masking colour 101±3

526

Index

maws 185±6 MaxFish 60 meat preservation and chitosan 361 tenderisation using marine enzymes 387 mechanical aeration devices 496 mechanical separation see flesh-bone separators mechanised fish filleting 65 medical applications bioactive compounds 450±9 chitin and chitosan 344, 354±7 FPH 240±1 marine enzymes 387, 388 see also health Mediterranean caviar 183±4 megrim 291 Melancor-NH 426 melanins 414, 425±6 melting point 290 membrane disruption 381 membrane filtration 206, 382 menhaden oil 260, 261, 262 menopause 335 mentaiko 183 mercaptan derivatives 349±50 mesophilic temperature range 468 metallocollagenases 377 metalloproteinases 118 methane 467 problems of bio-gas production 477±9 see also bio-gas methanol 465±6 supercritical methanol method 476±7 microemulsions 464±5 microfiltration 207, 208, 209±10 microorganisms algal oil 268±9, 270 antimicrobial activity of chitin and chitosan 358±60 antioxidants from marine bacteria 408 chitosan and preservation of food 360±2 FPH as growth medium 242 growth/activity and deterioration of seafood 48, 53, 56 microbial fermentation for hydrolysates 442 pigments from algae 424±5, 427, 428 role in composting 501±2 milk 329 milt 4, 6, 7, 9, 185 minced fish see fish mince

minerals xxii, xxiii±xxiv, 214 content of fish mince/surimi 216 minke whale 293 modified atmosphere packaging 33 molecular weight 347 monosodium glutamate (MSG) 324 monounsaturated fatty acids (MUFAs) 23 marine mammal oils 266, 271 MSI-1436 451±2 muscle 69±70, 73 fatty acids in 25±6, 27 isoelectric precipitation and solubilisation of muscle proteins 75±84 myofibrillar proteins 70, 211 solubility 78, 79 myogen-aggregation phenomenon 205 myosin 70, 207, 208 degradation and washing cycles 213 N-acetylation 347±8 N-alkylation 348±9 National Environmental Law Center (NELC) (US) 86 natural savoury flavours 324 neurological system 240 neutralisation 269 Neutrase 124, 126 Newlase A 122, 126 nitric oxide 455 nitrogen 492, 493 nitrogen, phosphorus and potassium (NPK) composition 508±9 Norway 3±4 regulation of disposal of waste at sea 174 Norway pout 178 Norwegian reference meal 439 nucleotides 318±19, 444 nutrients, soil 364, 506 nutrition feeds made from seafood processing wastes 443±4 properties of collagen and gelatin 292±5 properties of fish mince/surimi 216±17 o-phtaldehyde (OPA) method 127 obesity 451 ocean disposal 173±4, 460±1, 486, 487 ocean trawlers 47, 50±2 see also on-board handling octopus roe 185

Index odour problems 489, 491, 496, 500 off-flavour 363 oleaginous microorganisms 268±9, 270 oleic acid 23 oligomers, chitin and chitosan 340±1 applications 344, 353±64 preparation 350±3 omega-3 fatty acids xxii, xxiii, 22, 23, 75, 258±61, 444 see also marine oils omega-6 fatty acids 23, 258±9 `Omega Bread' 25 on-board handling 16, 32, 47±64, 173±4, 189±90 conservation and stabilisation 52±6 deterioration of marine biomass 47±9 future trends 61 processing 53, 56±9 and sorting 16, 49±52 utilisation of by-products from gadiform species 59±61 waste from 173±4 optimisation of extraction process 298 Oregon seafood plant 86 organic solvent extraction process 419 ornithine decarboxylase (ODC) 455 osmometry 127 osteocalcin 330, 334±5 osteoporosis 328, 335±6 ovariectomised rats 335±6 overfishing 144 oxidation of lipids 30±2, 35, 95±6, 97, 115±18, 156±7, 397 antioxidants see antioxidants deterioration of marine biomass 49, 53, 56 implications for by-product valorisation 32±4 oyster 260 packaging 33±4 papain 121, 125 particle settling velocity 83±4 particle size 84, 85, 86 passive aeration composting 495±6 passively aerated windrow system (PAWS) 497±501 application to composting of seafood wastes 497±8 environmental impact 500±1 peat as a bulking agent 497, 498±500, 501 pastilles 297 peat 497, 498±500, 501, 503, 507

527

pelagic species 144, 145 Penzim 387, 389 pepsin soluble collagen (PSC) 129±31, 132, 282, 285 pepsins 121, 376, 386 peptides 48, 251 fish bone peptides 333±6 peptide size and characterisation of hydrolysates 127±8 pH chemical processing methods for protein recovery 161±2 see also pH-shift processing and composting 493 vermicomposting 505, 506 and protein gel texture 79±80 solubility of fish mince 200 wash water and surimi/fish mince properties 214 pH-shift processing 75±84, 152±61, 199±200, 205±6, 215±16 pH-stat technique 127 pharmaceutical applications bioactive compounds 450±9 gelatin 297 see also medical applications phenolic compounds 400±1, 402, 500 phlorethols 400±1 phlorotannins 400±1 phosphate 218 phospholipases 378 phospholipids 31, 73±5, 156 phosphorus 508±9 in fish bones 330±1 photography 297 PhotoProtective Technologies 425±6 phycocyanin 424±5 phycoerythrin 424±5 physical quality of compost 507±8 phytoene 414±15 pigments 413±32 carotenoids 414±22 chlorophylls 414, 422±4 economic, environmental and safety considerations 426±7 future trends 427±8 melanins 425±6 phycocyanin and phycoerythrin 424±5 types and sources 414 pigs 444 gelatin from skin of 293, 295 plants antioxidants from 397, 398 growth and propagation 241±2

528

Index

pollack 290, 405±6, 463 polyphenoloxidases 380±1 polyphenols 400 polyphosphates 101 polysaccharides 403 polyunsaturated fatty acids (PUFAs) 23±5, 26, 29, 31 concentration of n±3 PUFA 111 health benefits 24±5, 109±11, 271±2 marine oils 258±78 algal oil 268±9 fish oils 261±5 marine mammal oils 266±7 see also fish oils; lipids; marine oils potassium 508±9 potatoes 362 power law 252 prawns 319 precipitation 78 enzyme concentration by 382±3 isoelectric precipitation and solubilisation 75±84, 152±61 preservation methods 16 chitin, chitosan and 360±2 on-board 52±6, 176±8 pretreatments, in collagen manufacture 282±3 processing 66±7 chemical processing methods see chemical processing methods on-board 53, 56±9 physical methods and flesh recovery 92±8 recovery of by-products from seafood processing streams 65±90 isoelectric precipitation and solubilisation 75±84, 152±61 protein recovery from surimi processing water 84±7, 145±6, 149±52, 154±5, 205±6, 209±10 profitability analyses 60 proline 324 collagen and gelatin 288, 289, 290, 291, 292, 293, 294 propanal 406, 407 propyl gallate (PG) 34, 397 Protamex 124, 307 protamine 11 medical and pharmaceutical applications 455±6 proteases 107, 118, 332 enzymatic hydrolysis using added proteases 119±27 marine enzymes xxiii, 11, 14, 376±7

protease-catalysed hydrolysis 111±13, 115 protein hydrolysates antioxidants 404±6 fish protein hydrolysates (FPH) see fish protein hydrolysates protein powders 249±57, 440±1 as bioactive ingredients 250 emulsions containing 251±5 flow properties analysis 251±2 viscoelastic properties 253±4 functional properties 250±1 future trends 256 thermal properties 254±5 protein±protein interactions 71±3 proteins xxii±xxiii, 3±21, 69±73, 211 biological activities of enzymatically hydrolysed proteins 128±9 characteristics of by-products from surimi wash water 207±8 chemical processing methods 144±67 content in by-product fractions 5±8 enzymatic extraction methods 118±29, 135, 230±3, 442 autolysis vs enzymatic hydrolysis 118 enzymatic hydrolysis with added enzymes 119±27 quantification of proteolysis extent 127±8 extraction of carotenoid pigments using 419 fish frame protein 206±7 fish protein concentrates (FPC) 11, 146±8, 490, 491 fish protein hydrolysates (FPH) see fish protein hydrolysates fish protein isolates see fish protein isolates in fish wastes 462±3 functional properties of enzymatically hydrolysed proteins 128 isoelectric precipitation and solubilisation 75±84, 152±61 mincing and 95, 97 protein-rich by-products 3±21 future trends 17 implications for by-products valorisation 16±17 overview 4±11 physical and chemical properties 11±16 recovery from surimi processing water 84±7, 205±6

Index water-insoluble proteins 209±10 Protemax 232 proteoglycans 454±5 proteolysis 48, 160 marine proteolytic enzymes xxiii, 11, 14, 376±7 proteolytic enzyme inhibitors 152 quantification of proteolysis extent 127±8 psychrophilic temperature range 468 puffer fish 291 purification 383±4 pyrolysis 464±5 pyruvate 414 quality of compost products 507±9 drying methods and quality of seafood flavour 322±4 fish mince and 99±103 improvement for collagen and gelatin 298±9 rancidity 30±4, 35, 363 red algae 424 red caviar 182 red fish 175 red hake, flavour from 313±15, 320 refiner 202 refrigeration technology 52±5 see also chilling; freezing/freeze storage regulations regulatory status of chitin and chitosan 364±5 waste disposal 174, 426, 436±7, 460±1 response surface regression (RSREG) 126 ribonucleotides 324 rigor mortis 213 roe 4, 6, 7, 8, 9, 12 production using marine enzymes 386 using 181±5 rosemary 34 rotary evaporated flavour products 322±4 rotary screen dehydrator 202 rotary screen-treated protein 207, 208 roundnose grenadier 178 Rozym 386 RSW (refrigerated sea water) 54, 55, 176±7 safety chitin and chitosan 364±5

529

consumers' concerns and collagen and gelatin 280 pigments 426±7, 428 saithe 5, 6, 7 salmon 5, 6, 7, 290, 426 composition of by-products 463 enzyme-catalysed process for lipid extraction 112±13 oil 260, 262±3, 264 roe 182±3 salmon caviar 182 salt soluble collagen 282 sand eel 178 sarcoplasmic proteins 70 and gel formation 160±1 solubility 78, 79 sardine 260 Sargassum horneri 399 Sargassum micracanthum 398, 399 saturated fatty acids 23 sausage casings 295 scales 129±33 Schiff base formation 349 screw cylinder flesh-bone separators 93 screw press 202±3 scytonemin 408 sea clam processing by-product 316±19 sea cucumber 294 roe 185 sea lettuce 424 sea urchin 294 roe 184±5 seafood flavour 304±27 aqueous extraction 305 enzymatic hydrolysis 306±8 enzyme-assisted seafood flavours from processing by-products 308±19 clam flavour 316±19 flavour from white fish frames 313±15 lobster flavour 309±13 fermentation 305±6 flavour-imparting compounds and chemistry 319±24 concentration, drying methods and flavour quality 322±4 cooking juice vs hydrolysate 321 formulation of seafood flavour extract 324 hydrolysis conditions 321±2, 323 lobster 319±20 red hake 320 species-specific compounds 324 future trends 325

530

Index

seafood nuggets 222 seafood patty 222 seafood sausage 223 seal 293 blubber oils 265, 267±8, 271±2 seasonal variation and fatty acid composition 28, 30 functional properties of fish mince/ surimi 212 properties of protein-rich by-products 11±16 seed germination tests 508 seeding (inoculation) 502 selective gears 175, 189 semi-continuous digesters 474, 478 sensory properties, and seafood flavour clam 317±18 drying method and 322±4 lobster 311±12, 313 red hake 314±15 separation 306 sequential batch anaerobic composting (SEBAC) digester 468 serine 324 serine collagenases 377 serine proteinases 118 sesquiterpenequinones 408 sexual maturity 212 shark 292 cartilage 131±3, 453 liver oil 261±2, 263 skin collagen 453 shellfish 185, 304 composting wastes from 494±5 enzymatic methods for by-products processing 133±4 preparation of chitin and chitosan from by-products 345±6 seafood flavour 308 wastes from processing 108, 109 and pigments 426±7 shrimp alkaline phosphatase (SAP) 387, 388 shrimp fishery 65 by-catch 144±5, 172, 174 side chains 70±3 silage, fish 9±10, 56, 57, 118, 441±2, 486, 490, 491 silent cutter/mixer 203 single cell oils (SCOs) 268±9, 270 size exclusion chromatography 128, 383 skin 4, 6, 7, 8, 12, 109 deskinning using marine enzymes 386

enzymatic extraction of collagen 129±31, 132 fatty acids 27 using 188 skipjack tuna 260 slurry ice (binary ice) 177 smooth muscle 69±70 sodium chloride 324 bio-gas production 477±8 soil conditioner 509 soil enrichment 364 soil nutrients 364, 506 sole 291 solid waste treatment 468±71 solubilisation calcium solubilisation using fish bone peptide 333±5 isoelectric precipitation and solubilisation 75±84, 152±61 marine enzymes 381 solubility chitin 341±3 N-alkylation for improved solubility 348±9 chitosan 341±3 of fish mince and pH 200 FPH 234, 235 proteins 251 solubles, fish 438, 440 sonolysis 352 sorbitol 218 sorting 16, 49±52, 61, 175±6 see also on-board handling soy protein hydrolysate 324 speciality ingredients 66 species fatty acid composition 28±9 and functional properties 11±16 fish mince/surimi 211±12 and storage stability of fish mince/ surimi 217 taste active compounds specific to 324 traceability of by-products 134±5, 136 sphagnum peat 498, 499 Spirulina 423±4 sprats 178 spray-dried flavour products 322±4 spreading (wastes) 489, 491 squalamine 451±2, 456 stabilisation carotenoid pigments 419 collagen and gelatin 287 Staphylococcus aureus 359 starches 97

Index starfish 294 stick water 438, 440 stomachs 185 storage by-product fractions 15±16 stability of fish mince/surimi 217±19 comparison between fish mince and surimi 218±19 factors affecting 217±18 mince from surimi by-products 219 storage modulus 253±4 striated muscle 69±70 strictaketal 402 string lettuce 423±4 stroma proteins (connective tissue) 70 sturgeon caviar 29, 181±2 sugars 97, 218 sulphited phlorotannins 400±1 super-absorbent hydrogel (SAH) 82 supercritical methanol method 476±7 supplements, food see food supplements surface/interfacial properties 236±7 surimi 8, 58, 96, 196±228 by-products 204±5 functional properties 210±16 machinery for preparation 200±4 manufacturing 198±200 conventional method 198±9 new technology 199±200 nutritional characteristics 216±17 protein recovery from surimi processing water 84±7, 145±6, 149±52, 154±5, 205±6 alkaline conditions 162 characteristics of by-products 207±8 water-insoluble proteins 209±10 storage stability 217±19 comparison with fish mince 218±19 utilisation 219±23 swim bladders 185±6 synthetic colorants 420±2 tablets 297 tarako 183 taste active compounds 324 taste active free amino acids (TAFAA) 318 tea extract 34 temperature anaerobic digestion 468 composting and 492, 493 vermicomposting 505, 506 storage/processing and fish mince/ surimi 213

531

wash water 214 terpenoid phenols 407±8 tert-butylhydroquinone (TBHQ) 397 tetraprenyltoluquinols 401±2 thermal gravimetric (TG) analysis 255 thermophilic temperature range 468 thrombosis 454, 455 tilapia 291, 295 lipase from stomach and intestine of 378 time, storage/processing 213 tissue engineering 452±3 titanium dioxide 101±3 tocopherols 402 tongues 92±3 using 179±81 total suspended solids (TSS) 464 traceability 134±5, 136, 190 transesterification 114, 465±7 transglutaminases (TGases) 379±80, 386±7 TGase modification and gelatin 298±9 triacylglycerols (TAG) 22, 26, 31, 73, 74, 265±6, 268 reducing bodily concentrations of 272 see also fatty acids; fish oils; lipids; marine oils Trichoderma harzianum 360 trimethylamine oxide (TMAO) 95 trimethylamine oxide (TMAO) demethylase 209 trimmings (cut-offs) 4, 5, 6, 7, 8, 186 trinitrobenzenesulphonic (TBNS) acid method 127 tropomyosin 70 troponin 70 trout 80±2 trypsin 14, 121, 376, 387 tryptophan 149 tumour cell inhibition 356 tuna 260 tuna intestinal enzymes (TICE) 331±2 tuna pyloric caeca crude proteinase (TPCCP) 122, 332 tusk 5, 6, 7 ulcer healing 356±7 ultrafiltration 299, 382 ultraviolet (UV) radiation 425 Umamizyme 124, 126 underutilised fish parts 179±89 belly flap/trimmings 186 fins and skins 188 frames 187±8

532

Index

heads, cheeks and tongues 179±81 liver 186±7 maws 185±6 milt 185 roe 181±5 stomachs 185 underutilised species 144, 145, 171±9, 189±90 by-catch, by-products and discards 171±3 key drivers for using 173±4 upgrading of 177±9 using 174±9 United Kingdom 4 unsaturated fatty acids 23±4, 403 upflow anaerobic filter reactor 469 upflow anaerobic sludge blanket (UASB) reactor 468, 469, 477 V cut 201 vacuum packaging 33±4 Valorga digester 470 value-added foods 66 vegetable oil 419 vermicomposting 502, 503±7 process parameters 504±6 seafood wastes 506±7 viscera 4, 5, 6, 7, 12, 109 fatty acids 27 gutting 16, 50±1 sorting 52 viscoelastic properties 253±4 viscosity 290±1 vitamin A 417 vitamin E 29±30, 32 volatile compounds 320 von Post classification system 499 warm-water fish 281, 288, 291 warmed-over flavour (WOF) 363 washing 99, 150 and functional properties of surimi and fish mince 213 effect of wash water and washing conditions 214 storage stability of fish mince/surimi 217±18 surimi manufacture 198±9 waste 108 alternatives for uses 488±90, 491 characteristics of seafood wastes 487

from primary processing 91±2 see also by-products waste disposal 489, 491 composting see composting problem of 450±1 regulations 174, 426, 436±7, 460±1 at sea 173±4, 460±1, 486, 487 wastewater characteristics 463, 464 treatment anaerobic digesters 468, 469 chitin and chitosan 364 water aqueous extraction of seafood flavour 305 content and composting 492, 493 vermicomposting 505 content of seafood wastes 462±3 dewatering 151, 199 properties in aquatic foods 68±9 protein±water interactions 70±3 purification using chitosan 364 used in surimi/fish mince preparation 214 water-binders 163 water gel desserts 295±6 water-holding capacity 13, 68±9 water-insoluble proteins 209±10 water jet deboning 207 water-soluble chitin (WSC) 354 water-soluble protein 205±6 wax esters 265±6 weight control 355 wet reduction process 438 whale blubber oils 265±6, 272 white fish frames, fish flavour from 313±15 white muscle 70, 73 whiteness of fish mince 97, 99±103 hydrogen peroxide 99±101, 102 titanium dioxide 101±3 windrows 495±6 PAWS 497±501 wound healing 354±5 xanthan gum 102±3 xanthophylls xxii, xxiii yeast 324 zeaxanthin 416, 417